Equipment needed for the methods described in this chapter.
\r\n\tIt is an exceedingly complex condition often with contradictory findings. Diagnosis has been controversial since Leo Kanner's narrow autism to the broader autism spectrum of today. Screening and diagnostic instruments are also problematic and have limitations and broader instruments are better for example the DISCO. The National Institute for Clinical Excellence recommends no specific instruments and states that diagnosis is a clinical task and the instruments are only to be used as adjunctive. While genetic factors are highly significant, there is huge complexity in the genotype and involves a vast array of genes of small effect. Environmental factors in autism are equally complex and controversial, ranging from prenatal and perinatal factors to drugs, testosterone, environmental toxins and fever inducing factors, etc. The natural history and the effect of intervention and treatment on the outcome is very important. Huge resources are devoted to research on autism with increasing numbers of publications.
",isbn:"978-1-83881-012-2",printIsbn:"978-1-83881-005-4",pdfIsbn:"978-1-83881-013-9",doi:null,price:0,priceEur:0,priceUsd:0,slug:null,numberOfPages:0,isOpenForSubmission:!1,hash:"b9c36a2454fac16e70ba00562cb6f009",bookSignature:"Dr. Michael Fitzgerald",publishedDate:null,coverURL:"https://cdn.intechopen.com/books/images_new/9634.jpg",keywords:"History, Cognitive Phenotype, Aq, CARS, Epidemiology, Risk, Neurochemistry, Brain Function, Drug Vaccinations, Testosterone, Long Term, Effect of Interventions",numberOfDownloads:103,numberOfWosCitations:0,numberOfCrossrefCitations:0,numberOfDimensionsCitations:0,numberOfTotalCitations:0,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"September 18th 2020",dateEndSecondStepPublish:"October 16th 2020",dateEndThirdStepPublish:"December 15th 2020",dateEndFourthStepPublish:"March 5th 2021",dateEndFifthStepPublish:"May 4th 2021",remainingDaysToSecondStep:"4 months",secondStepPassed:!0,currentStepOfPublishingProcess:4,editedByType:null,kuFlag:!1,biosketch:"Prof Michael Fitzgerald was the winner of the “Excellence in Psychiatry” award 2017 and was nominated as one of the top 4 Psychiatrists by Hospital Professional News Ireland – Top 100 Professionals in Ireland 2017. The first Professor of Child and Adolescent Psychiatry in Ireland, specializing in Autism, Aspergers Syndrome, and ADHD. He was the first Psychoanalyst recognized by the International Psychoanalytic Association founded by Sigmund Freud to work in the Republic of Ireland.",coeditorOneBiosketch:null,coeditorTwoBiosketch:null,coeditorThreeBiosketch:null,coeditorFourBiosketch:null,coeditorFiveBiosketch:null,editors:[{id:"205005",title:"Dr.",name:"Michael",middleName:null,surname:"Fitzgerald",slug:"michael-fitzgerald",fullName:"Michael Fitzgerald",profilePictureURL:"https://mts.intechopen.com/storage/users/205005/images/system/205005.jpg",biography:"Professor Michael Fitzgerald was the first Professor of Child and\nAdolescent Psychiatry in Ireland (Retired). Specialising in autism spectrum\ndisorders, he has diagnosed over 5000 patients. He has a\nlarge number of peer-reviewed publications and has written,\nco-written, and co-edited 34 books with Japanese, Dutch, and\nPolish translations. Professor Simon Baron-Cohen of the University\nof Cambridge described one of his books on autism as 'the\nbest book on autism” and described him as an 'exceptional scholar.” He has lectured\nextensively throughout the world, including the Royal Society/British Academy\nand the British Library in London. He was the overall winner of the 'Excellence in\nPsychiatry” Award 2017 and was nominated as one of the top four psychiatrists by\nHospital Professional News Ireland—Top 100 Professionals in Ireland 2017.",institutionString:"Trinity College",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"2",totalChapterViews:"0",totalEditedBooks:"3",institution:{name:"Trinity College Dublin",institutionURL:null,country:{name:"Ireland"}}}],coeditorOne:null,coeditorTwo:null,coeditorThree:null,coeditorFour:null,coeditorFive:null,topics:[{id:"16",title:"Medicine",slug:"medicine"}],chapters:[{id:"74828",title:"An Observationally and Psychoanalytically Informed Parent-Toddler Intervention for Young Children at Risk of ASD: An Audited Case Series and Convergences with Organicist Approaches",slug:"an-observationally-and-psychoanalytically-informed-parent-toddler-intervention-for-young-children-at",totalDownloads:15,totalCrossrefCites:0,authors:[null]},{id:"74695",title:"Associations between Monocyte Cytokine Profiles and Co-Morbid Conditions in Autism Spectrum Disorders",slug:"associations-between-monocyte-cytokine-profiles-and-co-morbid-conditions-in-autism-spectrum-disorder",totalDownloads:31,totalCrossrefCites:0,authors:[null]},{id:"74353",title:"L1-79 and the Role of Catecholamines in Autism",slug:"l1-79-and-the-role-of-catecholamines-in-autism",totalDownloads:38,totalCrossrefCites:0,authors:[null]},{id:"74867",title:"Exposure to Xenobiotics and Gene-Environment Interactions in Autism Spectrum Disorder: A Systematic Review",slug:"exposure-to-xenobiotics-and-gene-environment-interactions-in-autism-spectrum-disorder-a-systematic-r",totalDownloads:20,totalCrossrefCites:0,authors:[null]}],productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"},personalPublishingAssistant:{id:"185543",firstName:"Maja",lastName:"Bozicevic",middleName:null,title:"Ms.",imageUrl:"https://mts.intechopen.com/storage/users/185543/images/4748_n.jpeg",email:"maja.b@intechopen.com",biography:"As an Author Service Manager my responsibilities include monitoring and facilitating all publishing activities for authors and editors. From chapter submission and review, to approval and revision, copyediting and design, until final publication, I work closely with authors and editors to ensure a simple and easy publishing process. I maintain constant and effective communication with authors, editors and reviewers, which allows for a level of personal support that enables contributors to fully commit and concentrate on the chapters they are writing, editing, or reviewing. I assist authors in the preparation of their full chapter submissions and track important deadlines and ensure they are met. I help to coordinate internal processes such as linguistic review, and monitor the technical aspects of the process. As an ASM I am also involved in the acquisition of editors. Whether that be identifying an exceptional author and proposing an editorship collaboration, or contacting researchers who would like the opportunity to work with IntechOpen, I establish and help manage author and editor acquisition and contact."}},relatedBooks:[{type:"book",id:"5498",title:"Autism",subtitle:"Paradigms, Recent Research and Clinical Applications",isOpenForSubmission:!1,hash:"7a4a04bc1ec60da290315a53de5043b8",slug:"autism-paradigms-recent-research-and-clinical-applications",bookSignature:"Michael Fitzgerald and Jane Yip",coverURL:"https://cdn.intechopen.com/books/images_new/5498.jpg",editedByType:"Edited by",editors:[{id:"205005",title:"Dr.",name:"Michael",surname:"Fitzgerald",slug:"michael-fitzgerald",fullName:"Michael Fitzgerald"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"8430",title:"Neurodevelopment and Neurodevelopmental Disorder",subtitle:null,isOpenForSubmission:!1,hash:"696c96d038de473216e48b199613c111",slug:"neurodevelopment-and-neurodevelopmental-disorder",bookSignature:"Michael Fitzgerald",coverURL:"https://cdn.intechopen.com/books/images_new/8430.jpg",editedByType:"Edited by",editors:[{id:"205005",title:"Dr.",name:"Michael",surname:"Fitzgerald",slug:"michael-fitzgerald",fullName:"Michael Fitzgerald"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"7835",title:"Autism Spectrum Disorders",subtitle:"Advances at the End of the Second Decade of the 21st Century",isOpenForSubmission:!1,hash:"2cfcf44e79e12e620251aaa9d08a4a3e",slug:"autism-spectrum-disorders-advances-at-the-end-of-the-second-decade-of-the-21st-century",bookSignature:"Michael Fitzgerald",coverURL:"https://cdn.intechopen.com/books/images_new/7835.jpg",editedByType:"Edited by",editors:[{id:"205005",title:"Dr.",name:"Michael",surname:"Fitzgerald",slug:"michael-fitzgerald",fullName:"Michael Fitzgerald"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"6550",title:"Cohort Studies in Health Sciences",subtitle:null,isOpenForSubmission:!1,hash:"01df5aba4fff1a84b37a2fdafa809660",slug:"cohort-studies-in-health-sciences",bookSignature:"R. 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NSCs were initially identified in the subventricular zone of the mouse brain (Temple, 1989) and subsequently in various regions of adult brains from human and mouse (Taupin & Gage, 2002). NSCs can be derived in vitro from embryonic stem (ES) cells and induced pluripotent stem (iPS) cells by employing specific culture conditions. NSCs and their progeny can be expanded for several passages as tridimensional floating aggregates named neurospheres or as monolayer cultures. This in turn allows derivation of neurons of different parts of the nervous system (Gaspard & Vanderhaeghen, 2011). NSCs derived from ES/iPS cells of various genetic backgrounds represent invaluable tools for the investigation of neurogenesis, development of neurologic diseases models, and screening of new drugs to treat neurological diseases.
