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",isbn:"978-1-80356-552-1",printIsbn:"978-1-80356-551-4",pdfIsbn:"978-1-80356-553-8",doi:null,price:0,priceEur:0,priceUsd:0,slug:null,numberOfPages:0,isOpenForSubmission:!0,isSalesforceBook:!1,isNomenclature:!1,hash:"4c2e03f295fbc697350f0bf3bf89a14f",bookSignature:"Associate Prof. Murat Eyvaz, Dr. Ahmed Albahnasawi, M.Sc. Ercan Gürbulak and MSc. Mesut Tekbaş",publishedDate:null,coverURL:"https://cdn.intechopen.com/books/images_new/11840.jpg",keywords:"Aridity and Drought, Precipitation and Evapotranspiration, Land Use, Human Activity, Desertification, Desert, Soil Structure, Water Treatment, Water Scarcity, Irrigated Agriculture, Remote Sensing, Climate Change",numberOfDownloads:47,numberOfWosCitations:0,numberOfCrossrefCitations:0,numberOfDimensionsCitations:0,numberOfTotalCitations:0,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"March 10th 2022",dateEndSecondStepPublish:"May 11th 2022",dateEndThirdStepPublish:"July 10th 2022",dateEndFourthStepPublish:"September 28th 2022",dateEndFifthStepPublish:"November 27th 2022",dateConfirmationOfParticipation:null,remainingDaysToSecondStep:"14 days",secondStepPassed:!0,areRegistrationsClosed:!1,currentStepOfPublishingProcess:3,editedByType:null,kuFlag:!1,biosketch:"Dr. Murat Eyvaz has co-authored many journal articles and conference papers and has taken part in many national projects. He serves as an editor in 51 journals and a reviewer in 125 journals indexed in SCI, SCI-E, and other indexes. He has four patents on wastewater treatment systems. Dr. Eyvaz's research interests include applications in water and wastewater treatment facilities, electrochemical treatment processes, and filtration systems at the lab.",coeditorOneBiosketch:"Dr. Albahnasawi is a pioneering researcher in environmental sciences and engineering, he has co-authored numerous journal articles and conference papers on water and wastewater treatment, and waste remediation. Recently, his research interests are the application and designing of Microbial Fuel Cell integrated with Fenton oxidation for industrial wastewater treatment/solid waste management and monitoring of organic micropollutants by both chromatographic and spectrophotometric analyses.",coeditorTwoBiosketch:"Dr. Gurbulak is a pioneering researcher in environmental sciences and engineering. He has co-authored numerous journal articles and conference papers on water and wastewater treatment, and advanced waste remediation technologies. His research interests are the application and designing of hydrothermal processes for industrial wastewater treatment/solid waste management and monitoring of organic micropollutants by both chromatographic and spectrophotometric analyses.",coeditorThreeBiosketch:"Dr. Tekbaş is a pioneering researcher in environmental sciences and engineering, he has co-authored numerous journal articles and conference papers on water and wastewater treatment, and advanced waste remediation technologies. His research interests are the application and designing of supercritical water oxidation processes for wastewater treatment/solid waste management and electrochemical analyses.",coeditorFourBiosketch:null,coeditorFiveBiosketch:null,editors:[{id:"170083",title:"Associate Prof.",name:"Murat",middleName:null,surname:"Eyvaz",slug:"murat-eyvaz",fullName:"Murat Eyvaz",profilePictureURL:"https://mts.intechopen.com/storage/users/170083/images/system/170083.png",biography:"Dr. Murat Eyvaz is an associate professor in the Environmental Engineering Department, Gebze Technical University, Turkey. His research interests include applications in water and wastewater treatment facilities, electrochemical treatment processes, filtration systems at the lab and pilot-scale, membrane processes (forward osmosis, reverse osmosis, membrane bioreactors), membrane manufacturing methods (polymeric membranes, nanofiber membranes, electrospinning), spectrophotometric analyses (UV, atomic absorption spectrophotometry), chromatographic analyses (gas chromatography, high-pressure liquid chromatography). He has co-authored many journal articles and conference papers and has taken part in many national projects. He serves as an editor and reviewer for many indexed journals. Dr. Eyvaz has four patents on wastewater treatment systems.",institutionString:"Gebze Technical University",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"5",totalChapterViews:"0",totalEditedBooks:"6",institution:{name:"Gebze Technical University",institutionURL:null,country:{name:"Turkey"}}}],coeditorOne:{id:"323629",title:"Dr.",name:"Ahmed",middleName:null,surname:"Albahnasawi",slug:"ahmed-albahnasawi",fullName:"Ahmed Albahnasawi",profilePictureURL:"https://mts.intechopen.com/storage/users/323629/images/system/323629.png",biography:"Dr. Ahmed Albahnasawi is a post-doctorate fellow in the Environmental Engineering Department, Gebze Technical University, Turkey. His graduate work focused on the investigation of the treatability of the sequential anoxic-aerobic batch reactors followed by ceramic membrane for textile wastewater treatment. Based on his Ph.D. research, Dr. Albahnasawi published three journal articles and participated in three international conferences. His research interests include the application and design of a microbial fuel cell integrated with Fenton oxidation for industrial wastewater treatment/solid waste management and monitoring of organic micropollutants by both chromatographic and spectrophotometric analyses.",institutionString:"Gebze Technical University",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"0",totalChapterViews:"0",totalEditedBooks:"0",institution:{name:"Gebze Technical University",institutionURL:null,country:{name:"Turkey"}}},coeditorTwo:{id:"176699",title:"M.Sc.",name:"Ercan",middleName:null,surname:"Gürbulak",slug:"ercan-gurbulak",fullName:"Ercan Gürbulak",profilePictureURL:"https://mts.intechopen.com/storage/users/176699/images/system/176699.png",biography:"Dr. Ercan Gürbulak is a research associate in the Environmental Engineering Department, Gebze Technical University, Turkey. He received his bachelor’s degree in Environmental Engineering from Marmara University, Turkey, in 2005. He completed his MSc and Ph.D. at Gebze Technical University in 2008 and 2019, respectively. His research interests include the application and design of hydrothermal processes for industrial wastewater treatment/solid waste management and monitoring of organic micropollutants by both chromatographic and spectrophotometric analyses.",institutionString:"Gebze Technical University",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"1",totalChapterViews:"0",totalEditedBooks:"0",institution:{name:"Gebze Technical University",institutionURL:null,country:{name:"Turkey"}}},coeditorThree:{id:"189677",title:"MSc.",name:"Mesut",middleName:null,surname:"Tekbaş",slug:"mesut-tekbas",fullName:"Mesut Tekbaş",profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002bRIbNQAW/Profile_Picture_1644828776099",biography:null,institutionString:"Gebze Technical University",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"0",totalChapterViews:"0",totalEditedBooks:"0",institution:{name:"Gebze Technical 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vectors",isOpenForSubmission:!1,hash:"c9e622485316d5e296288bf24d2b0d64",slug:"anopheles-mosquitoes-new-insights-into-malaria-vectors",bookSignature:"Sylvie Manguin",coverURL:"https://cdn.intechopen.com/books/images_new/3092.jpg",editedByType:"Edited by",editors:[{id:"50017",title:"Prof.",name:"Sylvie",surname:"Manguin",slug:"sylvie-manguin",fullName:"Sylvie Manguin"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}}]},chapter:{item:{type:"chapter",id:"49866",title:"Smart Delivery Systems with Shape Memory and Self-Folding Polymers",doi:"10.5772/62199",slug:"smart-delivery-systems-with-shape-memory-and-self-folding-polymers",body:'Polymers are a group of materials that are versatile in their nature spanning from industrial materials such as synthetic plastics to biopolymers. Synthetic or naturally occurring, their properties depend on the nature of the constituent monomers, the sequence of the monomers, the length of the whole polymer, and the type of bonds they contain in their structure. In the past 20 years, a new group of polymers is recognized to exhibit interesting properties, namely shape memory and self-folding. Polymers that exhibit these properties are now the subject of intensive research for their potential applications in biomedical area and drug delivery. In this chapter, we will first introduce the principles of shape memory and then give a summary of the work done in this field. In the following section, we introduce the concept of self-folding and polymer origami, which is a subsection of self-folding. Recent research done in this exciting area is then presented, and future outlook on shape memory and self-folding polymers is specified.
The main characteristics of shape memory materials (SMMs) in general are that they show a plastic deformation (temporary shape) when an external stimulus is applied and are able to recover to their original shape from the temporary shape. This is also called “shape memory effect” (SME) [1].
One of the classes of shape memory materials is shape memory polymers (SMPs), which respond to external stimulus by offering mechanical action. Network elasticity of the shape memory polymer determines if SMP remembers one or more shapes [2]. Dual-shape memory polymers, which are going to be specified later on, are the first SMPs.
Until the external (shape memory-driving) stimulus is applied to trigger the shape recovery, the temporary shape of SMP is stable.
Temperature, light, electric field, magnetic field, pH, specific ions, or enzyme can be used as stimulus in order to change the shapes of shape memory polymers. The shape change can occur rapidly from temporary shape to the permanent one [3–5]. Figure 1 summarizes the relationship between polymer composition and structure, stimulus, and shape memory function.
In late 1970s, CdF Chimie Company (France) developed the first shape memory polymer called polynorbornene [6]. One year later, it was named as Norsorex by Nippon Zeon Company (Japan) and was commercialized [7]. Other commercial shape memory polymers which are named Kurare TP-301 and Asmer were produced by Kurare Corporation (Japan) and Asahi Company (Japan), respectively [8]. Widely used polyurethane (PU)-based SMP was developed by Mitsubishi Heavy Industries [4].
Another important branch of shape memory materials is shape memory alloys (SMAs). However, shape memory alloys have disadvantages among other types of shape memory materials, such as high manufacturing cost, toxicity, limited recovery, and complicated surgical problems. Shape memory polymers, ceramics, and hydrogels are preferred recently for their advantages compared to shape memory alloys [9].
The shape memory effect can be originated by combining polymer structure and morphology by processing and programming methods. The material properties are not related with SME [10].
The unique properties and major advantages of SMP materials are summarized below:
SMPs are lightweight and allow substantially higher elongations, which have properties for various technical applications [11].
SMPs have wide range of glass transition temperatures from −70°C to +100°C.
Since they have a wide range of glass transition temperatures, their stiffness can be tailored. Also, SMPs can be processed easily [12].
SMPs have shape recovery property up to 400% of plastic strain. (Shape memory alloys have 7–8%).
Between the glassy and rubbery states of SMPs, large reversible changes of elastic modulus can be observed (as high as 500 times) [13].
Shape memory polymers have high elastic deformation (strain up to more than 200% for most of the materials). They have low density and biodegradable materials.
Most of the SMPs are biocompatible materials which allow them to be used in potential medical applications.
SMPs are easy to process. They are applicable to molding or extrusion. They have low cost [13].
Shape memory polymers have low thermal conductivity compared to shape memory alloys. This feature can be useful if shape memory foams are used a as insulation materials [14,15].
They can use many external stimuli and triggers. There are many alternative ways to actuate shape recovery such as light, magnetic field, electricity, and water [16].
As mentioned previously, shape memory polymers and their composites can recover their original shapes after large deformation when subjected to an external stimulus [17]. This external stimulus can be temperature [18,19], magnetism [20–22], moisture [23], or light [18,24].
Among these shape memory polymers, the thermoresponsive SMPs are the most common type [25,26]. Thermally induced shape memory polymers have a wide range of applications in biomedical fields, [27] such as drug delivery, implanting, suture, and wound healing [28].
The general thermomechanical cycle of thermoresponsive shape memory polymers consists of the following steps at a macroscopic level:
Programming
Storage
Recovery [29].
This cycle is depicted in Figure 1. The first step is the fabrication of the shape memory polymers into an original shape. Second, the polymer is heated above the thermal transition temperature (
Thermomechanical cycle of thermoresponsive shape memory polymers.
At the molecular level, shape memory polymers offer shape memory effect by its two components, which are cross-links and switching segments. These SMPs can include chemically cross-linked networks or physically cross-linked copolymers. Physically cross-linked copolymers are usually made of linear block copolymers which have hard and switching segments. Their permanent shape is stabilized by hard segments, whereas temporary shape is fixed by switching segments [30]. Cross-links are formed by covalent bonds and physical interactions (i.e., molecular entanglements) [5,31]. For covalently cross-linked shape memory polymers, reshaping the materials is hard since the shape is fixed by covalent bonds. These types of polymers are called thermosets [32].
Polymer includes two separate domains in physical cross-linking case. The domain consists of two parts which are hard and reversible switching segments. The hard segment, which acts as net point, is related to the highest thermal transition temperature. Reversible switching segment or molecular switches are related to the second highest thermal transition temperature. These types of polymers are called thermoplastics [33]. Figure 2 shows the hard and soft segments during the shape memory process. The lines show the net points and square dots show the switching segments.
Hard and soft segments during shape memory effect.
Rousseau et al. reported that epoxy polymers (neopentyl glycol diglycidyl ether NGDE) have tunable glass transition temperatures by changing the cross-link density and chain flexibility. Ranging up to 89oC, excellent shape fixity and recovery were observed [34].
Zhou et al. reported that PDLLA/HA (poly D,L-lactide co-glycolide/hydroxyapatite) composites have desirable shape memory effects and the amount of HA particles in composites played an important role during the shape memory recovery. Since PDLLA has remarkable properties such as shape memory effect, biodegradability, biocompatibility, easier availability, and osteoconductivity of HA, these composites have potential in biomedical applications such as minimally invasive surgery (MIS) and bone and tissue repair [35].
Lendlein et al. developed a thermoresponsive shape memory polymer which consists of oligo(ε-caprolactone) diol (OGL) and crystallizable oligo(p-dioxanone) (ODX) as switching and hard segments, respectively. A fiber of this thermoplastic SMP was used to close the wound by loosely knotting the fiber and then when temperature increased to
In another study, Ashby et al. reported that poly(ε-caprolactone) (PCL) and poly(octylene adipate-co-meso-2,5-diazodipate) shape memory polymers were actuated by switching temperature. This new developed SMP would have potential applications in the biomedical field [36].
When an external stimulus is applied on SMP, the temporary shape becomes permanent shape. In order to obtain the temporary shape again, another external force or stress is required. This procedure shows that shape memory behavior is nonreversible or “one-way”, and it has a wide range of applications [37].
The most basic procedure of polymer shape memory effect is described in Figure 1. First, the shape memory polymer is heated to a transition temperature or deformation temperature (
The thermal transition temperature, which is associated with switching domains, has various types. The melting transition (
If we take a look at the macroscopic shape of shape memory polymer, when it has a permanent shape, its molecular chains are thermodynamically stable because of the highest entropy of SMP conformation. When SMP is heated above
One-way shape memory effect.