Derived from ES cells, NSCs and their progeny, neural progenitors, are routinely studied in vitro by a method called neurosphere culture system (Reynolds & Weiss, 1992). Neurospheres can measure 100-300 mm. The zonal distribution of different cell types that compose human and murine neurospheres resembles an outside-in brain structure with nestin-positive progenitor cells in the periphery and GFAP+ and β-tubulin III-positive cells in the centre (Moors et al., 2009; Campos et al., 2004).
Currently, there are two methods that allow derivation of neurospheres from mouse ES cells. Both of these methods utilize fibroblast growth factor 2 (FGF-2), epidermal growth factor (EGF), retinoic acid, and other supplements such as B27 and N2. FGF-2 is a critical component in neural differentiation protocols as it promotes proliferation of neuroprogenitor cells (Yoshimura et al., 2001). For selecting and expanding neural progenitor cells, culture media are supplemented with B-27 (containing retinoic acid) and N2 (which has a subset of component of B-27 that include insulin and transferrin).
In one method of derivation, ES cells are cultured in a specific medium supplemented with EGF, FGF-2 and N2. After 4-5 days, 50-80% of the cells undergo neural lineage specification. On day 7, these cells are dissociated and plated in suspension in uncoated plates in a differentiation medium supplemented with N2, B27, mouse EGF and human FGF-2. After a few days of culturing, neurospheres can be obtained (Conti et al., 2005).
Alternatively, in a second method, mouse ES cells are co-cultured for approximately one week with the PA6 stromal cell line to achieve efficient neuronal differentiation because of the stromal cell-derived inducing activity (SDIA) (Kawasaki et al., 2000). After 8 days of SDIA treatment, mouse ES cells are separated from PA6 stromal cells and cultured in suspension in serum-free medium supplemented with FGF-2. Under these conditions neurospheres appear within 4 days (Morizane et al., 2006).
Exemplifying stages of the neurosphere generation process are shown in figure 1.
The ability of mouse ES cells to spontaneously generate neurospheres and mature neurons in short time and in few steps, represents the strength for a powerful and reliable in vitro model to apply research strategies oriented to study the physiopathology of the biochemical and epigenetic mechanisms leading to the neurogenesis.
We recently observed that mouse ES cells when grown in simple culture conditions spontaneously form neurospheres. In this chapter, we describe a simple method for derivation of neurospheres from mouse ES cells without using FGF-2 and EGF and without co-culturing ES cells with stromal cells.
Generation of neurospheres from mES cells. a) Microphotograph of a mES colony; b) typical clusters of cells generated after culturing R1 mES for three passages in a DMEM/FBS medium; c) bright field of a neurosphere; d) progenitors migrating from neurospheres. [Images taken by authors.] Scale bar 1s 100 mm.
The reagents and equipment needed for the methods described in this chapter can be found listed in the tables below.
Equipment | Company | Catalog # |
0.22-μm 500 ml bottle top filter | Corning | 430513 |
0.22-μm 250 ml filter system | Corning | 430767 |
10 cm cell culture dish | Corning | 439167 |
24-well plates (Transwell-Clear permeable support 0.4 μm | Fischer | 07-200-154 |
6-well ultra low attachment plate | Costar | 3471 |
15-ml polypropylene conical tubes | BD Falcon | 352096 |
50-ml polypropylene conical tubes | BD Falcon | 352070 |
Transfer pipettes | Samco | 262-1S |
0.22-μm bottle top filters | TPP | 99505 |
0.45-μm bottle top filters | Nalgene | 165-0045 |
12 mm circular cover slips | Fisher | 01-472A |
Hemocytometer | Hycor Kova | 87144 |
1000-ml media bottles | ||
Kimwipes | Kimberly-Clarks | 34155 |
Humidified tissue culture incubator (37°C, 5% CO2) | - | - |
Centrifuge | - | - |
Water bath (37°C) | - | - |
Scanning confocal microscope | Olympus | FluoView 100 |
DM IRM inverted microscope (fitted with Leica HS N Plan BD 50X oil immersion objective) | Leica | - |
75W xenon lamp-based monochromator | Ushio, Japan | - |
CCD camera | Orca (Hamamatsu, Shizouka, Japan) | - |
535 ± 40nm bandpass filter | Omega Optical | - |
500 nm longpass dichromatic mirror | - | - |
Simple PCI | Compix, Inc (Cranberry, PA) | - |
Equipment needed for the methods described in this chapter.
Reagent | Company | Catalog # |
R1 ES cells | Mutant Mouse Regional Resource Centers | 011979-MU |
Non-essential amino acid solution (100X) | Millipore | TMS-001 |
Nucleosides (100X) | Millipore | ES-008-E |
L-glutamine (100X) | Millipore | TMS-002-C |
Penicillin/Streptomycin (100X) | Millipore | TMS-001 |
2-Mercaptoethanol (100X) | Millipore | ES-007-E |
Sodium pyruvate (100mM) | Cellgro | 25-00-CI |
Trypsin-EDTA (100X) | Gibco | 15400 |
Dulbecco’s phosphate buffered saline (10X) (PBS) | Sigma | P-3813 |
Gelatin from porcine skin (Type A) | Sigma | G1890 |
Dulbecco’s modified Eagle’s medium (DMEM) | Millipore | SLM-220-B |
Fetal bovine serum (ES cell grade) (FBS) | Gibco | 16141-079 |
Knockout Serum Replacement (KSR) | Invitrogen | 10828028 |
Neurobasal medium | Invitrogen | 21103-049 |
Phosphate buffered saline (PBS) | Gibco | 14040141 |
Poly-D-lysine (PDL) hydrobromide | Sigma | P6407 |
Leukemia inhibitory factor (LIF) (107) | Millipore | ESG1107 |
B27 | Invitrogen | 17504-044 |
N2 supplement | Invitrogen | 17502-048 |
FGF-basic Recombinant Mouse | Invitrogen | PMG0035 |
Laminin | Sigma | L2020 |
BisBenzimide H 33342 trihydrochloride | Sigma | B-2261 |
Mounting solution | Gelvatol | - |
Paraformaldehyde (16% in premixed PBS buffer at pH7 [below]) | Electron Microscopy Sciences | 15710-6 |
PBS buffer at pH7 (1X) | Roche | 11666789001 |
10% Normal Donkey Serum (NDS) | Jackson ImmunoResearch | 711-165-152 |
0.01% TritonX-100 | Fluka | BP151-100 |
Tubulin III | R&D Systems | MAB1195 |
Rabbit Anti-NMDAR1 | Abcam | Ab68144 |
Bovine albumin | Sigma | A-6003 |
Fura-2, AM cell-permeant | Invitrogen | F-1221 |
Cy3 AffiniPure Donkey Anti-Rabbit | Jackson ImmunoResearch | 711-165-152 |
AffiniPure Donkey Anti-Mouse | Jackson ImmunoResearch | 715-175-151 |
Reagents needed for the methods described in this chapter.
The setup for the reagents and equipment listed is as follows.
Reagent | Setup |
ES medium | For 500 ml of ES medium, add the following components: 400 ml DMEM, 75 ml FBS, 5 ml Pen/Strep, 5 ml 2ME (100 mM), 5 ml L-glutamine, 5 ml NEAA, 5 ml nucleosides, 5 ml 2ME, and 50 μl LIF. |
EB medium | For 500 ml of EB medium, add the following components: 450 ml DMEM, 25 ml KSR, 5 ml sodium pyruvate, 5 ml Pen/Strep, 5 ml 2ME (100 mM), 5 ml L-glutamine, and 5 ml NEAA. |
DMEM/FBS medium | For 500 ml of feeder cells medium, add the following components: 440 ml DMEM, 50 ml FBS, 5 ml NEAA, and 5 ml Pen/Strep. |
N2 medium | For 100 ml of N2 medium, add 96 ml DMEM/F12, 1 ml N-2, 1 ml Pen/Strep, 1 ml L-glutamine, 1 ml NEAA, 4 μl FGF-2, and 100 μl laminin. |
B27/Neurobasal medium | For 500 ml of B27/Neurobasal medium, add the following components: 480 ml Neurobasal medium, 10 ml B27, 5 ml L-glutamine 100 X, and 5 ml Pen/Strep. |
Gelatin 0.1% | Dissolve 100 mg of gelatin in 90 ml warm (~ 60 ºC) distilled water. Sterilize by autoclaving and store at room temperature. |
25x FGF stock solution (100 μg ml-1) | Dissolve 25 mg recombinant mouse FGF in 250 ml of sterile PBS. Store at ~20 °C. |
5x Poly-D-Lysine | Dissolve 50 mg poly-D-lysine in 333.3 ml distilled water and rinse the package with the water. Filter the solution with a 0.22-μm bottle top filter. Aliquot 5 ml into 15-ml conical tubes and store at -20 °C. |
Hepes-buffered salt solution (HBSS) | Prepare stocks of the composition described in table 4 below. To obtain the HBSS basic, combine the solutions in the following order: 10.0 ml Solution #1, 1.0 ml Solution #2, 1.0 ml Solution #3, 1.0 ml Solution #5, 86.0 ml distilled H2O, 1.0 ml Solution #4. Prior to use, prepare the following solution: 9.8 ml HBSS basic, 0.1 ml Stock #5, 0.1 ml Stock #6. The final HBSS composition is 137 mM NaCl, 5.4 mM KCl, 0.6 mM Na2HPO4, 0.6 mM KH2PO4, 20 mM KH2PO4, 1.3 mM CaCl2, 1.0 mM MgSO4, 10 mM NaHCO3, 5.5 mM glucose. |
Setup of reagents needed for the methods described.
Stock | Preparation |
1 | In 90ml of distilled H2O, dissolve 8.0 g NaCl. 0.4 g KCl, and fill to 100 ml with distilled H2O. |
2 | In 90ml of distilled H2O, dissolve 0.41 g Na2HPO4 (anhydrous), 0.80 g KH2PO4, and fill to 100 ml with distilled H2O |
3 | Add 0.72 g CaCl2 to 50ml of distilled H2O |
4 | Add 1.23 g MgSO4x7H2O to 50ml of distilled H2O |
5 | Add 4.76 g Hepes to 10ml of distilled H2O |
6 | Add 0.9 g Glucose to 10ml of distilled H2O |
7 | Add 0.84 g NaHCO3 to 10ml of distilled H2O |
Preparation of the stock solutions needed to prepare HBSS. The combination of these stock solutions in described in table 3.
Equipment | Setup |
Gelatin-coated plate | Add 5 ml of 0.1% (wt/vol) gelatin solution into a plate so that it covers the entire bottom of the plate. Incubate the plate for 20 min at room temperature. |
Poly-D-lysine (PDL) cover slips | Autoclave cover slips. Place sterilized cover slips into the wells of a 24-well plate. Dilute the 5 x poly-D-lysine to 1 x with sterile distilled water plate except for the outermost wells. Add 0.5–1 ml of the 1 x poly-D-lysine solution to cover the surface of the cover slips. After an overnight incubation, remove the PDL and wash the plates with sterile deionized water and dry in a cell culture hood. |
Setup for the equipment needed for the methods described.
The generation of neurospheres and subsequent neuronal differentiation from ES cells is achieved through the stages described in the protocols below.
Place R1 ES cells (Nagy et al., 1993) on 10 cm gelatin-coated dishes with 12 ml of ES medium in a humidified chamber in a 5% CO2/air mixture at 37°C. Change the medium every day.
When the cells reach 80-90% confluence split them to two dishes as follows: wash the cells with 1x PBS twice, then add 2 ml of 1x trypsin-EDTA. Rock the dish to ensure that the solution covers all the cells. Incubate the dish for 5 min at 37°C. Pipette up and down to break up clumps and obtain a good dissociation of cells. Pipette 8 ml ES medium to inhibit further tryptic activity and transfer to 15-ml conical tubes. Spin at 1000 rpm for 3 min. Aspirate the medium from cells and resuspend the pellet in 6 ml of fresh ES medium.
Transfer 3 ml of cell suspension into a new 10 cm gelatin-coated dish containing 9 ml of fresh ES medium.
Aspirate the medium, add 2 ml of 1x trypsin-EDTA and incubate for 5 min at 37 °C. Dissociate the cells by thoroughly pipetting several times with a P1000 Pipetman. Monitor the trypsinization under an inverted microscope. If the separation of the cells is not complete, continue the treatment with trypsin for additional 2-3 min.
Transfer the cell suspension into a 15-ml sterile conical tube. Pellet the cells centrifugation for 5 min at 1200 rpm; then resuspend the cell pellet in 10 ml EB medium.
Count the cells with a cell counter and plate 5x105 ES cells per well in 6-well ultra low attachment plates in EB medium. Embryoid bodies should appear by overnight incubation.
Culture plates for 7 days. Change the medium every other day as follows: Collect the EBs in suspension from the plate and transfer into a 15 ml conical tube. Leave the conical tube for 15 minutes to allow the EBs to settle to the bottom of the tube. Aspirate the medium and add 12 ml of fresh EB medium. Transfer the EBs suspension into a new 6-well ultra low attachment plates.
Collect EBs and remove the medium as described in the previous step.
Count the number of EBs and prepare an EBs suspension in Feeder medium at a density of 40-50 EBs/ml.
Transfer 40-50 intact EBs to non-coated 10 cm cell culture dish containing 12 ml DMEM/FBS medium.
Culture until cells reach confluence, changing medium every 2-3 days.
Aspirate the medium, add 2 ml of trypsin-EDTA solution and incubate for 5 min at 37°C. Dissociate cells thoroughly by pipetting with a P1000 Pipetman. Pipette 8 ml Feeder medium and transfer the cell suspension to a 15-ml conical tube.
Centrifuge for 5 min at 1200 rpm. Aspirate the medium and resuspend the cells in 12 ml DMEM/FBS. Culture the cells until confluence.
Trypsinize as described in section 4.2 and culture cells for other two passages.
Transfer the neurospheres floating in the culture medium into 50-ml conical tubes and allow them to settle for 10 minutes. Collect the supernatant containing neurospheres. Trypsinize cells attached to the bottom of the plate as described in section 6.2 and culture to generate new neurospheres.
Resuspend the neurospheres in the appropriate volume of N2 medium and culture for differentiation in a 24-well plate (15-20 neurospheres/well) containing poly-D-lysine-coated cover slips. Replace a half volume medium every other day.
At day 7 exchange N2 medium to B27/Neurobasal medium and let the cells differentiate for 10 more days with a medium change every other day.
Selective immunocytochemistry can be performed according to standard procedures as described in the following sections.
Plate the neurospheres on PDL-treated cover slips, and culture according to experimental protocols with the same conditions described above.
Fix mouse neurospheres-derived neurons for 20 minutes at room temperature in a 4% paraformaldehyde solution (prepared from stock 16% Electron Microscopy Sciences, 15710-6 in 1X Premixed PBS Buffer at pH 7: Roche, 11666789001)
After three 10-minutes washes in PBS at room temperature, perform a blocking step for 1 hour in 10% of Normal Donkey Serum (NDS, Jackson ImmunoResearch, 017-000-121) supplemented with 0.01% TritonX-100 (Fluka, BP151-100).
Incubate the samples overnight with primary antibodies diluted in 10% of NDS in order to detect the expression of Tubulin III (R&D Systems, MAB1195) and of the ubiquitous NMDA receptor subunit NR1. (Rabbit Anti-NMDAR1, Abcam ab68144; 1/250).
After three 10-minutes washes in PBS at room temperature, use fluorescently labeled secondary antibodies for detection (Cy3 AffiniPure Donkey Anti-Rabbit [Jackson ImmunoResearch, 711-165-152] or Cy5 AffiniPure Donkey Anti- Mouse [Jackson ImmunoResearch, 715-175-151]).
After three 10-minutes washes in PBS at room temperature, treat the cells with bisBenzimide H 33342 trihydrochloride (Sigma B-2261) for 2 minutes, to stain nuclei with 405nm fluorescence.
After two 10-minutes washes, in PBS remove the cover slip from the plates and mount on glass slide with mounting solution (Gelvatol).
Images can be acquired using a laser scanning confocal microscope (FluoView 1000, Olympus). The microscope is equipped with spectral detector technology that provides precise wavelength separation of the emitted light.
For intracellular Ca2+ concentration ([Ca2+]i) measurements, incubate cells for 45 min at 37°C with 5 µM fura-2-AM (cell permeant; Invitrogen, F-1221) and 5 µg/ml bovine serum albumin (Sigma A-6003) in Hepes-buffered salt solution (standard HBSS).
Then, wash cells 3 times for 5 min each with standard HBSS and place the cover slips onto a perfusion chamber on a DM IRM inverted microscope (Leica) fitted with a Leica HC N PLAN BD 50X oil immersion objective. [Ca2+]i can be monitored in single cells using excitation light provided by a 75W xenon lamp-based monochromator (Ushio, Japan). Emitted light can be detected using a CCD camera (Orca; Hamamatsu, Shizouka, Japan). Alternatively, illuminate cells with 340 and 380 nm light for fura-2. Emitted fluorescence can be passed through a 500 nm longpass dichromatic mirror and a 535 ± 40 nm bandpass filter (Omega Optical).
Analyze acquired data using software such as Simple PCI (Compix, Inc., Cranberry, PA) as the 340/380 ratio. Measure fluorescence in 15–25 individual neurons for each cover slip. Subtract background fluorescence, determined from three or four cell-free regions of the cover slips, from all signals prior to calculating the ratios. Choose the excitation light exposure time and a neutral density filter to avoid saturation of the fluorescence signal.