Basically, shape memory polymers are able to change their shapes between original and temporary shape under applied stress which is a type of one-way shape memory effect. Another type of one-way shape memory effect is triple shape memory effect. The main difference of triple shape memory effect (TSME) is having one intermediate shape between its original and temporary shapes. If there is more than one intermediate shape, it is called multiple shape memory effect for a more complicated motion generation [40]. Polymers blends, grafting and blocking copolymers, SMP hybrids, and polymer laminates are some of the ways of managing the triple shape memory effects [41]. Figure 4 shows the basic procedure of triple shape memory effect. As mentioned in the figure, there are two different thermal transition temperatures which are
Kumar et al. reported that grafted polymer network contains crystallizable poly(ethylene glycol) (PEG) side chains, and poly (ε-caprolactone) (PCL) shows triple shape memory effect [43]. The triple shape memory behavior of crystalline polyurethane was observed by Hu et al. [44]. Also, polyalkenemer and polyolefin-based polymer blends were used by Cuevas et al. in order to develop triple shape memory polymers which were two semicrystalline polymers (poly(cyclooctene) (PCO) and polyethylene (PE)) [42]. Xie et al. developed polymeric bilayers that consisted of two dual-shape memory polymers, and this new shape memory polymer bilayer shows triple shape memory effect [45]. Furthermore, Xie reported that perfluorosulphonic acid ionomer (PFSA) is able to show tunable shape memory effect which includes dual, triple, and quadruple without any change in the material composition [46].
Triple shape memory effect.
Most of the typical SMPs show one-way shape memory effect. A different type of SME which is the two-way shape memory effect can be observed in liquid crystalline elastomers and photo-actuated deformation polymers [47].
2W-SME can be quite useful; however, its high cost represents an obstacle for its widespread use. In 2W-SME process, a crystallization-induced elongation and melting-induced contraction is observed during cooling and heating, respectively. This condition originated from the formation of liquid crystalline structure [48]. The comparison of 2W-SME and 1W-SME is shown in Figure 5 briefly.
Chen et al. reported that the two-way shape memory behavior of SMP laminate was observed by bending upon heating from 25°C to 60°C and reverse bending upon cooling from 60°C to 25°C. Laminated layers were prepared from shape memory exhibiting polyurethane [49].
Zhou et al. developed self-folding poly(octylene adipate) (POA). Besides showing conventional one-way irreversible shape memory, this SMP is able to form one-way reversible shape memory during heating and two-way reversible shape memory upon heating and cooling cycles. The chemical cross-links of this semicrystalline elastomer are responsible for the memorization of the original shape, whereas the physical network constructs the temporary shape [50].
Comparison of two-way shape memory effect and one-way shape memory effect.
Certain magnetic materials tend to respond to magnetic fields and are capable of maintaining their magnetic properties after the external magnetic field is removed. Such magnetic particles are called as ferrimagnetic of ferromagnetic materials which are iron, nickel, cobalt, and some of their alloys [51]. Some magnetic materials such as iron oxides (Fe3O4, Fe2O3, etc.), when below a certain nanometer size, do not have any remaining magnetization once the external magnetic field is removed. These are superparamagnetic materials and can be preferred over ferromagnetic ones depending on the application.
Shape memory can be induced by the application of an external magnetic field due to magnetic heating. So magnetic induction is a type of thermal induction, where temperature change is driven by magnetic field due to the presence of magnetic material in the nanocomposite.
The magnetic SMP has several advantages:
By using magnetic heating, possible danger of overheating can be eliminated.
Since heat is not given to the system externally, any complex-shaped device can be actuated virtually.
This method is convenient for medical devices which can be actuated inside the human body, since they can be controlled in a wireless manner [52].
Hilt et al. reported that magnetic field can be used in order to heat the SMPs by remote heating. This heating method relies on magnetic nanoparticles within the polymer matrix in order to create heat via power loss when exposed to an alternating magnetic field [53].
According to the study of Razzaq et al., by changing the magnetic field parameters and amount of the Fe3O4 in the polyurethane polymer matrix, shape recovery of the helical bended strip of polyurethane filled with 20% volume microsized magnetite was observed [54].
Magnetically inducing method is an indirect method. So this method can be useful if shape memory polymer cannot be actuated by direct heating methods. However, Zhou et al. reported that for cross-linked poly(ε-caprolactone) polymers, magnetite composite shows better reactivity to hot water than alternating magnetic field [55].
In the research study of Schmidt, polymer network composites were implemented with different contents of superparamagnetic nanoparticles (Fe3O4) and butyl acrylate cross-linked with oligo (ε-caprolactone) dimethacrylate. As it can be seen in Figure 3, the permanent shape is deformed by heating above
Zhou et al. reported that poly (D,L-lactide) /magnetite (Fe3O4) nanocomposites show excellent shape memory effect when an alternating magnetic field was applied as a stimulus. Since PDLLA is biocompatible, these nanocomposites show potential for application as magnetically controlled smart implants in biomedical field [57].
According to the study of Puig et al., 8 wt% oleic acid-stabilized magnetic nanoparticles exhibited a temperature increase of 25°C at its surface when exposed to an alternating magnetic field in biomedical applications. Also, this temperature was enough to actuate the shape memory of the nanocomposite [58].
As observed, shape memory effect is usually induced by thermal stimulation by heating above the transition temperature of the polymers, such as the glass transition temperature or melting temperature. However, some other stimulating sources, such as electric field, may also be used in order to trigger polymers with shape memory [59].
Electricity can be used as a stimulus which enables resistive actuation of shape memory polymer filled with conductive fillers. By passing an electrical current, shape memory effect can be actuated easily in these nanocomposites. To date, most of the studies regarding electroactive SMPs composite are focused on thermoplastic SMP resins [60]. As a result of these, in recent studies, most of the electroactive shape memory polymers are well-dispersed carbon nanotube (CNT)-reinforced thermoplastic polyurethane (TPU) resin nanocomposites [61–63].
According to the study of Leng et al., thermoset styrene-based shape memory polymer nanocomposite filled with different amounts of nanosized carbon powder shows that with increasing nanocarbon powder amount, electrical conductivity increases. Also, it was reported that 10 vol.% nanocarbon powder shows good electroactive shape recovery property [64].
Zhou and coworkers fabricate cross-linked poly(ε-caprolactone) (cPCL) and conductive multiwalled carbon nanotubes (MWNTs). These nanocomposites exhibit excellent shape memory properties under the direct thermal and electrical stimulations. Therefore it was reported that this composite might be a promising prospect in biomedical applications [65].
Jung et al. obtained electroactive shape memory polymer composites by using polyurethane and three kinds of fillers. When polyurethane-multiwalled carbon nanotube composite is lightly coated with polypyrrole (PPy), new nanocomposite shows good electroactive shape memory properties when an electric voltage is applied [66].
As a trigger mechanism, water can be used for shape transition of shape memory polymers or shape memory polymer composites. Since solvent or water molecules can penetrate into the amorphous areas of shape memory polymers, this can result in a plasticizing effect on SMP molecules. Therefore the flexibility of the molecule increases and shape recovery is observed [67].
Chen et al. reported that water-induced poly(methacrylic acid)-grafted clay and thermoplastic polyurethane composite was developed with shape memory effect. Also, this new polymer–clay composite responds to pH changes and mechanically adaptive properties when water is exposed [68].
The polymer composite composed of carbon nanotubes and shape memory polyurethane (SMPU) was developed by Luo et al. The new and developed composite has shape memory-enhanced water-sensing property which can be used in smart polymer applications such as sensory materials [69].
Aqueous environments can be used as a trigger of shape memory effect. Nöchel et al. used different grafted copolymer networks (named CLEG) which consist of different ratios of the hydrophobic cross-linker poly(ε-caprolactone) diisocyanoethyl methacrylate (PCLDIMA) and hydrophilic poly(ethylene glycol) monomethyl ether monomethacrylate (PEGMA). The shape memory properties of this new hydrogel were studied by bending and uniaxial elongation experiments in the aqueous environments. As a result, CLEG has dual-shape capability and degradability and could be promising materials in biomedical applications [70].
Paakinaho et al. reported that the shape memory property of orientation-programmed PDLLA in an aqueous environment at 37°C was triggered by the combined effect of water molecules and thermal activation [71]. Mendez et al. developed new water-activated biomimetric nanocomposites by adding rigid cotton cellulose nanowhiskers (CNWs) into a rubbery polyurethane matrix. At a specific concentration of CNW, aqueous swelling and shape memory behavior were observed [72].
Self-folding is the ability of a material to fold and unfold without the external effects. Also without any external interference, such as human control, self-folding can be attributed to the self-assembly mechanism. These mechanisms can be patterned templates or thin films which can be folded, curved, or rolled-up to become spirals, tubes, and cylindrical tubes [73,74]. Self-folding can occur spontaneously or in response to stimuli such as light, pH [75], temperature, magnetic field, or solvent [76,77]. In Figure 6, self-folding mechanism which is provided by hinges is shown briefly.
Illustration of 3D and 2D self-folding mechanism.
Self-folding films have become attractive recently since there is a variety of polymers which are sensitive to different stimuli, and they can fold by responding external signals. Many polymers are able to change their properties with pH and temperature. For biotechnological applications, biodegradable and biocompatible polymers are favorable choices and are frequently encountered. Polymers can also undergo considerable and reversible changes of volume that allow design of systems with reversible folding [78]. Thermoresponsive self-folding films can be designed using continuous thermal expansion, melting shape memory transition of polymers which demonstrate the low critical solution temperature (LCST) in solutions [79].
Luchnikov et al. reported that a polymer bilayer consisting of a polystyrene (PS) layer on top of a poly(4-vinyl pyridine) (P4VP) layer is formed on the polished surface on the silicon wafer or other substrates (e.g., a glass slide) by means of dip or spin coating. Rolling, in this system, is achieved due to the swelling of the P4VP in a water solution of an acid, due to protonation of the pyridine rings [80]. In another study of Ionov, self-rolling due to swelling was observed. Fully biodegradable polysuccinimide shows self-rolling response with its polymer bilayers, and this results in the formation of microtubes [81].
Ionov et al. demonstrated a self-folding polymeric bilayer consisting of poly(methyl methacrylate) copolymer and poly(N-isopropylacrylamide) copolymer which can encapsulate an oily liquid. This new developed bilayer can be expressed as an anisotropic capsule, and the folding behavior is observed due to the swelling difference of the layers of the polymer film. Self-healing materials and drug delivery design are featured possible application areas of this approach [82]. A representative illustration of self-folding polymeric bilayer is shown in Figure 7.
Self-folding procedure of thermoresponsive polymeric bilayer.
Ionov et al. studied two different sets of thermoresponsive polymeric bilayer behaviors to different stimuli. Polymeric bilayers were selected as one passive layer which is hydrophobic or random copolymer, and the active layer is the thermoresponsive hydrogel. Thermoresponsive hydrogels show swelling and shrink response to increased or decreased temperature, whereas passive layers prevent swelling of the active layer. In this manner, nonuniform expand/shrink behavior is achieved. In other words, folding and unfolding can be observed due to swelling [83].
Huang et al. developed a heat-responsive poly lactic acid (PLA) structure which would provide good uses for customized deployable/retractable and biodegradable implant devices for individual patients [84].
Lee et al. developed that self-folding oral delivery device which is capable of providing enhanced mucoadhesion, drug protection, and targeted multidirectional delivery. The device is composed of three layers which are backing layer, a foldable bilayer, and a mucoadhesive layer entrapped with drugs. The swelling bilayer was made of pH-sensitive hydrogel cross-linked poly(methyacrylic acid) (PMAA) which swells during contact with body fluid whereas non-swelling layer is poly(hydroxyethyl methacrylate) (PHEMA), and it acts as a diffusion barrier in order to minimize drug leakage in the intestine. When mucoadhesive drug layer is attached on the bilayer, self-folding device attaches to the mucus first and then curls into the mucus due to the different swelling of the bilayered structure, leading to enhanced mucoadhesion [85].
Temperature-stimulated liquid crystal elastomer–polystyrene bilayers which can show complex shape changing behavior such as helical twisting, reversible folding, and patterned wrinkles were developed by Agrawal et al. Tailoring was achieved by changing film thickness and overall aspect ratio [86].
Huck et al. reported that microscale, quasi-2D composite objects are fabricated which can respond to the addition of salt and pH changes [87].
Schenning et al. developed accordion-like striped actuators made of liquid crystal polymer films, which show response to the pH or temperature. These actuators would be used in medical systems and microrobotics [88]. Another liquid crystal shape memory actuator was developed by Park et al. which shows different swelling behavior between its UV exposed and non-UV exposed sides [89].
Visible light-induced graphene oxide/poly(vinylidenefluoride-hexafluoro propylene) (PVDF-HFP) nanocomposite films were produced by Yu et al. Tumbler movement was observed on the films due to the photothermal effect of graphene oxide and shape memory effect of PVDF-HFP. This behavior can be improved for full-plastic devices actuated by visible light [90].
Ionov et al. developed self-assembled porous scaffolds with uniaxial tubular pores. By producing rectangular polymer bilayers which include hydrophopic (poly methyl-methacrylate-co-benzophenoneacylate) and stimuli-responding hydrophilic polymer (poly N-isopropylacrylamide-co-acylic acid co-benzophenoeacylate), pH-dependent rolling of bilayers was observed. Also, these bilayers are capable of controlled self-assembly [91].
Origami word is originated from the Japanese words ‘ori’ as in to ‘fold’ and ‘kami’ as the ‘paper.’ It is the art of paper folding from Japan and has been known worldwide [92]. Today, origami is not only the art of paper but also can be used for technological purposes by creating three-dimensional objects. In order to obtain these objects, fold pattern sequences should be provided to develop the desired object from a flat sheet [93].
Origami folding sequence of a cubic structure (a), illustration of polymer origami self-folding (b).
Origami enables the creation of complex structures unlike bilayers, which can only bend in one direction or fold multiple steps. The shape of self-folded structures depends on the initial shape of the film, the radius of curvature (depending on the layer thickness), and the presence of a substrate [94]. A typical origami folding structure is depicted in Figure 8(a). Space structure solar arrays, automobile airbags, shopping bags and cartons, photovoltaic cells, and biomedical are some of the uses of polymer origami structures [93].
As mentioned above, different stimuli can be used to trigger the material’s self-folding behavior as well as origami folding. Gracias et al. reported that self-folding functional microgrippers that combine a swellable, photo-cross-linked N-isopropylacrylamide-co-acrylic acid soft hydrogel with a non-swellable and hard-segmented polymer (polypropylene fumarate, PPF) show thermomechanical response to hydrogel where they can be used in surgical areas [95].