Neural induction from pluripotent stem cells often yield heterogeneous cell populations that can hamper quantitative and comparative analyses. There is a need for improved differentiation and enrichment procedures that generate highly pure populations of neural stem cells (NSC), glia and neurons. The method described here allows an efficient generation of neurospheres from mES cells. On day-in vitro 16 after neural induction about 50% of the cells derived from neurospheres show a neural morphology, a significant expression of neuronal markers such as Tuj1 and the ubiquitous NMDA receptor subunit NR1 (figure 2), and a functional NMDAR-dependent calcium influx mediated by exogenous glutamate administration (figure 3).
Neurosphere differentiation. a) Immunostaining of a neurospheres with antibody specific to NMDA receptor (NR1 sub-unit, green). b-c) Staining of neurosphere-derived neurons with (b) NMDA receptor (NR1 sub-unit, red) and (c) β-tubulin III (green). d) staining of astrocytes differentiated from neurospheres with GFAP (red). Nuclei were counterstained with DAPI (blue). [Images taken by authors.]
Analysis of calcium influx in neurons differentiated from neurospheres indicated by glutamate. a) Administration of 10μM glutamate (glu) in neurons causes an increase in [Ca2+]i (340/380 = 0.32 ± 0.05a). b) Glu-induced increase in [Ca2+]i in R1 cells is blocked by treatment with MK801, an NMDAR-specific antagonist, suggesting that NMDARs are the predominant glutamate-sensitive receptors in neurospheres-derived neurons. [Data generated by authors.]
Furthermore, the rest of the cellular population show typical astroglial morphology. This chapter illustrates the utility mouse ES cells as a simple system able to develop populations of viable NSC and neurons. The method described here will enable downstream studies that require consistent and defined cell populations to study and characterize the physiopathology of the neuronal differentiation.
This procedure allows generation of a cell line derived from mouse ES cells which continually produces neuropheres in the absence of FGF-2 and EGF and coculturing. By generating neurospheres via this method, one can use them for a variety of purposes for further experiments with neurons.
The genus Triticum occupies a special position among cereals due to their different levels of ploidy and their adaptation to widely differing ecological and geographical regions of the world. The wealth of studies available on the origin of polyploid wheat provides the opportunity to comparatively analyze their genomes and those of putative donors, gaining insight into the reorganization of chromosomes in the process of evolution, domestication, and breeding.
Interest in chromosome organization and evolution in common wheat diploid progenitors and wild wheat is primarily because these species are a valuable source of new genes that were lost in the process of domestication.
The first allopolyploid of the Emmer wheat group is Triticum dicoccoides Koern. (2n = 28, genome BBAA), which arose as a result of hybridization of the diploid species T. urartu Thum. ex Gandil. and Aegilops speltoides Tausch (the most probable donor of the B-genome) [1]. Hexaploid wheat (T. aestivum L.) appeared about 7–10 thousand years ago as a result of a second round of hybridization between tetraploid wheat and the wild species Ae. tauschii Coss. (donor of the D-genome).
Wheat of Timopheevi group (tetraploids—T. araraticum Jakubz., T. timopheevii Zhuk., T. militinae Zhuk. et Migusch., and hexaploid T. zhukovskyi Menabde et Ericzjan) also came about through the hybridization of T. urartu and Ae. speltoides, but in another time scale. They have a genome designated as GGAtAt or GGAtAtDD, pointing at their partial homology with the genomes of wheat species in the Emmer group [2].
Diploid progenitors and species of the Timopheevi group are the source of new genes for the resistance to biotic and abiotic stresses. They are regularly involved in hybridization with common wheat, either directly or through synthetic polyploid forms. In this regard, knowledge on the structure and evolution of chromosomes of common wheat, its cultural and wild relatives is particularly relevant.
Genome sequencing methods actively displace cytogenetic analysis in current research. However, in working with hybrid material and introgressed lines of common wheat, we argue that the relevance of cytogenetic methods endures in the post-genomic era.
This review gives a brief description of the cytogenetic methods that remain relevant at this time and their use in the study of chromosomal rearrangement during wheat evolution and breeding.
Classical cytogenetic methods such as chromosome banding are currently relevant for wild species of plants as well as for polyploid species. Genome duplication within polyploids in some cases makes it difficult to assess the reorganization of chromosomes during evolution and hybridization.
The development of chromosome banding techniques allowed for the identification of the chromosomes not only in morphology but also in individual-specific patterns. Descriptions of the results of chromosome banding are based on the chromosomal region (band) and the intensity of staining, which differs from the neighboring regions. There are several methods of chromosome banding, namely, C-, N-, F-, Hy-, G-, Re-, and AgNOR-banding [3]. The most common method of staining used in the analysis of cereal genomes is C-differential staining (C-banding), first demonstrated by Pardu and Gal [4]. This method identifies the regions of constitutive heterochromatin after denaturation of the chromosomes and subsequent processing by Giemsa reagent. The karyotypes of many cereal crops, including polyploid wheat and their wild relatives were characterized based on C-banding [3, 5, 6, 7, 8]. The use of C-banding allows the study of chromosomal rearrangement during evolution and breeding. For example, Badaeva with co-authors [9] used C-banding to analyze 460 samples of polyploid wheat and 39 forms of triticale (x Triticosecale Wittmack) from 37 countries. Fifty-eight main types of chromosomal rearrangements were identified. The results obtained by the authors showed that chromosomes of the B genome are more often involved in chromosomal rearrangements than chromosomes of A and D genomes.
Thus, it is clear that studies like these are necessary for a better understanding of the laws of evolutionary processes in the plant world. C-banding is also currently used to characterize hybrid material and wheat cultivars, especially when other methods of analysis do not reveal chromosome polymorphism [10].
In addition to the differential staining, a specific pattern on chromosomes can be obtained by hybridization in situ. Hybridization in situ is a direct method of localizing DNA sequences on chromosomes. It is based on the ability of denatured DNA molecules to form duplexes with homologous DNA sequences of chromosomes on a slide. In situ hybridization was first performed on animal chromosomes [11] and later applied to plants chromosomes [12]. Over its 50-year history, this method has undergone significant changes aimed at increasing the sensitivity in the detection of labeled probes. This is primarily due to the development of simpler and more efficient DNA tagging systems and better visualization of the hybridization signal. Currently, fluorescence in situ hybridization (FISH) is used to study the distribution of individual DNA sequences on chromosomes. Genomic in situ hybridization (GISH) is commonly used to identify alien DNA or to study the genomic composition of wheat amphiploids and hybrids.
Most often, various repetitive DNA sequences are used as probes for FISH. This is not surprising since repeats are the largest and most rapidly evolving part of the genome. According to the latest sequencing data, repetitive DNA accounts for about 80% of the cereal genome [13, 14]. Groups of repeats with similar structure, formed by amplification from a common original sequence, are called families. Families of repeats differ in their structure, the size of the monomer (from one to several thousand nucleotide pairs), the number of copies, and the type of proliferation.
Transposable elements are the most common repeat elements and account for more than 90% of the entire fraction of cereal repetitive DNA. All families of transposable elements are united into two larger categories—classes, according the mechanism of transposition (retrotransposons and DNA transposable elements). The current detail classification of transposable elements was described by Wicker et al. [15]. Mostly, the transposable elements are dispersed on chromosomes.
There are families of repeats whose members are organized in tandem and assembled into one or more loci. Depending on the length of the repeating unit (monomer), tandem repeats are divided into microsatellites (monomer length 1–6 bp), minisatellites (from 10 to 60 bp), and satellites (average monomer length from 100 to 700 bp) [16].
In fact, each chromosome has an individual “pattern” of repeats, which can be used effectively for marking and identification of individual chromosomes, and the whole genome.
This group of repetitive DNA sequences is well studied in plants, especially cereals, and is widely used as markers in genomic research and in identifying chromosomes. According to their distribution on chromosomes, the repetitive sequences can be classified as centromeric, subtelomeric or intercalary. In combination, they generate a diagnostic “pattern” on the chromosome. Tandem repeats, such as microsatellites and satellites, and genes of ribosomal RNA are most frequently used for marking the chromosomes of wheat and its relatives.
Microsatellites are repeats with motifs from 1 to 6 bp. In plant genomes, they are also referred to as simple sequence repeats (SSRs) [17]. Microsatellites are used extensively as PCR markers for mapping chromosomes of many plant species and for gene labeling in applied research. Microsatellites are also used as cytogenetic markers. There are a few studies in which the distribution of various microsatellites on T. aestivum chromosomes has been examined in detail using FISH [18, 19]. For example, the dinucleotide probes (AT)10 and (GC)10 recorded no signal on chromosomes. This confirms the earlier hypothesis that the wheat genome does not contain extended clusters of these microsatellites [20, 21]. A dispersed distribution on chromosomes was established for probes (AC)8 and (GCC)5. The large microsatellite blocks detected by the probes (AGG)5, (CAC)5, (ACG)5, (AAT)5, and (CAG)5 were found mainly in the pericentromeric regions of the B genome. Strong intercalary signals were detected after hybridization with the probe (ACT)5 on a number of chromosomes of A and B genomes. Molnar and co-authors [22] investigated the distribution of microsatellites (ACG)n and (GAA)n on the chromosomes of Ae. biuncialis Vis (2n = 4x = 28, UbUbMbMb) and Ae. geniculata Roth. (2n = 4x = 28, UgUgMgMg) and on the chromosomes of their diploid progenitors: Ae. umbellulata Zhuk (UU) and Ae. comosa Sm. In Sibth.& Sm. (MM). They concluded that the break points for intergenomic translocations are often localized in regions saturated with microsatellite repeats. Thus, a number of studies have demonstrated that probes based on microsatellites can be useful for the identification of chromosomes and for a better understanding of the principles of chromatin organization in cereals. An important methodologically significant result was obtained by Cuadrado and Jouve [23]. They found that labeled oligonucleotides with a repeating mono-, di-, tri-, or tetra-nucleotide motif have the unexpected ability to detect the corresponding SSR loci even on nondenatured chromosomes, which in some cases can greatly facilitate and accelerate cytological analysis.
The (GAA)n microsatellite is the most widely used marker for the identification of chromosomes. The first works on its localization in the genome of cereals were carried out at the end of the twentieth century [24]. The GAA microsatellite was used as a marker for identification and sorting of polyploid wheat chromosomes [25, 26]. Phylogenetic studies using the GAA microsatellite were previously problematic due to the deficiency of hybridization signals on the A- and D-genomes and the presence of a number of major hybridization sites on the B-/G-genomes [19, 26].
In recent years, additional publications using GAA microsatellites for the identification of chromosomes of the A-genome of diploid wheat species and for phylogenetic analysis have appeared. Two works published in 2012 included data on the karyotypic analysis of single samples of T. monococcum L. and T. urartu using the oligonucleotide probe (GAA)9 or GAA fragments obtained by PCR from genomic DNA of wheat [27, 28]. We performed a comparative analysis of the A-genome chromosomes in a diploid and polyploid wheat species consisting of two evolutionary lineages, Timopheevi and Emmer, using the pTm30 probe cloned from the T. monococcum genome and containing (GAA)56 microsatellite sequences (Figure 1) [29].
FISH with probes (GAA)n (green) and pTa71 (red) on the chromosomes of diploid Triticum species: (a) T. monococcum (acc. K-18140), (b) T. boeoticum Boiss (acc. K-25811), (c) T. boeoticum (acc. PI427328), and (d) T. urartu (acc. IG45298).
Up to four pTm30 sites located on 1AS, 5AS, 2AS, and 4AL chromosomes have been revealed in the wild diploid species, although most accessions contained one to two (GAA)n sites (Figure 1). The (GAA)n loci on chromosomes 2AS, 4AL, and 5AL found in T. dicoccoides were retained in T. durum Desf. and T. aestivum. In species of the Timopheevi lineage, only one large (GAA)n site has been detected in the short arm of the 6At chromosome [29].
It was shown that changes in the distribution of (GAA)n sequences on the A-genome chromosomes of diploid and polyploid wheats are associated with chromosomal rearrangements/modifications involving mainly the NOR (nucleolus organizer region)-bearing chromosomes, throughout the evolution of wild and domesticated species.
Satellite DNA repeating units are longer than 100 bp. These sequences are characterized by a high copy level (104–106) and form clusters of repeats, the length of which is rather difficult to estimate by high-throughput sequencing of genomes due to the “ejection” of the main part of tandem repeats during this process. Earlier studies of cereal genomes using pulsed field gel electrophoresis made it possible to estimate the length of the tandem repeat regions in a cluster as 90–600 kb [30]. Satellite DNA can comprise up to 5% of the genome and is the cause of significant differences in the content of heterochromatin DNA blocks in closely related species. Due to the high copy numbers of satellite DNA in the chromosomal locus, they are well detected in the FISH assay.
We can distinguish the following families of satellite DNA, whose units (in the form of cloned DNA sequences or PCR fragments) have been successfully used for the analysis of the genome of wheat and Triticeae species, including the study of the reorganization of genomes during evolution:
A family of repeats pAs1/Afa/pHcKB6/dpTa1 [31, 32], localized predominantly in the subtelomeric and intercalary chromosome regions of Ae. tauschii, D-genome of T. aestivum, species of the genus Hordeum, Elymus, and several other species.
The family of repeats 120 bp/pSc119.2 [33], widely distributed in subtelomeric and intercalary regions of chromosomes in many species of the tribe Triticeae (Figure 2) and in the closely related tribe Avenae [34]. This family of repeats was first isolated from Secale cereale L. and described as one of the families of telomeric rye heterochromatin [35].
The family of repeats 350 bp/pSc200/pSc74 and pSc250, which are the main tandem repeats of telomeric heterochromatin in rye Secale cereale [36]. During evolution, these sequences were amplified in the genome of individual species of Secale, as well as in certain species of the genera Agropyron and Dasypyrum of the Triticeae tribe.
pAesKB52/pGC1R-1/Spelt52 are tandem repeats of subtelomeric regions of chromosomes Ae. speltoides, Ae. longissima Schweinf, & Musch L., and Ae. sharonensis Eig (Figure 2) [37].
Spelt1 is a genome-specific sequence associated with telomeric heterochromatin of Ae. speltoides (Figure 2). Sequences of this family have not yet been detected by hybridization methods in the genomes of other Triticeae species, with the exception of T. monococcum (weak hybridization signal) and polyploid species formed with the participation of Ae. speltoides [37].
FISH to mitotic metaphase chromosomes. (a and c) Ae. speltoides, (b) Ae. longissima, and (d) hybrid line (T. aestivum × Ae. speltoides). Probe combinations used were (a) pSc119.2 (green) and Spelt52 (red), (b) pSc119.2 (red) and Spelt52 (green), and (c and d) pSc119.2 (red) and Spelt1 (green).
The probes, pSc119.2 and pAs1, are most often used for intraspecific identification of Triticeae tribe chromosomes by the FISH method. Thus, simultaneous hybridization of two DNA probes (pSc119.2 and pAs1) makes it possible to identify 17 (out of 21) chromosomes of the genome of common wheat [34, 38].
The combination of pSc119.2 and Spelt52 probes is effective for the study of all Aegilops species of the Sitopsis section (putative donors of the B/G genomes of polyploid wheats) (Figure 2). The probe combination pSc119.2 and Spelt1 is effective for only Ae. speltoides chromosome identification (Figure 2) [37]. The S-genomes of Aegilops species within the Sitopsis section are very similar to the common wheat B-genome. Accordingly, GISH with the DNA of these species is difficult on hybrid wheat lines. Therefore, we used FISH with the Spelt1 and Spelt52 probes to identify the Aegilops genetic material in the investigated lines (Figure 2). Simultaneous hybridization with probes pSc119.2 and pAs1 allows identification of wheat chromosomes.
The different level of homology within various families of tandem repeats depends on the rate of homogenization of repeats within the cluster, within each genome and species. The Spelt1-family is highly conserved, according to the sequencing of 10 Spelt1 sequences isolated from different accessions of Ae. speltoides, which shows a high level of homology (98%) [37]. It should be noted that more often a high level of interspecific polymorphism of satellite DNA families was demonstrated. This suggests that it may be possible to obtain efficient probes for the identification of chromosomes of the studied species by cloning DNA sequences of the repetitive families from these species.
Reports of the discovery of new families of highly repetitive DNA sequences are still emerging despite the existence of a large number of cereal tandem repeats already described. A previously unknown class of repeating DNA sequences named “Fat” was identified in the genome of common wheat [39]. Fat repeats are organized in clusters but with a dispersed distribution throughout the genome. The Fat-element content varies considerably across the genomes of different cereal species. The highest intensity of hybridization was found in the D-genome of wheat and Aegilops and in the S genome of Agropyron. This sequence was not found in oats or domesticated barley Hordeum vulgare L, but was present in minimal amounts in other species of the genus Hordeum. Based on this, it was concluded that the Fat-element first appeared in the evolution of cereals after the divergence of oats, during the separation of domestic barley from related grasses. The most intensive hybridization of Fat-repeats in the form of a large cluster of signals characterizes the chromosomes of the 4th homoeological group of wheat and Aegilops. This sequence is found only on the chromosomes of the D-genome of wheat and Aegilops, enabling it to be used as a FISH marker for identifying chromosomes and studying chromosome reorganization during evolution.
To discover new probes, 2000 plasmid wheat clones were examined by Komuro et al. [40]. Among them, 47 clones produced strong discrete signals on wheat chromosomes. Especially, valuable is combining pTa-535, pTa-713, and pTa-86 (pSc119 homolog) sequences, which allows to completely identify all 21 wheat chromosome pairs.
In addition, it seems promising to use oligonucleotides synthesized for various sites in the above-mentioned families of repeats identified in high-throughput sequencing, including sequencing data on individual chromosomes of wheat made for the identification of chromosomes. These probes have been shown to provide an easier, faster and more cost-effective method for the FISH analysis of wheat and hybrids [41, 42].