In a study, Baroud et al. manually cut geometric shape of polymethylsiloxane membrane layer placed on a hydrophobic surface. When a drop of water is added on a membrane and allowed to evaporate, as water volume decreases, thin membrane sheet wraps the liquid and forms 3D shape which shows the interaction between elasticity and capillary [96]. The illustration of this procedure is shown in Figure 8(b).
Gracias et al. developed three-dimensional (3D) microfabricated nanoliter containers by thermally actuating the 2D structure to fold into a 3D structure which is analogous to polymer origami. In this case, 2D template has smart hinges. Researchers suggested that this new fabrication method would be used for developing microscale biomedical devices in the future [97].
Ryu demonstrated photo-origami which actuates both flat and curved surfaces of polymer films in order to obtain 3-dimensional objects at a macroscopic scale via “localized photo induced stress relaxation” mechanism. Photo-origami is driven with amorphous, covalently crosslinked polymer which includes pentaerythritol tetra(3-mercaptopropionate) (PETMP), 2- methylene-propane-1,3-di(thioethylvinylether) (MDTVE), and ethylene glycol di(3-mercaptopropionate (EGDMP)). [93]. Gracias et al. developed photo-cross-linked polyethylene glycol (PEG)-based bio-origami hydrogel bilayers which can self-fold in aqueous solutions. These new developed bilayers conserve different kinds of tissues by self-folding which would be improvable considering the tissue engineering area [98].
The most important use of shape memory polymers is as drug carriers. Some drugs due to their stability of low solubility in the blood stream become more bioavailable when delivered in a carrier system. This may also allow the sustained release of the drug which can be desired if the drug is desired to be eluted over a long period of time. There are several drug delivery systems developed such as liposomal systems of biodegradable polymeric systems. Polymers offer a wide range of adjustable properties to be tailored for the desired drug elution system. Smart polymers are a new class of materials where novel drug delivery systems can be designed with. However, there are some criteria such as drug loading capacity, aqueous environment conditions, drug release pattern, and degradation behavior which determine the suitability of shape memory polymers as matrix materials for drug release or drug carrier applications [99].
Based on the requirements defined by a specific application, such polymer systems will allow selecting one specific polymer out of a larger number of related materials without the need to change to a totally different material chemistry. In addition to adjustable thermal properties, polymer systems can also be helpful to realize demanded mechanical properties or desired degradation rates of SMP implants [100].
For biomedical applications of SMPs, the capability to change their shape should be established at relevant conditions, i.e.,
About the controlled drug release ability of shape-memory polymers, Ohya et al. reported that chemically cross-linked networks of branched oligo(ε-caprolactone) polymer can perform sustained release of theophylline drug over a month without initial burst release in Phosphate Buffer Saline (PBS) at 37°C [102].
According to the study of Ameer et al., hydroxyl-dominant (HD) polydiolcitrates (HD polymers) can perform the subsequent release of hydrophobic dichlorofluororescein (DCF) drug. This means polydiolcitrate elastomers could benefit from smart biodegradable materials for tissue engineering applications [103].
Such systems are ideal for minimally invasive surgery where implants are inserted through a small incision, and after self-anchoring, they start to release a pharmacological agent. Dobrzyński et al. developed a shape memory polymer based on L-lactide, glycolide, and trimethylene carbonate in order to obtain double layer systems for paclitaxel drug delivery that paves the way for drug eluting stents [104].
Lendlein et al. reported that oligo (caprolactone-co-glycolide) dimethacrylate was combined with drug, and its activation was obtained between 28-42°C where shape memory effect was pronounced at body temperature. In this study, diffusion-controlled drug release was observed independent from polymer degradation [105].
Gong et al. reported that biodegradable polymeric cross-linked poly(ε-caprolactone) (cPCL) and poly(sebacic anhydride) (PSA) were developed, and release accumulation of drug can be enhanced by adding PSA into cPCL matrix. This shape memory polymer could be used as potential drug-eluting stents in biomedical field [106].
During the past decade, biomedical applications of shape memory materials became a very active area of research [107].
As mentioned in the previous section, the progress in surgical techniques, especially in minimally invasive surgery, allows these materials to be used widely in biomedical applications of polymers.
These smart materials are enabled to insert a bulky device in the body through a small keyhole incision in a temporarily fixed shape. After a precise position by the surgeon, such smart materials gain their application-relevant shape on demand [108].
Zhang et al. demonstrated a new nanofibrous, biomimetic, shape memory, and biodegradable poly(D,L-lactide-
Nelson et al. developed hydrogel bilayers composed of N-isopropylacrylamide with polyethyleneglycol diacrylate. By using the expansion difference of these layers due to swelling, self-folding behavior was observed. These new devices are switchable and show reversible shape transition against external stimuli. These layers were designed for different purposes in order to demonstrate microstructures which can preserve magnetic alginate microbeads when they have folded structures. After exposing to NIR laser source, microbeads are capable of being released from the microstructures. So, this method offers a solution for drug and cell delivery systems [110].
Tai et al. demonstrated an origami technique by constructing 3D spherical structure from 2D parylene-C (PA-C) film for intraocular implantation application [111]. Maitland et al. developed an SMP adapter in order to reduce the hemodynamic stress which arises from impingement of dialysis needle in an arteriovenous graft [112].
The most effective areas of biomedical applications of shape memory polymers are intravascular stents, treating aneurysm occlusion and clot removal.
Intravascular stents are predominantly used in main arteries or peripheral arteries, in the treatment of cardiovascular diseases (CVDs) which is the number one cause of death globally: more people die annually from CVDs than from any other cause [113]. First-generation stents are metallic, whereas second-generation ones are polymer-coated drug-eluting metallic stents. New-generation stents are biodegradable polymers that are also drug eluting. The potential advantages of pure polymer stents compared to bare metal stents include increased biocompatibility, biodegradability, increased drug loading, enhanced compliance matching, reduced cost, ease of fabrication for patient-specific devices, molecular surface engineering, and the use of shape memory effect [114].
Metallic stents are effective in hindering acute occlusion and reducing late restenosis after coronary angioplasty; however, many concerns still remain. Compared with metallic stents, poly-L-lactic acid (PLLA) stents are biodegradable and can deliver drugs locally, and these were developed by Igaki and Tamai. It was reported that feasibility, safety, and effectiveness of PLLA-biodegradable stents were verified in humans [115].
Wagner et al. reported that shape memory thermoplastic polyurethane is developed as a self-expandable stent, and it can be used as a drug delivery system [116].
A polymer system consists of
Chen et al. developed shape memory chitosan-based films (chitosan/PEO/glycerol) which are used to produce biodegradable stents. Chitosan cross-linked with epoxy compound stent’s most remarkable property shows rapid expansion from a crimped state by using the hydration in an aqueous environment as a stimulant. By using minimally invasive surgery techniques, this stent can be placed into an artery. In another study of Chen and coworkers, genipin cross-linked chitosan-based stents were developed. The possibility of using this newly developed genipin stent as a drug delivery vehicle was also examined by loading an anti-proliferation drug, sirolimus. Findings showed that the genipin stent with enhanced mechanical strength can be used as an attractive stent platform for local drug delivery [118,119].
Maitland et al. performed the design and fabrication of an SMP stent (MM5520 thermoplastic polyurethane) which was triggered by photothermal actuation at 40–45°C [120].
Wang and coworkers developed fully biodegradable polymeric stent that can self-expand at body temperatures (37°C), using the concept of elastic memory. This self-expansion is necessary in fully polymeric stents, to overcome the problem of elastic recoil following balloon expansion in a body vessel. Bilayered biodegradable stent prototypes were produced from poly-L-lactic acid and polyglycolic acid (PLGA) polymers [121].
An aneurysm is a formation of balloon-like bulge in an artery. This bulge is formed when a part of artery wall becomes weak which results in unusual widening. Aneurysms can occur in any of the artery; however, the most common ones are aortic, cerebral (in the brain), popliteal (in the leg, behind knee), mesenteric (in the intestine), and splenic artery aneurysm (in the spleen). Almost 13,000 Americans die annually from aortic aneurysms. Surgery is the possible option for the treatment of large aortic aneurysms [122,123].
Shape memory polymer foams are the most attractive materials for aneurysm treatment by using them as a filling device in the artery [124]. Wilson and his coworkers showed that the shape memory polymer foam which includes hexamethylene diisocyanate (HDI), N,N,N’,N’-tetrakis(2-hydroxypropyl)ethylenediamine (HPED), and triethanolamine (TEA) can be laser deployed in an
In the study of Wong et al., shape memory polymer CalomerTM was investigated as a candidate for aneurysm coils. In this work, shape memory polymer coils spread inside a simulated aneurysm model proved that the typical hemodynamic forces do not prevent the shape recovery process [126].
Maitland et al. developed shape memory polyurethane foam with high porosity which makes it convenient for intracranial aneurysm treatments. Adding 4% tungsten into this foam improves radioopacity. Inherent radioopacity makes the material to be visualized by using conventional patient imaging modalities such as fluoroscopy in order to deliver it safely [127].
Another shape memory polymer foam based on hexamethylenediisocyanate, triethanolamine, and tetrakis(2-hydroxyl propyl)ethylenediamine was developed by Maitland et al. which is found to be suitable for aneurysm treatment [128].
Raymonda et al. developed a non-cytotoxic, non-mutagenic, and poorly thrombogenic polyurethane-based foam called cold hibernated elastic memory (CHEM) for endovascular procedures. The shape memory property and possibly modifying
When blood does not flow smoothly in the blood vessels, it can begin to coagulate and/or blood clots. A blood clot, or thrombus, may continue to grow, blocking the blood supply to certain parts of the body and causing damage to tissues and organs. Some medical intervention is often required to remove the clots. It is estimated that each year thrombosis affects as many as 900,000 people in the USA and kills up to 100,000 [130,131].
Maitland et al. reported that a prototype endovascular electromechanical clot extraction device was fabricated using a combination of shape memory polymer and shape memory nickel–titanium alloy (nitinol). This preliminary study suggests that SMP–nitinol device may have an application in the treatment of acute stroke or other thromboembolic diseases [132].
Also, Maitland and coworkers aimed to evaluate the feasibility of utilizing a system of SMP acrylates for a thrombectomy device by determining an optimal cross-link density that provides both adequate recovery stress for blood clot removal and sufficient strain capacity to enable catheter delivery. They have reported an acrylic SMP system with glass transitions above body temperature in the range of 65–75°C with tailorable recovery stresses that were controlled by varying the cross-link density. From the four different material compositions evaluated, devices with 15 mole% bisphenol A(BPA) gave the most favorable outcome [133].
Wilson et al. reported that coil, umbrella, and microgripper-shaped polyurethane microactuators are used to treat stroke by activating this shape memory polymer with laser to remove clots. The actuation of the devices was obtained between 65 and85°C [134].
In this chapter, novel smart polymeric materials, i.e., shape memory polymers, self-folding polymers, polymer origami structures, and their potential applications are summarized. Shape memory polymers are the class of smart materials which can fix their shape after applying a deformation by cooling below their transition temperature. Then, by reheating the above transition temperature, shape recovery to the original shape is observed. They can be produced from polymer material or blending with network-based polymers.
Due to the their excellent biocompatibility, SMPs are promising building blocks of biomedical applications such as polymer vascular stents with shape memory polymers as the drug delivery system, smart surgical suture, laser-activated SMP microactuators to remove clots in a blood vessel and implants for minimally invasive surgery.
Today, SMP development continues rapidly, in the case of clinical applications. Enhancing biomedical applications beyond medical devices would be achieved by blending the shape memory polymers with compatible materials.
When minimally invasive surgery application is taken into account, implants should be able to carry out complex movements. These movements have to be performed by a material which can perform predetermined shift many times such as SMPs. Also, SMPs would be required for individual patients since they are compatible against specific conditions.
Shape memory polymers are considered to be a future strategy which may prevent undesired complications during the treatment in biomedical applications compared to shape memory alloy-based materials due to their biodegradability. Since SMPs are versatile materials, they would continue to develop as a result of their promising potential applications.
Past efforts in epidemiological (nuclear power industry, atomic bomb explosions, nuclear reactor accidents, etc.) and clinical (diagnostic imaging, radiation oncology, radiation therapy planning etc.) research strongly contributed to the current understanding of ionizing radiation effects on human organs, tissues, and cells [1, 2]. In principle radiation biology is based on effects of instantaneous (10−18 s) [3], stochastic damaging interactions of ionizing radiation with cells, a main target being the genetic material, i.e. chromatin in the cell nucleus [4]. In this context, radio-sensitivity and radio-resistance as opposing terms describe the extent of individual cellular susceptibility or ‘response’ upon radiation exposure which are highly dependent on physical (e.g., radiation type, dose, dose rate, etc.), chemical (e.g., hydroxyl radicals, etc.) and biological (e.g., developmental and proliferative state of the affected cell type) factors. As the overall organismal radiation response results from the entirety of all individual radiation responses on the single cell level, deeper understanding of the underlying, complex molecular mechanisms and dynamics of radiation induced DNA damaging and repair on the cellular level is highly relevant for fundamental and applied radiation biology (for review see [1, 2, 5]).
\nHence, cytometric analyses based on fluorescence microscopy have become the method of choice to study damaging effects of ionizing radiation and DNA repair. This has contributed a lot to today’s knowledge. However, conventional fluorescence microscopy is limited to average lateral resolutions around 200 nm laterally and 600 nm axially [6] and thus is limited to the bulk analysis of molecular cellular processes and structures. In parallel, super-resolution fluorescence microscopy techniques have rapidly evolved during the last few decades and turned out to be powerful tools to study cellular structures and molecular architectures on the nanoscale (for review see [6, 7, 8]). Methods based on stochastic reversible photobleaching [9, 10, 11, 12, 13, 14, 15] of single molecules called Single Molecule Localization Microscopy (SMLM) [16] reach effective resolutions down to 10 nm and have become popular among modern super-resolution imaging techniques as their realization is highly practical and straightforward using established specimen preparation methods of standard fluorescence microcopy [17]. As such resolutions allow the detection of single molecules, such as nucleosomes [18], proteins [19, 20], receptors and junction proteins [21, 22], or even single chromatin loops [23] etc., super-resolution microscopy opens new avenues for the research of radiation induced damaging and repair processes [5, 24].
\nWith this article, we attempt to introduce the novel SMLM approach to radiation biophysics and radiation biology. We start with a brief summary on the basics of ionizing radiation, induction of DNA damage and damage repair mechanisms, to follow up with some standard radiobiology analysis methods. We further provide an overview of the working principles of selected sub-diffraction microscopy techniques with a focus on SMLM. Finally, the successful application of localization microscopy in radiation biology research is demonstrated along examples of current works.