An important and well-studied family of tandem repeats is the family of ribosomal RNA genes (rRNA). A detailed analysis of the monomers and cluster organization was carried out for these genes. Their localization on chromosomes in various species of cereals and possible mechanisms of evolutionary variability, including the processes of divergence and homogenization, were described by Hillis et al. [43]. There are two classes of rRNA-genes in the cereal genome: genes encoding 5S rRNA and 45S rRNA. 45S and 5S rDNA are located independently of each other, even in cases when they are both localized on one arm of a chromosome.
The 45S rDNA of cereals contains a coding region and a nontranscribed spacer sequence. The gene region includes three DNA sequences encoding 18S, 5.8S, and 26S rRNA, which are separated by internally transcribed spacer sequences. Polymorphism exists in the number of 45S rDNA loci in the Triticeae species genomes. The “major” loci of these genes are located on the short arms of homoeologous chromosome groups 1, 5, and 6. Nucleolus-forming regions are found on chromosomes 1A, 1B, 6B, and 5D of T. aestivum [44]. In addition, minor loci of 45S rDNA are also present, in which active RNA synthesis is not observed.
The genes encoding the 5S rRNA have the smallest repeating unit length among the ribosomal genes (320–500 bp). The repeating unit of 5S rDNA contains a 120 bp conserved coding region and a variable nontranscribed spacer sequence. There are from 1000 to 4000 copies of 5S rRNA genes per haploid genome in cereals. Two subfamilies of 5S rDNA are distinguished in the Triticeae genomes, depending on the length of the spacer: 5SDna1 (200–345 bp) and 5SDna2 (350–380 bp). Hybridization of 5S-repeats on chromosomes of various Triticeae species showed that in most species, they are located in homoeological groups 1 and 5. An analysis of the chromosome distribution of 5SDna1 and 5SDna2 subfamilies showed that the short units of 5S rDNA have preferential localization on the chromosomes of homoeologous group 1, while the long units are located on group 5. It was shown that 5S rRNA genes with a monomer length of 290 bp are located on chromosomes 1B and 1D of common wheat, and genes with a monomer length of 410 bp are located on chromosomes 5B and 5D [45]. Further work on the isolation and sequencing of individual monomers led to division of the 5S rRNA genes into a larger number of subfamilies [46].
Analysis of chromosome 5B sequencing data, as well as individual BAC-clones containing 5S rDNA, showed that long and short types of subunits can be located on one chromosome, but they form separate clusters interrupted by the insertion of mobile elements [47].
The presence of conservative (coding) and polymorphic (noncoding) sequences in rDNA promoted their widespread use as molecular markers in phylogenetics. 5S and 45S rDNA are also widely used as cytogenetic markers for FISH due to their large copy number and localization in certain regions of chromosomes. A number of phylogenetic studies using individually cloned copies of 45S and 5S rDNA have been carried out for wheat and its relatives [48, 49]. An interesting fact is that among Triticum and Aegilops, two species (T. timopheevii and Ae. speltoides) lost the 5S rDNA locus on the chromosome of homoeologous group 1 (1G and 1S, correspondently) during evolution [50].
Another class of repetitive DNA, widely represented in the genome of plants, is transposable genetic elements (TEs), which are divided into two classes: class I elements (retrotransposons) and class II elements (DNA transposable elements).
At present, it seems likely that the diverse TEs, which have a mainly dispersed chromosomal localization, are the major contributors to the observed interspecies differentiation of chromosomes revealed by genomic in situ hybridization (GISH). GISH, a method based on the hybridization of labeled genomic DNA of one species to metaphase chromosomes of another species or hybrid, is widely used to assess the degree of genome homology. GISH serves as a unique approach to studying the formation of genomes of polyploid species and revealing the nature of their relationship, the analysis of introgression of alien genetic material, and the localization of break points in intergenomic translocations in remote hybrids [51, 52].
The development of BAC (bacterial artificial chromosome) libraries containing clones with very large inserts (>100 kb) of genomic DNA has opened up new possibilities for studying the reorganization of genomes by BAC in situ hybridization (BAC-FISH). The localization of BAC clones on chromosomes is mainly connected with families of TEs in their composition. Thus, carrying out BAC-FISH on wheat chromosomes showed a different BAC localization in the genome depending on which family of TEs or other repeats were present in them [53, 54].
It is interesting to note the differential amplification of individual members of retrotransposon families belonging to the subclass Ty3-gypsy-retrotransposons in the genomes of diploid species, which retains genomic specificity in allopolyploid wheats (Figure 3) [55, 56].
Thus, FISH analysis of a BAC clone (BAC_2383A24) demonstrated its predominant localization to chromosomes of the B-genome of allopolyploid wheats and its putative diploid progenitor Ae. speltoides (Figure 3).
FISH to mitotic metaphase chromosomes of (a) Ae. speltoides and T. urartu, (b) T. urartu and Ae. tauschii, and (c) T. aestivum. With the probe combinations: (a) BAC clone 2383A24 (green), (b) BAC clone 112D20 (red), (c) BAC clone 2383A24 (green), and BAC clone 112D20 (red).
Analysis of the complete BAC_2383A24 nucleotide sequence revealed that three elements of the gypsy LTR retrotransposon family Fatima make up 47.2% of all the LTR retrotransposons in this BAC. Phylogenetic analysis, as well as FISH, showed that these Fatima elements are predominantly from the B genome of common wheat and its putative progenitor Ae. speltoides (Figure 3). Similar approaches, including hybridization with BAC clone 112D20, demonstrated that the Lila family of Ty3-gypsy-retrotransposons is predominantly from the D-genome and its progenitor Ae. tauschii. Multiple FISH with both clones allows the identification of all three subgenomes of hexaploid wheat (Figure 3).
Dating of the LTR retrotransposon insertion showed that TE proliferation mainly occurred in this diploid species before it entered into allopolyploidy [55, 57].
Genetic erosion, caused by modern agricultural breeding practices, has led to the observed decrease in genetic variation in crops, including common wheat T. aestivum. Wheat relatives—wild and cultivated cereals—are used as sources of effective genes for resistance to biotic and abiotic stresses and to increase genetic diversity.
Introgression of genes from related species to wheat depends on the level of divergence between the species involved in the cross. Species belonging to the primary gene pool have homoeologous genomes. This group includes wild and cultivated forms of T. turgidum and species of donors A and D of the genomes of common wheat: T. urartu, T. monococcum, and T. boeoticum, as well as Ae. tauschii. The transfer of genes from these species can be carried out by crossing, homoeologous recombination, backcrossing and selection, as well as through the development of synthetic amphiploids.
The secondary gene pool includes polyploid species of wheat and Aegilops which have at least one homoeologous genome with T. aestivum. The transfer of genes from these species to common wheat by means of homoeologous recombination is also possible if recombination has taken place between the target homoeologous chromosomes. This group also includes hexaploid species with GGAtAtDD genome: T. kiharae, Dorof. et Migusch., T. miguschovae; tetraploid species with GGAtAt genome: T. timopheevii, T. militinae, and T. araraticum; and diploid species of Aegilops from the Sitopsis section, which are close to the B genome of T. aestivum. Cytogenetic analysis of hybrids from crosses of common wheat with T. timopheevii showed homoeologous introgression of G genome fragments to practically all chromosomes of both the B genome and the D genome of common wheat [58]. It should also be noted that the extent of introgressive regions varies among wheat lines [59]. Genetic material from Ae. speltoides (SS genome), the putative progenitor of the B and G genomes of polyploid wheat, was successfully transferred to all three genomes of common wheat, but especially, as expected, in the chromosome of the B-genome [60, 61].
Species that do not carry the genomes A, B, and D, and those related to the tertiary gene pool, are considered more distant relatives of wheat. The transfer of genes from these species is difficult since it cannot be accomplished by recombination and therefore requires the use of other strategies. Currently, there are standard methods that facilitate the transfer of genes from species that do not have related genomes with common wheat. Some are based on the methods of chromosome engineering, and others manipulate the genetic control of meiotic recombination or employ genetic engineering. The transfer of genetic material in this case occurs both in the partly homoeologous group of chromosomes and into other groups [62].
The strategy used in cytological analysis of hybrids depends first on the nature of the relationships between the species involved in crossing. In instances where the donor species belongs to the tertiary gene pool with respect to T. aestivum, GISH is first used, which allows the estimation of the size and localization of the alien translocation. GISH can be used successfully to identify translocations of rye, wheatgrass, and Aegilops species (with the exception of the Sitopsis group) in the wheat genome (Figure 4).
GISH with Th. intermedium DNA (green) in combination with pAs1 probes (red) of common wheat cultivar Tulaikovskaya 5 (a) and GISH with S. cereale DNA (green) and with Ae. umbellulata DNA (red) of the triticale line with introgression from Ae. umbellulata (b).
However, GISH does not answer the questions: which wheat chromosome is replaced by an alien chromosome or which alien chromosome took part in the translocation. In addition, if the genomes of the crossed species are evolutionarily close, that is, if donor species refer to primary and secondary gene pools, then GISH will also be difficult. A similar problem occurs, for example, in the analysis of hybrids from the crossing of hexaploid wheat with Ae. speltoides (B-genome putative donor) or with the species Ae. tauschii which is a donor of the D genome. In this case, species-specific markers are used to identify chromosomes in hybrids or introgressive wheat lines, such as, for example, Spelt1 for the Ae. speltoides genome (Figure 2).
It should be noted that when identifying the alien introgressions in lines/varieties developed by remote hybridization of cereals, best results are usually obtained by combining different methodological approaches and using different chromosomal markers.
The wheat allopolyploids have long attracted the attention of researchers, both from the perspective of studying the processes of genome reorganization during amphiploidization and to develop new wheat lines for breeding.
To accomplish these fundamental and applied tasks, various approaches are used. In recent years, SNP markers and various technologies for their identification have been actively involved, as well as reference genome data for wheat and related species. This allows us to obtain more detailed information about the organization and evolution of the wheat genome and the structure of gene families present in reference genomes. Despite continued progress in deciphering the complex wheat genome, a complete understanding of the reorganization of the wheat genome during evolution can only be obtained by combining molecular methods of analysis with cytogenetic ones. The latter makes it possible to identify rearrangements of homoeological chromosomes in the process of evolution and breeding.
The first translocations in wheat varieties were detected by cytogenetic methods. Later, the molecular markers developed for these translocations allowed the use of marker-assisted breeding for selection of the desired genotypes.
One of the most successful used in selection is the translocation of the short 1R chromosomal arm to 1A and 1B of the wheat chromosome during breeding. At present, more than 300 soft wheat varieties carry the T1RS.1BL translocation [
It should be emphasized once again that, despite extensive development of molecular markers for genome analysis, including high-throughput genotyping, it is impossible to characterize the modern diversity within the genus Triticum without involving cytogenetic methods.
We are grateful to Carly Schramm for critical review of the manuscript. This work was supported by the Russian Foundation for Basic Research, project no. 17-04-00507 and the IC&G Budgetary project no. 0324-2018-0018.
The authors declare that they have no competing interests.
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',metaTitle:"Publication Agreement - Chapters",metaDescription:"IN TECH aims to guarantee that original material is published while at the same time giving significant freedom to our authors. For that matter, we uphold a flexible copyright policy meaning that there is no transfer of copyright to the publisher and authors retain exclusive copyright to their work.\n\nWhen submitting a manuscript the Corresponding Author is required to accept the terms and conditions set forth in our Publication Agreement as follows:",metaKeywords:null,canonicalURL:"/page/publication-agreement-chapters",contentRaw:'[{"type":"htmlEditorComponent","content":"The Corresponding Author (acting on behalf of all Authors) and INTECHOPEN LIMITED, incorporated and registered in England and Wales with company number 11086078 and a registered office at 5 Princes Gate Court, London, United Kingdom, SW7 2QJ conclude the following Agreement regarding the publication of a Book Chapter:
\\n\\n1. DEFINITIONS
\\n\\nCorresponding Author: The Author of the Chapter who serves as a Signatory to this Agreement. The Corresponding Author acts on behalf of any other Co-Author.
\\n\\nCo-Author: All other Authors of the Chapter besides the Corresponding Author.
\\n\\nIntechOpen: IntechOpen Ltd., the Publisher of the Book.
\\n\\nBook: The publication as a collection of chapters compiled by IntechOpen including the Chapter. Chapter: The original literary work created by Corresponding Author and any Co-Author that is the subject of this Agreement.
\\n\\n2. CORRESPONDING AUTHOR'S GRANT OF RIGHTS
\\n\\n2.1 Subject to the following Article, the Corresponding Author grants and shall ensure that each Co-Author grants, to IntechOpen, during the full term of copyright and any extensions or renewals of that term the following:
\\n\\nThe aforementioned licenses shall survive the expiry or termination of this Agreement for any reason.
\\n\\n2.2 The Corresponding Author (on their own behalf and on behalf of any Co-Author) reserves the following rights to the Chapter but agrees not to exercise them in such a way as to adversely affect IntechOpen's ability to utilize the full benefit of this Publication Agreement: (i) reprographic rights worldwide, other than those which subsist in the typographical arrangement of the Chapter as published by IntechOpen; and (ii) public lending rights arising under the Public Lending Right Act 1979, as amended from time to time, and any similar rights arising in any part of the world.
\\n\\nThe Corresponding Author confirms that they (and any Co-Author) are and will remain a member of any applicable licensing and collecting society and any successor to that body responsible for administering royalties for the reprographic reproduction of copyright works.
\\n\\nSubject to the license granted above, copyright in the Chapter and all versions of it created during IntechOpen's editing process (including the published version) is retained by the Corresponding Author and any Co-Author.
\\n\\nSubject to the license granted above, the Corresponding Author and any Co-Author retains patent, trademark and other intellectual property rights to the Chapter.
\\n\\n2.3 All rights granted to IntechOpen in this Article are assignable, sublicensable or otherwise transferrable to third parties without the Corresponding Author's or any Co-Author’s specific approval.
\\n\\n2.4 The Corresponding Author (on their own behalf and on behalf of each Co-Author) will not assert any rights under the Copyright, Designs and Patents Act 1988 to object to derogatory treatment of the Chapter as a consequence of IntechOpen's changes to the Chapter arising from translation of it, corrections and edits for house style, removal of problematic material and other reasonable edits.
\\n\\n3. CORRESPONDING AUTHOR'S DUTIES
\\n\\n3.1 When distributing or re-publishing the Chapter, the Corresponding Author agrees to credit the Book in which the Chapter has been published as the source of first publication, as well as IntechOpen. The Corresponding Author warrants that each Co-Author will also credit the Book in which the Chapter has been published as the source of first publication, as well as IntechOpen, when they are distributing or re-publishing the Chapter.
\\n\\n3.2 When submitting the Chapter, the Corresponding Author agrees to:
\\n\\nThe Corresponding Author will be held responsible for the payment of the Open Access Publishing Fees.
\\n\\nAll payments shall be due 30 days from the date of the issued invoice. The Corresponding Author or the payer on the Corresponding Author's and Co-Authors' behalf will bear all banking and similar charges incurred.
\\n\\n3.3 The Corresponding Author shall obtain in writing all consents necessary for the reproduction of any material in which a third-party right exists, including quotations, photographs and illustrations, in all editions of the Chapter worldwide for the full term of the above licenses, and shall provide to IntechOpen upon request the original copies of such consents for inspection (at IntechOpen's option) or photocopies of such consents.
\\n\\nThe Corresponding Author shall obtain written informed consent for publication from people who might recognize themselves or be identified by others (e.g. from case reports or photographs).
\\n\\n3.4 The Corresponding Author and any Co-Author shall respect confidentiality rights during and after the termination of this Agreement. The information contained in all correspondence and documents as part of the publishing activity between IntechOpen and the Corresponding Author and any Co-Author are confidential and are intended only for the recipient. The contents may not be disclosed publicly and are not intended for unauthorized use or distribution. Any use, disclosure, copying, or distribution is prohibited and may be unlawful.
\\n\\n4. CORRESPONDING AUTHOR'S WARRANTY
\\n\\n4.1 The Corresponding Author represents and warrants that the Chapter does not and will not breach any applicable law or the rights of any third party and, specifically, that the Chapter contains no matter that is defamatory or that infringes any literary or proprietary rights, intellectual property rights, or any rights of privacy. The Corresponding Author warrants and represents that: (i) the Chapter is the original work of themselves and any Co-Author and is not copied wholly or substantially from any other work or material or any other source; (ii) the Chapter has not been formally published in any other peer-reviewed journal or in a book or edited collection, and is not under consideration for any such publication; (iii) they themselves and any Co-Author are qualifying persons under section 154 of the Copyright, Designs and Patents Act 1988; (iv) they themselves and any Co-Author have not assigned and will not during the term of this Publication Agreement purport to assign any of the rights granted to IntechOpen under this Publication Agreement; and (v) the rights granted by this Publication Agreement are free from any security interest, option, mortgage, charge or lien.
\\n\\nThe Corresponding Author also warrants and represents that: (i) they have the full power to enter into this Publication Agreement on their own behalf and on behalf of each Co-Author; and (ii) they have the necessary rights and/or title in and to the Chapter to grant IntechOpen, on behalf of themselves and any Co-Author, the rights and licenses expressed to be granted in this Publication Agreement. If the Chapter was prepared jointly by the Corresponding Author and any Co-Author, the Corresponding Author warrants and represents that: (i) each Co-Author agrees to the submission, license and publication of the Chapter on the terms of this Publication Agreement; and (ii) they have the authority to enter into this Publication Agreement on behalf of and bind each Co-Author. The Corresponding Author shall: (i) ensure each Co-Author complies with all relevant provisions of this Publication Agreement, including those relating to confidentiality, performance and standards, as if a party to this Publication Agreement; and (ii) remain primarily liable for all acts and/or omissions of each such Co-Author.
\\n\\nThe Corresponding Author agrees to indemnify and hold IntechOpen harmless against all liabilities, costs, expenses, damages and losses and all reasonable legal costs and expenses suffered or incurred by IntechOpen arising out of or in connection with any breach of the aforementioned representations and warranties. This indemnity shall not cover IntechOpen to the extent that a claim under it results from IntechOpen's negligence or willful misconduct.