\nIonizing radiation penetrates through material and deposits enough energy to ionize molecules or atoms by liberating electrons. The effects of ionizing radiation on biological materials are highly dependent on the dose, the dose rate and type of radiation. In living cells, ionizing radiation hits all kinds of biomolecules, such as desoxyribonuleic acids (DNAs), aminoacids (proteins), lipids (membranes), carbohydrates, etc. However, most harmful consequences to living organisms show damages inflicted to their genomic DNA, especially in the form of DNA double-strand breaks (DSBs) [25, 26]. Especially follow-up effects of false strand repair may lead to significant dysfunctional development as for instance tumor genesis.
\nIonizing radiation (IR) includes all high energy/speed (> 1% speed of light) ions (e.g. carbon ions), atom nuclei (e.g. alpha particles), subatomic particles (e.g. beta particles, protons or neutrons) and high-energy electromagnetic waves (e.g. high energy ultraviolet (UV) rays, X-rays and gamma rays), that carry enough energy to directly or indirectly ionize atoms or molecules by liberating electrons from them, and to break molecular bonds [27].
\nThe most common types of ionizing radiation occurring under environmental circumstances are caused by radioactive decay and can be divided into three groups: alpha, beta and gamma radiation [27]. Alpha radiation is made up of particles comprising two protons and two neutrons (helium nucleus) that carry energies in the range of up to several MeV. Due to its large particle size, alpha radiation has the lowest penetration depth through biological materials and the highest energy deposition per distance traveled. Beta particles are made up of electrons or positrons, thus exerting higher penetration depths and lower energy depositions compared to alpha particles. Gamma rays are high energy electromagnetic waves that exhibit the highest penetration and compared to particles, lowest energy deposition per track unit in biomaterials among these three types of IR. Due to the dispersed and low energy deposition in tissue, gamma and beta radiation are often referred to as low linear energy transfer (LET) radiation, whereas alpha particles belong to high LET radiation [27].
\nFor clinical diagnosis and therapy in radiology or radiation oncology [28, 29], typically artificial radiation sources are applied, as for instance to produce X-rays in the energy range of keV to MeV, electrons and positrons, protons, and heavy ions (carbon or nitrogen). Like alpha particle, protons and heavy ions belong to high LET radiation. The advantage of protons and especially heavy ions is based on the characteristic absorbance with a Bragg peak at the end of the particle track where most of the particle energy is deposited. This energy positioning peak can exactly be localized in the tumor volume so that intact cells and tissues in the tumor surroundings are excluded from radiation damaging [30].
\nThe absorbed dose D of ionizing radiation is quantified by the amount of energy deposited per unit mass of the penetrated material and is measured in units of Joule per kilogram (J/kg) or Gray (Gy) [27]. It describes an universal energy absorption for all types of ionizing radiation and is most commonly used in radio-physical research, whereas a radiation type specific dose also called the equivalent dose H calculated by multiplication with a weighting factor WR (e.g. WR = 1 for gamma radiation and WR = 20 for alpha radiation) is often used in radio-biology, radio-medicine, or radiation protection and safety. The equivalent dose can be further weighted by a tissue weighting factor WT to result in the effective dose E, which describes radiobiological effects considering the used radiation type and the tissue/organ of interest. Both, the equivalent dose and effective dose are quantified in units of Sievert (Sv) and do not represent physically measurable quantities but rather a value based on clinical and epidemiological outcome that is typically used in radiation safety [31].
\nAmong all kinds of ionizing radiation induced biological effects, damages to chromatin especially the DNA molecules in the nucleus of cells are thought to be the most severe with respect to cellular survival and carcinogenesis [2, 5, 32, 33]. DNA base oxidation, single strand breaks (SSBs) and double strand breaks (DSBs) are the most common ionizing radiation induced damages to the DNA molecule, that affect genome integrity and DNA biochemistry [34].
\nDSBs of DNA belong to the most complex and severe types of DNA damages as they directly affect genome integrity and the way of cellular survival [35, 36, 37]. Single strand breaks (SSBs) induced by ionizing radiation and base damages occur more frequently than double strand breaks [34]. It can be estimated to about 40 DSBs/Gy and about 1,000 SSBs/Gy. SSBs are less severe to genome integrity as an intact template strand is still available for complementarity-aided, error-free repair of the lesion. But DSBs are also simply formed by two or more opposing SSBs in close proximity or combinations of different DNA damage types [26].
\nInduction of DSBs in native chromatin is rapidly followed up by phosphorylation of nearby histones of the H2A variant H2AX at serine residues at position 139 [38]. This results in the generation of plenty γH2AX molecules around a DSB damage site, where about 2 Mbp of DNA are usually phosphorylated [39]. This leads to the formation of focus structures of sizes in the range of micrometers, which can be visualized under a fluorescence microscope [40]. These phosphorylated histones serve as signal and anchor points for many downstream recruited proteins of certain DNA damage response and repair machineries [41]. As the number of γH2AX foci is quantitative for DNA damage, counting of specifically labeled foci has been established as a measure for dose-efficiency and correlated to cell survival [42].
\nSingle ionizing radiation induced DNA lesions can be caused by direct or indirect hits [43]. Ionizing radiation penetrating through a cell nucleus can hit and ionize atoms in a DNA molecule itself with a certain probability. However, the most prominent primary reaction underlying all ionizing radiation induced DNA damages is the radiolysis mediated formation of reactive oxygen species (ROS), e.g. •OH radicals, O2•- radicals and H2O2, which can further inflict reducing damage and thus lesion to the DNA [44]. Ionizing radiation, especially high LET radiation, is known for its property to efficiently induce highly complex damages to DNA. Such complex DNA damage sites composed of multiple lesions in close proximity on both strands are also termed locally multiple damage sites (LMDS) [45].
\nLiving organisms developed highly efficient and customized ways to repair the severe damages inflicted to their genome. The DNA DSB sites are rapidly (within seconds to minutes) recognized and marked by proteins of an initial response, which serve as signals and docking sites for more specialized proteins of DNA repair pathways. The fate of repair type depends on the concerted presence of pathway specific damage response proteins [1, 2, 46, 47, 48]. The main two ways by which cells respond to DNA double-strand breaks are non-homologous end joining (NHEJ; also called canonical NHEJ = cNHEJ) and homologous recombination (HR). NHEJ mediated DSB repair is fast and can be error-prone, but it can be flexibly performed throughout all cell cycle phases. HR works error-free, but is mostly restricted to late S and G2 phases as a homologous sister chromatid is required as a repair template [49, 50, 51, 52]. Recent data, however, have suggested that active genes may employ HR also in G1 phase, by utilizing the nascent RNA as a template for precise repair (reviewed in [53]). As the DNA-end resection is inhibited in G1 cells, an alternative model with cNHEJ taking the advantage of the same principle (RNA-templated repair) has also been proposed. Interestingly, DNA repair by HR is preferred in lower eukaryotic life forms, whereas NHEJ is predominantly observed in mammalian organisms. Alternative low abundant DSB repair pathways are the alternative end joining pathways (a-NHEJ; also called back-up EJ), micro-homology mediated EJ (MMEJ) and single strand annealing (SSA). One main difference between all DSB repair mechanisms is the extent of initial DNA end-resection at the damage site [26, 54, 55, 56, 57, 58, 59]. The DNA damage response (DDR) against DSBs is subject to intensive radiobiological investigation and fluorescence microscopy of in situ DSB repair proteins serves as state of the art biological dosimetry.
\nAfter the induction of a DSB, damage response proteins are rapidly recruited and accurately determine the fate of the DSB towards a repair pathway that best deals with the damage site in a certain genomic and cellular context. The chromatin remodeling p53-binding protein (53BP1) protects the break site from extensive end resection [60], thereby promoting repair by non-homologous end joining [61], whereas BRCA1 facilitates extensive end resection for repair by homologous recombination [52, 62].
\nThe NHEJ repair pathway is initiated with binding of the Ku70-Ku80 heterodimer complex to the DNA ends of the DSB site, which serves as a linkage between damage site and further damage response proteins [61, 63]. In a second step, DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is recruited to the Ku complex forming the DNA-PK complex. On-going recruitment of X-ray complex (XRCC4)/DNA Ligase IV (X4LIG4) complex and XLF to the DNA-PK complex forms the core NHEJ complex [64]. DNA-PKcs sterically protects the break site for repair and phosphorylates other repair proteins [65, 66] and H2AX [41]. Furthermore, DNA-PK auto-phosphorylation results in a conformational change of the core complex, thereby enabling DNA end processing by nucleases and dissociation of the DNA-PKcs subunit [67, 68]. Finally, ligation of the DNA ends is mediated by the X-ray complex (XRCC4)/DNA Ligase IV (X4LIG4) complex and XLF [69, 70, 71]. Artemis endonuclease [72, 73], polynucleotide kinase (PNK) [74], DNA polymerase (pol) μ and λ can be additionally involved in NHEJ repair depending on the chemical properties of the DNA damage site [75].
\nTo initiate repair by HR, the free damaged DNA ends at the DSB site must be sensed and bound by a protein complex comprised of MRE11, RAD50 and NBS1 (MRN complex) [76]. Next, the protein kinase Ataxia Telangiectasia Mutated (ATM) [77] is recruited to the MRN complex at the damage site [78], which auto-phosphorylates and phosphorylates components of the neighboring chromatin. Most prominent phosphorylations are those of the histone variant H2AX (γH2AX), one of the earliest and a very sensitive marker of cellular response to DSBs [38]. End resection is initiated by the single-strand endonuclease and exonuclease activity of the Mre11 protein [52, 79] of the MRN complex. RAD50 further stimulates Mre11 nuclease activity and Nbs1 interacts with CtIP [80], another protein that is essential for the initiation of MRN complex mediated end resection [81]. Exonuclease 1 (Exo1) and Dna1/BLM are recruited by CtIP to continue end resection [82, 83, 84] until it gets attenuated by RPA coating of resected ssDNA ends [85]. BRCA2 in combination with BRCA1 and PALB2 dismantles the ssDNA ends from RPA coats enabling binding and forming of the RAD51 nucleoprotein filament, which stimulates homology search and strand invasion [86]. Sister chromatid strand recombination via Holiday junctions is further facilitated by RAD54A and its paralog RAD54B [87, 88], finally resulting in conservative repair of the DNA lesion.
\na-NHEJ or b-NHEJ has been described in slightly different ways which are not well distinguished [56, 57, 58]. Mostly, in the presence of short micro-homologies (>4 bp) after CtIP-MRN mediated end resection, repair via an alternative end joining (MMEJ) can take place [89]. This is initiated by Poly(ADP-ribose) polymerase 1 (PARP1) and followed up by DNA polymerase θ (pol θ) mediated strand extension starting at the paired micro-homology site. Ligase1 and Ligase2 are supposed to perform the final ligation of DNA ends [90, 91].
\nWhen the damage site is flanked by larger regions with non-allelic sequence homologies, repair by single-strand annealing is also possible. The absence of Ku proteins and even more extensive end resection to expose the homologous regions as single strands are necessary for SSA repair [92]. Again, RAP binding to the resected ends promotes RAD52 mediated annealing of homologous regions. Nuclease XPF-ERCC1 trims the remaining non-homologous overhangs and DNA Ligase 1 connects the DNA ends [93].
\nSeveral studies indicate, that damaged genomic Alu elements use micro-homologies for single-strand annealing, thereby often leading to translocations [94, 95]. Such nonconventional damage repair processes might explain a significant portion of the observed deletion events associated with malignancies [59]. In fact, in vitro model systems could already demonstrate Alu mediate non-allelic homology dependent DSB repair [96].
\nRadio-sensitivity can be assessed on different scales ranging from whole organs and tissues over single cells to molecular markers and mechanisms. Based on clinical and experimental findings on DNA damage induction and response mechanisms, the dose effect of ionizing radiation on biological material is commonly described with a linear-quadratic model [97, 98]. At low dose ranges (below 1 Gy), radiation damages are supposed to linearly increase with the applied dose, whereas at higher doses the probability for multiple hits increases and complex DNA damage spots dominate.
\nIn this chapter, we summarize some established methods to study cellular and molecular effects of ionizing radiation. In the past, sophisticated assays were developed to detect and quantify radiation induced damages to the cell and nuclear DNA ranging from techniques to assess overall cell survivability, large-scale chromosomal damages and rearrangements over sensitive detection of DNA break sites to modern state of the art technologies that can visualize the formation of damage foci in situ with the help of suitable biomarkers.
Colony forming assays (CFAs) based on clonogenic survival (also called clonogenic assay) (see [99] and citations therin) as a method to quantify cell survival after radiation exposure was firstly described by Puck and Marcus in 1956 [100]. CFAs measure the ability of cells to divide after treatment with agents that impair cellular reproduction, e.g. radiation (Figure 1). Since then, colony forming assays were improved for many different cell types and are widely used as a “gold standard” in radiobiological studies. In practice, irradiated cells are plated at higher dilutions so that single cells are well separated. Upon incubation colonies form each originating from a single cell. Thereby, colonies comprising 50 cells or more are considered for estimating the survival fraction. Treatments such as exposure to ionizing radiation damages the reproductive survivability of cells and thus results in a lower number of colony formation events at the same number of plated cells [103].
A fraction of ionizing radiation induced DNA double strand breaks results in heavy genomic rearrangements that can be detected on metaphase chromosomes. False rearrangements of multiple centromeric regions between chromosomes can lead to dicentric, acentric, centric ring conformations [104, 105] that can be visualized under a conventional fluorescence microscope (Figure 2a). The good reproducibility and comparability lets the so called dicentric assay stand among the gold standards of biological dosimetry [106]. Nowadays dicentric assays are further developed towards biological dosimetry in the low dose range (< 500 mGy).
The micronucleus test is a method to assess and detect chromosomal breakages in interphase nuclei developed by Schmid et al. in 1975 [107]. Radiation damage can result in major chromosomal aberrations and loss on the centromeric region by wrong rearrangement of DNA double strand breaks (Figure 2a). These heavily damaged acentric chromosomes can form separated globular structures outside the main nucleus in interphase. As micronucleus formation can be readily detected in interphase nuclei, analysis can be performed much faster and serves as an efficient alternative for the analysis of instable chromosome aberrations [108].
In 1984, Ostling and Johanson published a micro-electrophoresis technique that could visualize DNA damages in single cells [109]. First, cells are embedded in agarose and lysed with non-ionic detergents under high salt concentrations, so that only nucleoids (supercoiled DNA loops attached to the nuclear matrix) remain. Ionizing radiation-induced breaks relax and locally unwind the supercoiled DNA structure, thereby partly linearizing the strand at the break site. When voltage is applied linearized DNA segments (SSB) protrude from the nucleoid and migrate faster towards the anode while the nucleoid core remains assembled. The nucleoid and its tail resemble a comet, when stained with 4′,6-diamidino-2-phenylindole (DAPI) or other quantitative DNA fluorescence dyes and visualized under the fluorescence microscope, thus leading to the term ‘comet assay’ [110, 111]. Alkaline variations of the comet assay [110] were introduced, that can detect DNA damages over an extended dose range (0.25 Gy to 2 Gy) than under neutral pH conditions (1 Gy - 3 Gy) [111]. Modern approaches extend the method by automatization of experimental procedures and image analysis [112, 113, 114], thereby enabling statistically robust high-throughput detection of DNA damages for potential clinical applications.
DNA damage response proteins like γH2AX, 53BP1, RAD51 etc. accumulate at initial damage sites and rapidly form foci-like structures in the nucleus (see for example Figure 2b). Antibody staining and fluorescence microscopy of such damage response proteins is an established tool to visualize and quantify DNA damage repair foci at single cell resolution. One advantage of this technique is the ability to assess molecular dynamics of DNA damage repair by visual observation of foci formation at different time points after irradiation.
(A) Example of colony formation after cell exposure to different doses of X-ray irradiation. (B) Typical survival curves for cell colonies after irradiation with different types of photon and particle radiation. Linear-quadratic cell survival curves are fitted and can be used to calculate the relative biological effectiveness. Note: These figures are modified and were originally published under CC BY license in [
(a) Example of a lymphocyte metaphase plate with centromeres highlighted by FISH. The cells were irradiated with 3 Gy X-rays. The big arrows show two dicentric chromosomes. The small arrow heads label the corresponding acentric fragments. (b) Typical examples of fibroblast nuclei (stained with a specific DNA dye) with γH2AX foci after exposure to high dose irradiation. The foci are labeled by specific antibodies.
Fluorescence microscopy of potent marker labels is a powerful analysis tool to assess cellular effects of ionizing radiation on the single cell level by optical examination. Due to past efforts, a myriad of fluorescent probes exists for the molecular labeling of almost any known biological target structure (e.g. specific antibodies against γH2AX, 53BP1, MRE11, RAD51 or other repair proteins as well as against heterochromatin or euchromatin etc.). This opens the door to analyze molecular mechanisms underlying fundamental biological functions by optical investigation, e.g. DNA damage response and repair dynamics upon ionizing radiation exposure [1, 2, 5].
\nA variety of novel super-resolution microscopy techniques were invented in the last few decades [115]. With the help of novel super-resolution microscopy techniques, the molecular effects of ionizing radiation in single cells can be studied on the nanoscale. Nano-labeled molecular structures can be resolved in biological specimens down to a precision of 10 nm (1/50 of the wavelength of visible light), which is in the range of single nucleosomes, antibodies, receptors, etc. (see for example [20, 21]).
\nIn order to improve the resolution in light microscopy, a prerequisite is to circumvent the diffraction limit of light, a physical phenomenon firstly described by Ernst Karl Abbe and John William Strutt, 3rd Baron Rayleigh, during the late 19th and early 20th century [115]. In diffraction limited fluorescence/light microscopy, the Abbe or Rayleigh criterion (Figure 3) is commonly used to define a resolution measure describing the minimal distance D between two point-like light sources with wavelength λ that can be resolved:
\nRayleigh criterion for the diffraction of two point-like light sources and single-molecule localization microscopy techniques to circumvent the diffraction limit. (A) The resolution limit of two adjacent point-like sources of light is defined by the distance between these two light points; the first intensity minimum of one light point overlaps with the main intensity maximum of the other light point. (B) The diffraction limited resolution of fluorescence microscopy illustrated by an example of three point-like signal sources within a distance below the resolvable range. (C) Working principle of SPDM by spectral isolation of labeling molecules. Here the spatial positions of three point-like fluorescent light sources can be separated by three different colors green, red and blue (from left to right). (D) Working principles of most single molecule localization microscopy methods rely on spectral modulation that switches most fluorophores into a dark state in a stochastic manner. Thereby, detection of only a sparse subpopulation of labels that are either totally isolated or lie apart at distances greater than the diffraction limit is possible. A series of acquisitions, each representing another stochastic sparse subpopulation of signals, can be summarized to result in a complete image below diffraction limit. (E) Minimal Jablonski diagram showing the electronic states and transitions involved in the intrinsic stochastic blinking of fluorophores. Note: These figures are modified and were originally published under CC BY license in [
Therein λ is the wavelength and NA the Numerical Aperture of the objective lens (NA = n sin(α/2); n = refraction index; α = lens aperture angle). Conventional fluorescence microscopy techniques that use objective lenses with high numerical aperture NA (≥ 1.4) are available today. In confocal laser scanning microscopes, they typically achieve resolutions down to 200 nm in lateral and 600 nm in axial direction. However, modern super-resolution microscopy using the same objective lenses circumvent this physical limit by sophisticated interaction with fluorescence signals so that they can visualize biological specimen down to resolutions in the order of 10 nm, which is in the range of single nucleosomes, antibodies, receptors, etc. [16].
\nA complete overview of super-resolution microscopy techniques is beyond the aim of this article. However, we want to mention some meanwhile very well established ones:
\nSophisticated near-field super-resolution methods, e.g. total internal reflection fluorescence (TIRF) microscopy (TIRFM) [118, 119] or near-field scanning optical microscopy (SNOM, NSOM) [120, 121], belong to the first techniques breaking the diffraction limit by novel techniques working in the optical near field of fine crystal tips probing the specimen without an microscope objective lens. Unfortunately, near-field techniques are technically restricted to the visualization of surfaces of cells, membranes or isolated organelles [122, 123, 124].
\nMore recently evolved far-field super-resolution fluorescence microscopy techniques use objective lenses available from establishes microscope manufactures and can be separated into two principle approaches. The first is based on the spatially modulated excitation of fluorophores, e.g. by point spread function engineering as in stimulated emission depletion (STED) [125] or by excitation through a series of illumination patterns as in structured illumination microscopy (SIM) [126]. A second group of super-resolution techniques is based on optical isolation of fluorescent molecules through switchable intensities [17] or intrinsic differences in spectral signatures [127]. The latter techniques often referred to as single molecule localization microscopy (SMLM) in general, can be practically implemented with customary microscope parts and standard objective lenses [16]. Spectral precision distance microscopy (SPDM) an early development of the 1990s [128] is the one and only localization microscopy method, that establishes optical isolation of molecular labels through constant differences in absorption and emission spectra of different fluorophores, that are applied in a combinatory labeling strategy [127, 129]. Most localization microscopy methods, however, rely on stochastic spectral modulations of single fluorophore molecules, such as photo-activated localization microscopy (PALM) [12], fluorescence PALM (FPALM) [13], stochastic optical reconstruction microscopy (STORM) [15, 130], direct STORM (dSTORM) [131], ground state depletion microscopy followed by individual molecule return (GSDIM) [132], SPDM with physically modifiable fluorophores (SPDMphymod) [14, 17], etc. In the following chapters, we will describe single molecule localization microscopy in more details as being applied in radiation biophysics and we will provide examples indicating wide applications in nano-probing biomolecules and molecular mechanisms.
\nSingle molecule localization microscopy is one the most popular super-resolution techniques, because it can be practically realized with standard optical setup and standard specimen preparation methods using commercially available fluorophore labels. Fundamental to all SMLM techniques is the stochastic sampling of signals. The intrinsic blinking nature of a variety of available fluorophores at excitation with high laser powers (in the range of several kW/cm2), enables SMLM with conventional dyes like GFP / YFP, Alexa488, Alexa568, etc. [16, 17, 133].
\nApart from conventional fluorescence, which is based on rapid, repetitive excitation (10–15 s) and red-shifted emission (10–9 s) between the ground state S0 and excited singlet state S1, fluorescent molecules additionally undergo intersystem crossing (ISC) [134] from S1 to dark triplet states T1 [135]. From there, fast relaxation (10–3 s) to the original S0 ground state enables re-entry to new cycles of normal fluorescence (Figure 3E, left). Further transition from the T1 state into a second dark state D also occurs, which takes longer (ms to min) to recover to the ground state S0 (Figure 3E, right) [116, 136]. This reversible photobleaching via the long lived dark state D results in a limited number of stochastically blinking fluorochromes at resolvable time scales that can be used in single molecule localization microscopy to determine sub-diffraction positions of single fluorescing molecules [133].
\nFor image acquisition, a time series of raw diffraction limited images (several hundreds to a few thousand) from the same region of interest are registered and efficiently searched for blinking events under a user-defined intensity threshold to discriminate signals from background. Then, the intensity profile of each blinking event is fitted by a Gaussian curve and the barycenter point of the signal source is calculated. Notably, the localization precision of such a point merely depends on its intensity/background ratio [137].
\nA major advantage of SMLM approaches lies in the data format. The point matrix containing the lateral x and y coordinates of each localization signal allows all kinds of mathematical and statistical analyses (Figure 4). Most prominent are analyses based on Ripley’s point-to-point distance information which can be used for the elucidation of signal densities, cluster formation, and spatial organization of labels [139]. Recently, novel mathematical approaches like persistent homology determinations were introduced to investigate topological similarities [138]. Computation of the coordinate matrix into an image with user-defined resolution and visual enhancements is then possible. If provided, multi-color analyses on the single molecule level can be performed to study more complex molecular mechanisms and dynamics.
\nGeneral workflow of single molecule localization microscopy and data analysis. Serial images are acquired from the same region of interest (i). The point-spread function of each blinking event in each single image is gauss fitted to estimate the intensity maximum (ii), which represents the idealized lateral coordinates of the signal source (iii). The result is a data table containing the coordinates of all detected signal points. The matrix representation of data allows mathematical and stiatistical analysis of clustering, distance distributions, signal densities, multi-color signal distributions, enhanced visualization and topology (iv). Note: These figures are modified and were originally published under CC BY license in [
Phosphorylated histone variant γH2AX molecules at the site of DSBs and their accumulation into γH2AX-foci are well-established markers of DNA damage response and repair. Most recent studies performed SMLM of γH2AX specific antibodies in HeLa cells that were exposed to different doses of γ-radiation and fixed at different time points after radiation exposure [140]. Quantitative analysis resulted in a linear quadratic increase in measured γH2AX localization signal points and cluster numbers with increasing doses of radiation exposure (Figure 5A, B). With increasing repair time, the number of γH2AX clusters decreases; thereby successfully demonstrating repair dynamics and cell recovery by γH2AX-cluster relaxation on the molecular level. As dose responses and molecular dynamics for γH2AX clusters and raw γH2AX signal points well correlate with past observations, this study can serve as a benchmark standard for future super-resolution radiobiology experiments.
\n(A) “Visualization of cluster formation from the SMLM image of cell nuclei after 2 Gy radiation exposure.
Similar studies indicated that the γH2AX cluster size remained constant during repair also at later times post irradiation, i.e., at later times only the number of clusters reduced. This typical size was about 400 nm in diameter after photon irradiation and nearly independent from dose or the cell types analyzed [140, 141]. For α-particle irradiation from radioactive decay [142], the γH2AX cluster size along the particle track was about 200–300 nm; this size could be also observed for γH2AX clusters induced by environmental stress as for instance the deficiency of folat during long time culturing [143].
\nAfter exposure to photon radiation (different doses and energies) the SMLM analysis of heterochromatin around γH2AX clusters using specific antibodies against H3K9me3 methylation sites, revealed a fast relaxation of the chromatin and slower re-condensation after finishing the repair processes [144]. The degree of relaxation was independent of the dose which is in good relation to the equally sized γH2AX clusters [140, 141]. In contrast the euchromatin density increased during repair followed by a decrease after finishing the repair processes [144]. However, in total the chromatin showed an increasing clustering during repair followed by a reduction of clusters dependent on the energy of the damaging photons (unpublished). In general it can be assumed that DNA damaging by ionizing radiation does not only induce a reorganization of chromatin at the damaged sites but may also induce long range chromatin rearrangements for repair processes. Whether such chromatin rearrangements are random or directed to improve repair protein recruitment will be subject of future investigations.
\nBeyond γH2AX cluster formation, foci and sub-foci clusters of repair proteins were investigated after photon or particle irradiation [141, 142, 145, 146, 147]. In the following, some typical examples are shown taken from ongoing projects:
53BP1 foci were investigated in differently radio-resistant cell types, the moderately radio-resistant neonatal human dermal fibroblast cell line (NHDF) and highly radio-resistant U87 glioblastoma cell line. Specimens of both cell types were exposed to high-LET 15N-ion radiation of doses of 1.3 Gy (in a 10° irradiation scheme) and 4.0 Gy (in a 90° irradiation scheme) at the particle irradiation facility of the Joint Institute for Nuclear Research, Dubna, Russia [145, 146].
At given time points up to 24 h post irradiation, SMLM of fluorescently tagged 53BP1 molecules was performed and the coordinate data of each labeled molecule were quantitatively evaluated [137, 139, 140]. Clusters of these tags were determined as sub-units of repair foci (Figure 6a) and the formation and relaxation of these clusters revealed a higher ratio of 53BP1 proteins being recruited into clusters in NHDF cells (less radio-resistant) as compared to U87 cells (more radio-resistant) with different levels of distribution prior to DNA damage induction. This relation of 53BP1 inside and outside particle track clusters (Figure 6b) remained different for both cell types during the repair time observed. This could be seen as a measure of the “just-in-time” availability of 53BP1 proteins but did not reflect the absolute number of 53BP1 proteins available. The speed of cluster formation and relaxation differed for the two cell types (Figure 6c) indicating the recruitment of the existing proteins in the cell nucleus (higher in U87 cells) rather than a de novo production [147].
\n(a) 2D density SMLM images of 53BP1 repair proteins. Typical examples are shown for fluorescently-labeled 53BP1 proteins in NHDF cells (a) and U87 cells (B) “after 1.3 Gy tangential 15N-irradiation (10° angle between the ion beam and the cell layer). The time values indicate the period post irradiation when the samples were taken as aliquots of the same irradiated culture and fixed. For comparison, examples of non-irradiated control cells are presented. The left columns are merged images of SMLM data and wide-field images. In the right columns the SMLM images clusters and cluster areas are shown. The scale bars equal to 1 μm.” (b) relative amounts of 53BP1 signals detected within (blue) and outside (orange) repair clusters. “Graphs: Mean values and margins given by the standard deviation are depicted in gray. The values are always normalized to the mean number of signals detected at a given time point. The data are presented for NHDF fibroblasts (A) and U87 cells (B) after 1.3 Gy tangential 15N-irradiation (10° angle between the ion beam and the cell layer). Images: The pointillist images represent examples of sections of cell nuclei with labelling points inside (blue) and outside (orange) clusters at the given time points. The samples were taken as aliquots of the same culture at different time points (from 5 min to 24 hrs) after irradiation. For comparison, examples of non-irradiated control cells are presented (= 0 min).” (c) Ripley distance frequency analysis. The relative frequencies of pairwise distances are presented for the aliquots of the irradiated cell samples at different time points post irradiaton (color label of curves); (A) NHDF and (B) U87cells irradiated under 10° irradiation angle. Note: These figures are modified and the parts of the text written in “...” are reproduced from the original figures which were originally published under CC BY license in [
A certain number of the clusters remained persistent, even longer than 24 h post irradiation (Figure 6b); thereby the number of these remaining clusters varied in each cell line. The heavily damaged cell nuclei maintained repair activity in order to process the complex damage patterns caused by high-LET 15N-radiation. This long-standing repair activity of 53BP1 proteins was shown in both cell types and the behavior of the cells could causatively be linked to the cell-type specific radio-resistance.
\nThe dynamics and cluster formation of tagged 53BP1 molecules showed that these clusters were embedded within a random distribution of points. After irradiation, a fast formation of 53BP1 clusters was observed (Figure 6c). During the early repair time of about 30 min - 1 h after radiation exposure some clusters were dispersed while others persisted and the amount of randomly distributed proteins was growing. The latter clusters that were persistent did not disappear until the end of the repair period being studied (24 h).
2. Another study performed two-color SMLM of immunostained γH2AX and Mre11 proteins [141] and revealed significantly delayed foci formation by Mre11 compared to γH2AX. While γH2AX clusters are already established at 30 min after radiation exposure (Figure 7, left), Mre11 is still ubiquitously distributed in the nucleus. Mre11 cluster formation is maximal at around 180 min after irradiation with significant association to γH2AX clusters (Figure 7, right).
Overview of the results obtained from SMLM measurements. Left panels show the data obtained after radiation exposure for MCF-7 breast cancer cell nuclei (“MCF-7”) in comparison to cell nuclei of CCD-1059SK fibroblasts (“Fibis”); right panels show the data obtained without radiation treatment, i.e., the natural occurrence of MRE11 clusters in these cells. The columns of each panel represent the mean values calculated from 20 nuclei each. The error bars on top of the column indicate the standard deviation. For each time step after the irradiation process, the data are given for cells exposed to 2 Gy ionizing radiation and for cells subjected to the same culturing procedure but not to radiation treatment. Level of significance between the corresponding values: *** = 0.1%. Note: These figures and their legends in “..” are slightly modified and were originally published under CC BY license in [
The reason to apply topological analyses is to record properties of point patterns, which are invariant under certain deformations of the object. Mathematically these deformations correspond to continuous transformations of the topological space defined by the structures. Here we have considered two properties, the number of “components” (explained below), which are independent from each other in such sense that connections between points only exist within the respective components and the number of “holes” of the structures inside the components (explained below, Figure 8a). In algebraic topology, these properties are called the Betti numbers for zero and one -dimensional simplicial complexes [148].
\nLeft: Illustration of the barcode data representation. (A) Continuously growing spheres, exemplarily depicted at 5 different scales α, around the point data illustrate the idea of the α-shape filtration. (B) As the growing spheres mutually embed the Centre of each-other the corresponding centres are connected by an edge. Whenever a triangle is formed, it is included in the complex as a face element. (C) Barcodes (Betti numbers) of dimension 0 (D0) and 1 (D1) corresponding to connected components and holes. Right: Heat map depicting the Jaccard indices averaged from components and holes for similarity of (non-)heterochromatin associated γH2AX clusters. Note: These figures are modified and were originally published under CC BY license in [
SMLM images as for instance of γH2AX foci/clusters are point-sets for which components and holes can be defined. A geometric relationship among the points is defined by growing spheres of radius α around each of them. Whenever two spheres mutually embed each-other’s center, these centers of the growing spheres are connected and the connected points belong to the same component. With increasing radii, the number of components is reducing. At the end of the procedure, a single component is remaining, the whole γH2AX cluster. For the definition of holes, a polygon is appropriate. Whenever the edges form a closed area, a hole is counted until another line closes a triangle separated from the original hole [147, 148].
\nThe results are presented as “barcodes” to track the formation and disappearance of components and holes with increasing α (Figure 8, left panel). These barcodes offer easy comparison of different sets of barcodes and their similarity can be calculated by the Jaccard index [149]. The Jaccard index results is a value between 0 and 1, where 0 is equal to no overlap of two bars and 1 describes two identical bars. Barcodes of different dimensions are defined as similar, if the averages of the individual similarity indices fulfill the Jaccard index conditions of similarity. Importantly, topological comparisons are independent of the scale so that it is possible to compare variably large clusters.
\nThe barcode transfers the examined structures into a form of visualization that is scale invariant. The formation and dissolution of small scaled complexes is recorded alongside the lifetime of large scaled complexes. Consequently, for γH2AX clusters, the barcodes contain bars representing components and holes in the nanometer but also in the micrometer scale ranges. In Figure 8 (right panel) a representative result of a heat map of Jaccard Indices is shown for SkBr3 breast cancer cells. 200 heterochromatin associated γH2AX clusters and 200 non-heterochromatin associated γH2AX clusters were selected by determining those with the highest and lowest heterochromatic densities in the environment and examined according to their topological similarity. For the average similarity for components and holes, heterochromatin associated γH2AX clusters showed a clear similarity whereas non-heterochromatin associated γH2AX clusters did not. This means that by topological analysis the heterochromatin associated γH2AX clusters could be discriminated as those clusters of high topological similarity [138]. The proximity of γH2AX clusters to heterochromatin seems to have a significant measurable impact on its structure. Interestingly, the non-heterochromatin associated γH2AX clusters and heterochromatin associated γH2AX clusters were more similar than the non-heterochromatin associated γH2AX clusters themselves. It can be clearly seen that the proximity to heterochromatin influences the structure of the clusters.
\nFor particle irradiated NHDF cells and U87 cells, the similarity values obtained by averaging of components and holes values for each 53BP1 cluster were determined and the clusters of the 10° irradiations scheme were compared. The Jaccard indices revealed values between 0.55 and 0.82 for U87 and NHDF cells. The broad frequency distribution did not show a peak for NHDF cells whereas for U87 cells a clear peak at 0.64 was found. If the clusters of the 90° irradiation scheme were compared, the peak was located at 0.63 for U87 cells. This value was the same, if the 10° with the 90° irradiation scheme was compared. For these two comparisons (90° vs. 90°, 10° vs. 90°), NHDF cells revealed a bimodal peak distribution where one peak was located at 0.67 and the other one at 0.72 (Figure 9). Thus, it can be concluded that in case of the more radio-sensitive NHDF cells a higher topological similarity in 53BP1 clustering was identified than the case the less radio-sensitive U87 cells.
\n“Normalized histograms of the frequencies of similarity values of barcodes (Jaccard indices) of 53BP1 clusters in NHDF and U87 cells irradiated under 10° or 90° irradiation angle and fixed 2h post irradiation. The distributions of the average similarity of dimension 0 and 1 barcodes of 53BP1 clusters in NHDF and U87 cells are shown. The similarity distributions of clusters in cells irradiated under an angle of 10° are shown in blue, the similarity distributions of clusters in cells irradiated under 90° are shown in orange, and the similarity distributions obtained when comparing clusters in cells irradiated with 10° to clusters in cells irradiated with 90° are depicted in green”. Note: These figures are modified and were originally published together with the cited figure legend under CC BY license in [
Alu short interspersed elements (SINEs) make up 11% of the human genome with over 1 million copies [150]; thereby making them ideal markers for assessing global chromatin architecture and dynamics by SMLM. Despite their involvement in many diseases of modern human [151, 152, 153, 154, 155, 156, 157, 158] and post-transcriptional regulation [159, 160, 161, 162, 163, 164, 165, 166], evidence grows that Alu elements are significantly regulating genome integrity and stability as a response to environmental stress. Concordantly, RNA Pol III transcriptional activation of Alu elements upon chemically and radiation induced DNA damage was observed [167] and epigenetic changes, such as DNA hypomethylation, in Alu elements are differently induced in human cell lines, when exposed to different types of radiation [168].
\nSMLM of irradiated breast cancer cells stained by combinatorial fluorescence in situ hybridization (COMBO-FISH) [169, 170] with a unique, short 17-mer oligonucleotide specific for genomic Alu elements (Figure 10A) resulted in a negative linear quadratic decline of the dose efficiency curve of localization signal points in the 0.5 Gy to 4 Gy dose range (Figure 10C) [170]. Furthermore, differential association of Alu signals with H3K9me3 heterochromatin between irradiated and non-irradiated cells could be revealed (Figure 10B). The heterochromatin relaxed after irradiation. However, the extension of this relaxation was independent of the dose. Alu dosimetry was also applied to the low dose range (< 0.5 Gy) (Figure 10D) [16], thereby opening new paths to study the molecular mechanisms underlying the controversial low dose radiation effects [171, 172, 173, 174, 175], which are difficult to assess due to a lack of appropriate biomarkers for the <0.5 Gy dose range [176].
\n(A) Single molecule localization microscopy analysis of Alu clustering and dose dependent effects of numbers of Alu labelling points after exposure to ionizing photon radiation. (B) Density distribution of heterochromatin labelling in concentric rings around the center of ALU clusters. The reduction of the density peak corresponding to heterochromatin relaxation around the Alu clusters was independent of the dose. (C) Linear quadratic dose response observed by SMLM of specific oligonucleotide nanoprobe labeling of Alu elements in SkBr3 cells after exposure to different doses of γ-radiation. (D) Linear dose response observed by SMLM of specific oligonucleotide nanoprobe labeling of Alu elements in SkBr3 cells after exposure to low doses of γ-radiation. Note: These figures are modified and were originally published under CC BY license in [
With this article, we have addressed scientists, researchers, and clinicians working in interdisciplinary fields, which are searching for a brief introduction to current radiobiology, its fundamental principles and methodologies. We would further like to have caught the attention of radiation biologists in laboratories, clinics, and industry by demonstrating novel super-resolution microscopy techniques that have the potential to drive radiobiology to a next generation. Single molecule localization allows geometrical and topological analyses on the meso- and nano-scale at the single-cell level in situ with the advantages of easy practice and the applicability to already existing experimental methods (e.g. immunostaining, FISH). As super-resolution microscopy techniques are still not a wide-spread routine in molecular biology laboratories, the long history of fluorescence microscopy data from radiobiological studies provides a solid basis for validation. We have shown that radiobiology can be an application of SMLM based nanoscopy and its versatile data analysis method which allow the investigation of new perspectives of DNA damage induction and repair. It can even help to discover novel markers of biological dosimetry as demonstrated by our recent studies assessing dose dependent effects on retrotransposon Alu availability. Nano-scaled analysis of repair foci architecture and dynamics by assessing foci like 53BP1, Mre11, etc. will give further insight into the molecular mechanisms of DNA damage response and fate of repair pathway of individual damage sites in single cells. Indeed, evidence grows that nanostructure and function of chromatin are highly interdependent aspects that govern the fundamentals of molecular genetics, such as cell type differentiation, gene expression, DNA damage repair and reproduction. Thus, super-resolution radiobiology could serve as a general proof of principle for many other molecular biology applications in future. Finally, we believe that single-molecule localization microscopy will develop to a standard application of radiation biology and might even add to the repertoire of diagnostic technologies in clinical facilities in the future.
\nThe successful collaborations with Felix Bestvater, German Cancer Research Center (DKFZ), Heidelberg, Christoph Cremer, Institute for Pharmacy and Molecular Biotechnology, Heidelberg University, Dieter W. Heermann, Institute for Theoretical Physics, Heidelberg University, Harry Scherthan, Institute for Radiobiology of the Bundeswehr, Munich, and Martin Falk, Institute of Biophysics, Czech Academy of Sciences, Brno, are gratefully acknowledged. The work was supported by the project grant (“Einflüsse strahleninduzierter, multipler und einzelner spezifisch-targetierter DNA-Strangschäden auf die übergeordnete meso- und nanoskalige Chromatinarchitektur und die Topologie von Reparaturfoci” (NANOSTRANG)) of the Federal Ministry of Education and Research.
\nThere is no conflict of interest for any of the authors.
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MRI is commonly used once treating brain, prostate cancers, ankle and foot. The Magnetic Resonance Imaging (MRI) images are usually liable to suffer from noises such as Gaussian noise, salt and pepper noise and speckle noise. So getting of brain image with accuracy is very extremely task. An accurate brain image is very necessary for further diagnosis process. During this chapter, a median filter algorithm will be modified. Gaussian noise and Salt and pepper noise will be added to MRI image. A proposed Median filter (MF), Adaptive Median filter (AMF) and Adaptive Wiener filter (AWF) will be implemented. The filters will be used to remove the additive noises present in the MRI images. The noise density will be added gradually to MRI image to compare performance of the filters evaluation. The performance of these filters will be compared exploitation the applied mathematics parameter Peak Signal-to-Noise Ratio (PSNR).",book:{id:"6144",slug:"high-resolution-neuroimaging-basic-physical-principles-and-clinical-applications",title:"High-Resolution Neuroimaging",fullTitle:"High-Resolution Neuroimaging - Basic Physical Principles and Clinical Applications"},signatures:"Hanafy M. Ali",authors:[{id:"213318",title:"Dr.",name:"Hanafy",middleName:"M.",surname:"Ali",slug:"hanafy-ali",fullName:"Hanafy Ali"}]},{id:"41589",doi:"10.5772/50323",title:"The Role of the Amygdala in Anxiety Disorders",slug:"the-role-of-the-amygdala-in-anxiety-disorders",totalDownloads:9671,totalCrossrefCites:4,totalDimensionsCites:28,abstract:null,book:{id:"2599",slug:"the-amygdala-a-discrete-multitasking-manager",title:"The Amygdala",fullTitle:"The Amygdala - A Discrete Multitasking Manager"},signatures:"Gina L. Forster, Andrew M. Novick, Jamie L. Scholl and Michael J. Watt",authors:[{id:"145620",title:"Dr.",name:"Gina",middleName:null,surname:"Forster",slug:"gina-forster",fullName:"Gina Forster"},{id:"146553",title:"BSc.",name:"Andrew",middleName:null,surname:"Novick",slug:"andrew-novick",fullName:"Andrew Novick"},{id:"146554",title:"MSc.",name:"Jamie",middleName:null,surname:"Scholl",slug:"jamie-scholl",fullName:"Jamie Scholl"},{id:"146555",title:"Dr.",name:"Michael",middleName:null,surname:"Watt",slug:"michael-watt",fullName:"Michael Watt"}]},{id:"26258",doi:"10.5772/28300",title:"Excitotoxicity and Oxidative Stress in Acute Ischemic Stroke",slug:"excitotoxicity-and-oxidative-stress-in-acute-ischemic-stroke",totalDownloads:7157,totalCrossrefCites:6,totalDimensionsCites:25,abstract:null,book:{id:"931",slug:"acute-ischemic-stroke",title:"Acute Ischemic Stroke",fullTitle:"Acute Ischemic Stroke"},signatures:"Ramón Rama Bretón and Julio César García Rodríguez",authors:[{id:"73430",title:"Prof.",name:"Ramon",middleName:null,surname:"Rama",slug:"ramon-rama",fullName:"Ramon Rama"},{id:"124643",title:"Prof.",name:"Julio Cesar",middleName:null,surname:"García",slug:"julio-cesar-garcia",fullName:"Julio Cesar García"}]},{id:"62072",doi:"10.5772/intechopen.78695",title:"Brain-Computer Interface and Motor Imagery Training: The Role of Visual Feedback and Embodiment",slug:"brain-computer-interface-and-motor-imagery-training-the-role-of-visual-feedback-and-embodiment",totalDownloads:1439,totalCrossrefCites:13,totalDimensionsCites:23,abstract:"Controlling a brain-computer interface (BCI) is a difficult task that requires extensive training. Particularly in the case of motor imagery BCIs, users may need several training sessions before they learn how to generate desired brain activity and reach an acceptable performance. A typical training protocol for such BCIs includes execution of a motor imagery task by the user, followed by presentation of an extending bar or a moving object on a computer screen. In this chapter, we discuss the importance of a visual feedback that resembles human actions, the effect of human factors such as confidence and motivation, and the role of embodiment in the learning process of a motor imagery task. Our results from a series of experiments in which users BCI-operated a humanlike android robot confirm that realistic visual feedback can induce a sense of embodiment, which promotes a significant learning of the motor imagery task in a short amount of time. We review the impact of humanlike visual feedback in optimized modulation of brain activity by the BCI users.",book:{id:"6610",slug:"evolving-bci-therapy-engaging-brain-state-dynamics",title:"Evolving BCI Therapy",fullTitle:"Evolving BCI Therapy - Engaging Brain State Dynamics"},signatures:"Maryam Alimardani, Shuichi Nishio and Hiroshi Ishiguro",authors:[{id:"11981",title:"Prof.",name:"Hiroshi",middleName:null,surname:"Ishiguro",slug:"hiroshi-ishiguro",fullName:"Hiroshi Ishiguro"},{id:"231131",title:"Dr.",name:"Maryam",middleName:null,surname:"Alimardani",slug:"maryam-alimardani",fullName:"Maryam Alimardani"},{id:"231134",title:"Dr.",name:"Shuichi",middleName:null,surname:"Nishio",slug:"shuichi-nishio",fullName:"Shuichi Nishio"}]}],mostDownloadedChaptersLast30Days:[{id:"29764",title:"Underlying Causes of Paresthesia",slug:"underlying-causes-of-paresthesia",totalDownloads:192666,totalCrossrefCites:3,totalDimensionsCites:7,abstract:null,book:{id:"1069",slug:"paresthesia",title:"Paresthesia",fullTitle:"Paresthesia"},signatures:"Mahdi Sharif-Alhoseini, Vafa Rahimi-Movaghar and Alexander R. Vaccaro",authors:[{id:"91165",title:"Prof.",name:"Vafa",middleName:null,surname:"Rahimi-Movaghar",slug:"vafa-rahimi-movaghar",fullName:"Vafa Rahimi-Movaghar"}]},{id:"63258",title:"Anatomy and Function of the Hypothalamus",slug:"anatomy-and-function-of-the-hypothalamus",totalDownloads:4558,totalCrossrefCites:6,totalDimensionsCites:12,abstract:"The hypothalamus is a small but important area of the brain formed by various nucleus and nervous fibers. Through its neuronal connections, it is involved in many complex functions of the organism such as vegetative system control, homeostasis of the organism, thermoregulation, and also in adjusting the emotional behavior. The hypothalamus is involved in different daily activities like eating or drinking, in the control of the body’s temperature and energy maintenance, and in the process of memorizing. It also modulates the endocrine system through its connections with the pituitary gland. Precise anatomical description along with a correct characterization of the component structures is essential for understanding its functions.",book:{id:"6331",slug:"hypothalamus-in-health-and-diseases",title:"Hypothalamus in Health and Diseases",fullTitle:"Hypothalamus in Health and Diseases"},signatures:"Miana Gabriela Pop, Carmen Crivii and Iulian Opincariu",authors:null},{id:"57103",title:"GABA and Glutamate: Their Transmitter Role in the CNS and Pancreatic Islets",slug:"gaba-and-glutamate-their-transmitter-role-in-the-cns-and-pancreatic-islets",totalDownloads:3478,totalCrossrefCites:3,totalDimensionsCites:9,abstract:"Glutamate and gamma-aminobutyric acid (GABA) are the major neurotransmitters in the mammalian brain. Inhibitory GABA and excitatory glutamate work together to control many processes, including the brain’s overall level of excitation. The contributions of GABA and glutamate in extra-neuronal signaling are by far less widely recognized. In this chapter, we first discuss the role of both neurotransmitters during development, emphasizing the importance of the shift from excitatory to inhibitory GABAergic neurotransmission. The second part summarizes the biosynthesis and role of GABA and glutamate in neurotransmission in the mature brain, and major neurological disorders associated with glutamate and GABA receptors and GABA release mechanisms. The final part focuses on extra-neuronal glutamatergic and GABAergic signaling in pancreatic islets of Langerhans, and possible associations with type 1 diabetes mellitus.",book:{id:"6237",slug:"gaba-and-glutamate-new-developments-in-neurotransmission-research",title:"GABA And Glutamate",fullTitle:"GABA And Glutamate - New Developments In Neurotransmission Research"},signatures:"Christiane S. Hampe, Hiroshi Mitoma and Mario Manto",authors:[{id:"210220",title:"Prof.",name:"Christiane",middleName:null,surname:"Hampe",slug:"christiane-hampe",fullName:"Christiane Hampe"},{id:"210485",title:"Prof.",name:"Mario",middleName:null,surname:"Manto",slug:"mario-manto",fullName:"Mario Manto"},{id:"210486",title:"Prof.",name:"Hiroshi",middleName:null,surname:"Mitoma",slug:"hiroshi-mitoma",fullName:"Hiroshi Mitoma"}]},{id:"35802",title:"Cross-Cultural/Linguistic Differences in the Prevalence of Developmental Dyslexia and the Hypothesis of Granularity and Transparency",slug:"cross-cultural-linguistic-differences-in-the-prevalence-of-developmental-dyslexia-and-the-hypothesis",totalDownloads:3601,totalCrossrefCites:2,totalDimensionsCites:7,abstract:null,book:{id:"673",slug:"dyslexia-a-comprehensive-and-international-approach",title:"Dyslexia",fullTitle:"Dyslexia - A Comprehensive and International Approach"},signatures:"Taeko N. Wydell",authors:[{id:"87489",title:"Prof.",name:"Taeko",middleName:"N.",surname:"Wydell",slug:"taeko-wydell",fullName:"Taeko Wydell"}]},{id:"58597",title:"Testosterone and Erectile Function: A Review of Evidence from Basic Research",slug:"testosterone-and-erectile-function-a-review-of-evidence-from-basic-research",totalDownloads:1331,totalCrossrefCites:2,totalDimensionsCites:2,abstract:"Androgens are essential for male physical activity and normal erectile function. Hence, age-related testosterone deficiency, known as late-onset hypogonadism (LOH), is considered a risk factor for erectile dysfunction (ED). This chapter summarizes relevant basic research reports examining the effects of testosterone on erectile function. Testosterone affects several organs and is especially active on the erectile tissue. The mechanism of testosterone deficiency effects on erectile function and the results of testosterone replacement therapy (TRT) have been well studied. Testosterone affects nitric oxide (NO) production and phosphodiesterase type 5 (PDE-5) expression in the corpus cavernosum through molecular pathways, preserves smooth muscle contractility by regulating both contraction and relaxation, and maintains the structure of the corpus cavernosum. Interestingly, testosterone deficiency has relationship to neurological diseases, which leads to ED. Testosterone replacement therapy is widely used to treat patients with testosterone deficiency; however, this treatment might also induce some problems. Basic research suggests that PDE-5 inhibitors, L-citrulline, and/or resveratrol therapy might be effective therapeutic options for testosterone deficiency-induced ED. Future research should confirm these findings through more specific experiments using molecular tools and may shed more light on endocrine-related ED and its possible treatments.",book:{id:"5994",slug:"sex-hormones-in-neurodegenerative-processes-and-diseases",title:"Sex Hormones in Neurodegenerative Processes and Diseases",fullTitle:"Sex Hormones in Neurodegenerative Processes and Diseases"},signatures:"Tomoya Kataoka and Kazunori Kimura",authors:[{id:"219042",title:"Ph.D.",name:"Tomoya",middleName:null,surname:"Kataoka",slug:"tomoya-kataoka",fullName:"Tomoya Kataoka"},{id:"229066",title:"Prof.",name:"Kazunori",middleName:null,surname:"Kimura",slug:"kazunori-kimura",fullName:"Kazunori Kimura"}]}],onlineFirstChaptersFilter:{topicId:"18",limit:6,offset:0},onlineFirstChaptersCollection:[{id:"81646",title:"Cortical Plasticity under Ketamine: From Synapse to Map",slug:"cortical-plasticity-under-ketamine-from-synapse-to-map",totalDownloads:14,totalDimensionsCites:0,doi:"10.5772/intechopen.104787",abstract:"Sensory systems need to process signals in a highly dynamic way to efficiently respond to variations in the animal’s environment. For instance, several studies showed that the visual system is subject to neuroplasticity since the neurons’ firing changes according to stimulus properties. This dynamic information processing might be supported by a network reorganization. Since antidepressants influence neurotransmission, they can be used to explore synaptic plasticity sustaining cortical map reorganization. To this goal, we investigated in the primary visual cortex (V1 of mouse and cat), the impact of ketamine on neuroplasticity through changes in neuronal orientation selectivity and the functional connectivity between V1 cells, using cross correlation analyses. We found that ketamine affects cortical orientation selectivity and alters the functional connectivity within an assembly. These data clearly highlight the role of the antidepressant drugs in inducing or modeling short-term plasticity in V1 which suggests that cortical processing is optimized and adapted to the properties of the stimulus.",book:{id:"11374",title:"Sensory Nervous System - Computational Neuroimaging Investigations of Topographical Organization in Human Sensory Cortex",coverURL:"https://cdn.intechopen.com/books/images_new/11374.jpg"},signatures:"Ouelhazi Afef, Rudy Lussiez and Molotchnikoff Stephane"},{id:"81582",title:"The Role of Cognitive Reserve in Executive Functioning and Its Relationship to Cognitive Decline and Dementia",slug:"the-role-of-cognitive-reserve-in-executive-functioning-and-its-relationship-to-cognitive-decline-and",totalDownloads:22,totalDimensionsCites:0,doi:"10.5772/intechopen.104646",abstract:"In this chapter, we explore how cognitive reserve is implicated in coping with the negative consequences of brain pathology and age-related cognitive decline. Individual differences in cognitive performance are based on different brain mechanisms (neural reserve and neural compensation), and reflect, among others, the effect of education, occupational attainment, leisure activities, and social involvement. These cognitive reserve proxies have been extensively associated with efficient executive functioning. We discuss and focus particularly on the compensation mechanisms related to the frontal lobe and its protective role, in maintaining cognitive performance in old age or even mitigating the clinical expression of dementia.",book:{id:"11742",title:"Neurophysiology",coverURL:"https://cdn.intechopen.com/books/images_new/11742.jpg"},signatures:"Gabriela Álvares-Pereira, Carolina Maruta and Maria Vânia Silva-Nunes"},{id:"81488",title:"Aggression and Sexual Behavior: Overlapping or Distinct Roles of 5-HT1A and 5-HT1B Receptors",slug:"aggression-and-sexual-behavior-overlapping-or-distinct-roles-of-5-ht1a-and-5-ht1b-receptors",totalDownloads:19,totalDimensionsCites:0,doi:"10.5772/intechopen.104872",abstract:"Distinct brain mechanisms for male aggressive and sexual behavior are present in mammalian species, including man. However, recent evidence suggests a strong connection and even overlap in the central nervous system (CNS) circuitry involved in aggressive and sexual behavior. The serotonergic system in the CNS is strongly involved in male aggressive and sexual behavior. In particular, 5-HT1A and 5-HT1B receptors seem to play a critical role in the modulation of these behaviors. The present chapter focuses on the effects of 5-HT1A- and 5-HT1B-receptor ligands in male rodent aggression and sexual behavior. Results indicate that 5-HT1B-heteroreceptors play a critical role in the modulation of male offensive behavior, although a definite role of 5-HT1A-auto- or heteroreceptors cannot be ruled out. 5-HT1A receptors are clearly involved in male sexual behavior, although it has to be yet unraveled whether 5-HT1A-auto- or heteroreceptors are important. Although several key nodes in the complex circuitry of aggression and sexual behavior are known, in particular in the medial hypothalamus, a clear link or connection to these critical structures and the serotonergic key receptors is yet to be determined. This information is urgently needed to detect and develop new selective anti-aggressive (serenic) and pro-sexual drugs for human applications.",book:{id:"10195",title:"Serotonin and the CNS - New Developments in Pharmacology and Therapeutics",coverURL:"https://cdn.intechopen.com/books/images_new/10195.jpg"},signatures:"Berend Olivier and Jocelien D.A. Olivier"},{id:"81093",title:"Prehospital and Emergency Room Airway Management in Traumatic Brain Injury",slug:"prehospital-and-emergency-room-airway-management-in-traumatic-brain-injury",totalDownloads:49,totalDimensionsCites:0,doi:"10.5772/intechopen.104173",abstract:"Airway management in trauma is critical and may impact patient outcomes. Particularly in traumatic brain injury (TBI), depressed level of consciousness may be associated with compromised protective airway reflexes or apnea, which can increase the risk of aspiration or result in hypoxemia and worsen the secondary brain damage. Therefore, patients with TBI and Glasgow Coma Scale (GCS) ≤ 8 have been traditionally managed by prehospital or emergency room (ER) endotracheal intubation. However, recent evidence challenged this practice and even suggested that routine intubation may be harmful. This chapter will address the indications and optimal method of securing the airway, prehospital and in the ER, in patients with traumatic brain injury.",book:{id:"11367",title:"Traumatic Brain Injury",coverURL:"https://cdn.intechopen.com/books/images_new/11367.jpg"},signatures:"Dominik A. Jakob, Jean-Cyrille Pitteloud and Demetrios Demetriades"},{id:"81011",title:"Amino Acids as Neurotransmitters. The Balance between Excitation and Inhibition as a Background for Future Clinical Applications",slug:"amino-acids-as-neurotransmitters-the-balance-between-excitation-and-inhibition-as-a-background-for-f",totalDownloads:19,totalDimensionsCites:0,doi:"10.5772/intechopen.103760",abstract:"For more than 30 years, amino acids have been well-known (and essential) participants in neurotransmission. They act as both neuromediators and metabolites in nervous tissue. Glycine and glutamic acid (glutamate) are prominent examples. These amino acids are agonists of inhibitory and excitatory membrane receptors, respectively. Moreover, they play essential roles in metabolic pathways and energy transformation in neurons and astrocytes. Despite their obvious effects on the brain, their potential role in therapeutic methods remains uncertain in clinical practice. In the current chapter, a comparison of the crosstalk between these two systems, which are responsible for excitation and inhibition in neurons, is presented. The interactions are discussed at the metabolic, receptor, and transport levels. Reaction-diffusion and a convectional flow into the interstitial fluid create a balanced distribution of glycine and glutamate. Indeed, the neurons’ final physiological state is a result of a balance between the excitatory and inhibitory influences. However, changes to the glycine and/or glutamate pools under pathological conditions can alter the state of nervous tissue. Thus, new therapies for various diseases may be developed on the basis of amino acid medication.",book:{id:"10890",title:"Recent Advances in Neurochemistry",coverURL:"https://cdn.intechopen.com/books/images_new/10890.jpg"},signatures:"Yaroslav R. Nartsissov"},{id:"80821",title:"Neuroimmunology and Neurological Manifestations of COVID-19",slug:"neuroimmunology-and-neurological-manifestations-of-covid-19",totalDownloads:41,totalDimensionsCites:0,doi:"10.5772/intechopen.103026",abstract:"Infection with SARS-CoV-2 is causing coronavirus disease in 2019 (COVID-19). Besides respiratory symptoms due to an attack on the broncho-alveolar system, COVID-19, among others, can be accompanied by neurological symptoms because of the affection of the nervous system. These can be caused by intrusion by SARS-CoV-2 of the central nervous system (CNS) and peripheral nervous system (PNS) and direct infection of local cells. In addition, neurological deterioration mediated by molecular mimicry to virus antigens or bystander activation in the context of immunological anti-virus defense can lead to tissue damage in the CNS and PNS. In addition, cytokine storm caused by SARS-CoV-2 infection in COVID-19 can lead to nervous system related symptoms. Endotheliitis of CNS vessels can lead to vessel occlusion and stroke. COVID-19 can also result in cerebral hemorrhage and sinus thrombosis possibly related to changes in clotting behavior. Vaccination is most important to prevent COVID-19 in the nervous system. There are symptomatic or/and curative therapeutic approaches to combat COVID-19 related nervous system damage that are partly still under study.",book:{id:"10890",title:"Recent Advances in Neurochemistry",coverURL:"https://cdn.intechopen.com/books/images_new/10890.jpg"},signatures:"Robert Weissert"}],onlineFirstChaptersTotal:17},preDownload:{success:null,errors:{}},subscriptionForm:{success:null,errors:{}},aboutIntechopen:{},privacyPolicy:{},peerReviewing:{},howOpenAccessPublishingWithIntechopenWorks:{},sponsorshipBooks:{sponsorshipBooks:[],offset:0,limit:8,total:null},allSeries:{pteSeriesList:[{id:"14",title:"Artificial Intelligence",numberOfPublishedBooks:9,numberOfPublishedChapters:87,numberOfOpenTopics:6,numberOfUpcomingTopics:0,issn:"2633-1403",doi:"10.5772/intechopen.79920",isOpenForSubmission:!0},{id:"7",title:"Biomedical Engineering",numberOfPublishedBooks:12,numberOfPublishedChapters:98,numberOfOpenTopics:3,numberOfUpcomingTopics:0,issn:"2631-5343",doi:"10.5772/intechopen.71985",isOpenForSubmission:!0}],lsSeriesList:[{id:"11",title:"Biochemistry",numberOfPublishedBooks:27,numberOfPublishedChapters:288,numberOfOpenTopics:4,numberOfUpcomingTopics:0,issn:"2632-0983",doi:"10.5772/intechopen.72877",isOpenForSubmission:!0},{id:"25",title:"Environmental Sciences",numberOfPublishedBooks:1,numberOfPublishedChapters:9,numberOfOpenTopics:4,numberOfUpcomingTopics:0,issn:"2754-6713",doi:"10.5772/intechopen.100362",isOpenForSubmission:!0},{id:"10",title:"Physiology",numberOfPublishedBooks:11,numberOfPublishedChapters:139,numberOfOpenTopics:4,numberOfUpcomingTopics:0,issn:"2631-8261",doi:"10.5772/intechopen.72796",isOpenForSubmission:!0}],hsSeriesList:[{id:"3",title:"Dentistry",numberOfPublishedBooks:8,numberOfPublishedChapters:129,numberOfOpenTopics:0,numberOfUpcomingTopics:2,issn:"2631-6218",doi:"10.5772/intechopen.71199",isOpenForSubmission:!1},{id:"6",title:"Infectious Diseases",numberOfPublishedBooks:13,numberOfPublishedChapters:107,numberOfOpenTopics:3,numberOfUpcomingTopics:1,issn:"2631-6188",doi:"10.5772/intechopen.71852",isOpenForSubmission:!0},{id:"13",title:"Veterinary Medicine and Science",numberOfPublishedBooks:10,numberOfPublishedChapters:103,numberOfOpenTopics:3,numberOfUpcomingTopics:0,issn:"2632-0517",doi:"10.5772/intechopen.73681",isOpenForSubmission:!0}],sshSeriesList:[{id:"22",title:"Business, Management and Economics",numberOfPublishedBooks:1,numberOfPublishedChapters:12,numberOfOpenTopics:2,numberOfUpcomingTopics:1,issn:"2753-894X",doi:"10.5772/intechopen.100359",isOpenForSubmission:!0},{id:"23",title:"Education and Human Development",numberOfPublishedBooks:0,numberOfPublishedChapters:0,numberOfOpenTopics:2,numberOfUpcomingTopics:0,issn:null,doi:"10.5772/intechopen.100360",isOpenForSubmission:!1},{id:"24",title:"Sustainable Development",numberOfPublishedBooks:0,numberOfPublishedChapters:11,numberOfOpenTopics:4,numberOfUpcomingTopics:1,issn:null,doi:"10.5772/intechopen.100361",isOpenForSubmission:!0}],testimonialsList:[{id:"6",text:"It is great to work with the IntechOpen to produce a worthwhile collection of research that also becomes a great educational resource and guide for future research endeavors.",author:{id:"259298",name:"Edward",surname:"Narayan",institutionString:null,profilePictureURL:"https://mts.intechopen.com/storage/users/259298/images/system/259298.jpeg",slug:"edward-narayan",institution:{id:"3",name:"University of Queensland",country:{id:null,name:"Australia"}}}},{id:"13",text:"The collaboration with and support of the technical staff of IntechOpen is fantastic. The whole process of submitting an article and editing of the submitted article goes extremely smooth and fast, the number of reads and downloads of chapters is high, and the contributions are also frequently cited.",author:{id:"55578",name:"Antonio",surname:"Jurado-Navas",institutionString:null,profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002bRisIQAS/Profile_Picture_1626166543950",slug:"antonio-jurado-navas",institution:{id:"720",name:"University of Malaga",country:{id:null,name:"Spain"}}}}]},series:{item:{id:"11",title:"Biochemistry",doi:"10.5772/intechopen.72877",issn:"2632-0983",scope:"Biochemistry, the study of chemical transformations occurring within living organisms, impacts all areas of life sciences, from molecular crystallography and genetics to ecology, medicine, and population biology. Biochemistry examines macromolecules - proteins, nucleic acids, carbohydrates, and lipids – and their building blocks, structures, functions, and interactions. Much of biochemistry is devoted to enzymes, proteins that catalyze chemical reactions, enzyme structures, mechanisms of action and their roles within cells. Biochemistry also studies small signaling molecules, coenzymes, inhibitors, vitamins, and hormones, which play roles in life processes. Biochemical experimentation, besides coopting classical chemistry methods, e.g., chromatography, adopted new techniques, e.g., X-ray diffraction, electron microscopy, NMR, radioisotopes, and developed sophisticated microbial genetic tools, e.g., auxotroph mutants and their revertants, fermentation, etc. More recently, biochemistry embraced the ‘big data’ omics systems. Initial biochemical studies have been exclusively analytic: dissecting, purifying, and examining individual components of a biological system; in the apt words of Efraim Racker (1913 –1991), “Don’t waste clean thinking on dirty enzymes.” Today, however, biochemistry is becoming more agglomerative and comprehensive, setting out to integrate and describe entirely particular biological systems. The ‘big data’ metabolomics can define the complement of small molecules, e.g., in a soil or biofilm sample; proteomics can distinguish all the comprising proteins, e.g., serum; metagenomics can identify all the genes in a complex environment, e.g., the bovine rumen. This Biochemistry Series will address the current research on biomolecules and the emerging trends with great promise.",coverUrl:"https://cdn.intechopen.com/series/covers/11.jpg",latestPublicationDate:"May 24th, 2022",hasOnlineFirst:!0,numberOfPublishedBooks:27,editor:{id:"31610",title:"Dr.",name:"Miroslav",middleName:null,surname:"Blumenberg",slug:"miroslav-blumenberg",fullName:"Miroslav Blumenberg",profilePictureURL:"https://mts.intechopen.com/storage/users/31610/images/system/31610.jpg",biography:"Miroslav Blumenberg, Ph.D., was born in Subotica and received his BSc in Belgrade, Yugoslavia. He completed his Ph.D. at MIT in Organic Chemistry; he followed up his Ph.D. with two postdoctoral study periods at Stanford University. Since 1983, he has been a faculty member of the RO Perelman Department of Dermatology, NYU School of Medicine, where he is codirector of a training grant in cutaneous biology. Dr. Blumenberg’s research is focused on the epidermis, expression of keratin genes, transcription profiling, keratinocyte differentiation, inflammatory diseases and cancers, and most recently the effects of the microbiome on the skin. He has published more than 100 peer-reviewed research articles and graduated numerous Ph.D. and postdoctoral students.",institutionString:null,institution:{name:"New York University Langone Medical Center",institutionURL:null,country:{name:"United States of America"}}},editorTwo:null,editorThree:null},subseries:{paginationCount:4,paginationItems:[{id:"14",title:"Cell and Molecular Biology",coverUrl:"https://cdn.intechopen.com/series_topics/covers/14.jpg",isOpenForSubmission:!0,editor:{id:"165627",title:"Dr.",name:"Rosa María",middleName:null,surname:"Martínez-Espinosa",slug:"rosa-maria-martinez-espinosa",fullName:"Rosa María Martínez-Espinosa",profilePictureURL:"https://mts.intechopen.com/storage/users/165627/images/system/165627.jpeg",biography:"Dr. Rosa María Martínez-Espinosa has been a Spanish Full Professor since 2020 (Biochemistry and Molecular Biology) and is currently Vice-President of International Relations and Cooperation development and leader of the research group 'Applied Biochemistry” (University of Alicante, Spain). Other positions she has held at the university include Vice-Dean of Master Programs, Vice-Dean of the Degree in Biology and Vice-Dean for Mobility and Enterprise and Engagement at the Faculty of Science (University of Alicante). She received her Bachelor in Biology in 1998 (University of Alicante) and her PhD in 2003 (Biochemistry, University of Alicante). She undertook post-doctoral research at the University of East Anglia (Norwich, U.K. 2004-2005; 2007-2008).\nHer multidisciplinary research focuses on investigating archaea and their potential applications in biotechnology. She has an H-index of 21. She has authored one patent and has published more than 70 indexed papers and around 60 book chapters.\nShe has contributed to more than 150 national and international meetings during the last 15 years. Her research interests include archaea metabolism, enzymes purification and characterization, gene regulation, carotenoids and bioplastics production, antioxidant\ncompounds, waste water treatments, and brines bioremediation.\nRosa María’s other roles include editorial board member for several journals related\nto biochemistry, reviewer for more than 60 journals (biochemistry, molecular biology, biotechnology, chemistry and microbiology) and president of several organizing committees in international meetings related to the N-cycle or respiratory processes.",institutionString:null,institution:{name:"University of Alicante",institutionURL:null,country:{name:"Spain"}}},editorTwo:null,editorThree:null},{id:"15",title:"Chemical Biology",coverUrl:"https://cdn.intechopen.com/series_topics/covers/15.jpg",isOpenForSubmission:!0,editor:{id:"441442",title:"Dr.",name:"Şükrü",middleName:null,surname:"Beydemir",slug:"sukru-beydemir",fullName:"Şükrü Beydemir",profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0033Y00003GsUoIQAV/Profile_Picture_1634557147521",biography:"Dr. Şükrü Beydemir obtained a BSc in Chemistry in 1995 from Yüzüncü Yıl University, MSc in Biochemistry in 1998, and PhD in Biochemistry in 2002 from Atatürk University, Turkey. He performed post-doctoral studies at Max-Planck Institute, Germany, and University of Florence, Italy in addition to making several scientific visits abroad. He currently works as a Full Professor of Biochemistry in the Faculty of Pharmacy, Anadolu University, Turkey. Dr. Beydemir has published over a hundred scientific papers spanning protein biochemistry, enzymology and medicinal chemistry, reviews, book chapters and presented several conferences to scientists worldwide. He has received numerous publication awards from various international scientific councils. He serves in the Editorial Board of several international journals. 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He is a member of the Turkish Biochemical Society, American Chemical Society, and German Genetics society. Dr. Ekinci published around ninety scientific papers, reviews and book chapters, and presented several conferences to scientists. He has received numerous publication awards from several scientific councils. 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He worked on the structure-function relationships of glycoconjugates and his main project was the investigations on the biological roles of the de-N-glycosylation enzymes (Endo-N-acetyl-β-D-glucosaminidase and peptide-N4-(N-acetyl-β-glucosaminyl) asparagine amidase). From 2002 he contributes to the understanding of the Blood-brain barrier functioning using proteomics approaches. He has published more than 70 papers. 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