\\n\\n4.2 Nothing in this Publication Agreement shall have the effect of excluding or limiting any liability for death or personal injury caused by negligence or any other liability that cannot be excluded or limited by applicable law.
\\n\\n5. TERMINATION
\\n\\n5.1 IntechOpen has a right to terminate this Publication Agreement for quality, program, technical or other reasons with immediate effect, including without limitation (i) if the Corresponding Author or any Co-Author commits a material breach of this Publication Agreement; (ii) if the Corresponding Author or any Co-Author (being an individual) is the subject of a bankruptcy petition, application or order; or (iii) if the Corresponding Author or any Co-Author (being a company) commences negotiations with all or any class of its creditors with a view to rescheduling any of its debts, or makes a proposal for or enters into any compromise or arrangement with any of its creditors.
\\n\\nIn case of termination, IntechOpen will notify the Corresponding Author, in writing, of the decision.
\\n\\n6. INTECHOPEN’S DUTIES AND RIGHTS
\\n\\n6.1 Unless prevented from doing so by events outside its reasonable control, IntechOpen, in its discretion, agrees to publish the Chapter attributing it to the Corresponding Author and any Co-Author.
\\n\\n6.2 IntechOpen has the right to use the Corresponding Author’s and any Co-Author’s names and likeness in connection with scientific dissemination, retrieval, archiving, web hosting and promotion and marketing of the Chapter and has the right to contact the Corresponding Author and any Co-Author until the Chapter is publicly available on any platform owned and/or operated by IntechOpen.
\\n\\n6.3 IntechOpen is granted the authority to enforce the rights from this Publication Agreement, on behalf of the Corresponding Author and any Co-Author, against third parties (for example in cases of plagiarism or copyright infringements). In respect of any such infringement or suspected infringement of the copyright in the Chapter, IntechOpen shall have absolute discretion in addressing any such infringement which is likely to affect IntechOpen's rights under this Publication Agreement, including issuing and conducting proceedings against the suspected infringer.
\\n\\n7. MISCELLANEOUS
\\n\\n7.1 Further Assurance: The Corresponding Author shall and will ensure that any relevant third party (including any Co-Author) shall, execute and deliver whatever further documents or deeds and perform such acts as IntechOpen reasonably requires from time to time for the purpose of giving IntechOpen the full benefit of the provisions of this Publication Agreement.
\\n\\n7.2 Third Party Rights: A person who is not a party to this Publication Agreement may not enforce any of its provisions under the Contracts (Rights of Third Parties) Act 1999.
\\n\\n7.3 Entire Agreement: This Publication Agreement constitutes the entire agreement between the parties in relation to its subject matter. It replaces and extinguishes all prior agreements, draft agreements, arrangements, collateral warranties, collateral contracts, statements, assurances, representations and undertakings of any nature made by or on behalf of the parties, whether oral or written, in relation to that subject matter. Each party acknowledges that in entering into this Publication Agreement it has not relied upon any oral or written statements, collateral or other warranties, assurances, representations or undertakings which were made by or on behalf of the other party in relation to the subject matter of this Publication Agreement at any time before its signature (together "Pre-Contractual Statements"), other than those which are set out in this Publication Agreement. Each party hereby waives all rights and remedies which might otherwise be available to it in relation to such Pre-Contractual Statements. Nothing in this clause shall exclude or restrict the liability of either party arising out of its pre-contract fraudulent misrepresentation or fraudulent concealment.
\\n\\n7.4 Waiver: No failure or delay by a party to exercise any right or remedy provided under this Publication Agreement or by law shall constitute a waiver of that or any other right or remedy, nor shall it preclude or restrict the further exercise of that or any other right or remedy. No single or partial exercise of such right or remedy shall preclude or restrict the further exercise of that or any other right or remedy.
\\n\\n7.5 Variation: No variation of this Publication Agreement shall be effective unless it is in writing and signed by the parties (or their duly authorized representatives).
\\n\\n7.6 Severance: If any provision or part-provision of this Publication Agreement is or becomes invalid, illegal or unenforceable, it shall be deemed modified to the minimum extent necessary to make it valid, legal and enforceable. If such modification is not possible, the relevant provision or part-provision shall be deemed deleted.
\\n\\nAny modification to or deletion of a provision or part-provision under this clause shall not affect the validity and enforceability of the rest of this Publication Agreement.
\\n\\n7.7 No partnership: Nothing in this Publication Agreement is intended to, or shall be deemed to, establish or create any partnership or joint venture or the relationship of principal and agent or employer and employee between IntechOpen and the Corresponding Author or any Co-Author, nor authorize any party to make or enter into any commitments for or on behalf of any other party.
\\n\\n7.8 Governing law: This Publication Agreement and any dispute or claim (including non-contractual disputes or claims) arising out of or in connection with it or its subject matter or formation shall be governed by and construed in accordance with the law of England and Wales. The parties submit to the exclusive jurisdiction of the English courts to settle any dispute or claim arising out of or in connection with this Publication Agreement (including any non-contractual disputes or claims).
\\n\\nLast updated: 2020-11-27
\\n\\n\\n\\n
\\n"}]'},components:[{type:"htmlEditorComponent",content:"
The Corresponding Author (acting on behalf of all Authors) and INTECHOPEN LIMITED, incorporated and registered in England and Wales with company number 11086078 and a registered office at 5 Princes Gate Court, London, United Kingdom, SW7 2QJ conclude the following Agreement regarding the publication of a Book Chapter:
\n\n1. DEFINITIONS
\n\nCorresponding Author: The Author of the Chapter who serves as a Signatory to this Agreement. The Corresponding Author acts on behalf of any other Co-Author.
\n\nCo-Author: All other Authors of the Chapter besides the Corresponding Author.
\n\nIntechOpen: IntechOpen Ltd., the Publisher of the Book.
\n\nBook: The publication as a collection of chapters compiled by IntechOpen including the Chapter. Chapter: The original literary work created by Corresponding Author and any Co-Author that is the subject of this Agreement.
\n\n2. CORRESPONDING AUTHOR'S GRANT OF RIGHTS
\n\n2.1 Subject to the following Article, the Corresponding Author grants and shall ensure that each Co-Author grants, to IntechOpen, during the full term of copyright and any extensions or renewals of that term the following:
\n\nThe aforementioned licenses shall survive the expiry or termination of this Agreement for any reason.
\n\n2.2 The Corresponding Author (on their own behalf and on behalf of any Co-Author) reserves the following rights to the Chapter but agrees not to exercise them in such a way as to adversely affect IntechOpen's ability to utilize the full benefit of this Publication Agreement: (i) reprographic rights worldwide, other than those which subsist in the typographical arrangement of the Chapter as published by IntechOpen; and (ii) public lending rights arising under the Public Lending Right Act 1979, as amended from time to time, and any similar rights arising in any part of the world.
\n\nThe Corresponding Author confirms that they (and any Co-Author) are and will remain a member of any applicable licensing and collecting society and any successor to that body responsible for administering royalties for the reprographic reproduction of copyright works.
\n\nSubject to the license granted above, copyright in the Chapter and all versions of it created during IntechOpen's editing process (including the published version) is retained by the Corresponding Author and any Co-Author.
\n\nSubject to the license granted above, the Corresponding Author and any Co-Author retains patent, trademark and other intellectual property rights to the Chapter.
\n\n2.3 All rights granted to IntechOpen in this Article are assignable, sublicensable or otherwise transferrable to third parties without the Corresponding Author's or any Co-Author’s specific approval.
\n\n2.4 The Corresponding Author (on their own behalf and on behalf of each Co-Author) will not assert any rights under the Copyright, Designs and Patents Act 1988 to object to derogatory treatment of the Chapter as a consequence of IntechOpen's changes to the Chapter arising from translation of it, corrections and edits for house style, removal of problematic material and other reasonable edits.
\n\n3. CORRESPONDING AUTHOR'S DUTIES
\n\n3.1 When distributing or re-publishing the Chapter, the Corresponding Author agrees to credit the Book in which the Chapter has been published as the source of first publication, as well as IntechOpen. The Corresponding Author warrants that each Co-Author will also credit the Book in which the Chapter has been published as the source of first publication, as well as IntechOpen, when they are distributing or re-publishing the Chapter.
\n\n3.2 When submitting the Chapter, the Corresponding Author agrees to:
\n\nThe Corresponding Author will be held responsible for the payment of the Open Access Publishing Fees.
\n\nAll payments shall be due 30 days from the date of the issued invoice. The Corresponding Author or the payer on the Corresponding Author's and Co-Authors' behalf will bear all banking and similar charges incurred.
\n\n3.3 The Corresponding Author shall obtain in writing all consents necessary for the reproduction of any material in which a third-party right exists, including quotations, photographs and illustrations, in all editions of the Chapter worldwide for the full term of the above licenses, and shall provide to IntechOpen upon request the original copies of such consents for inspection (at IntechOpen's option) or photocopies of such consents.
\n\nThe Corresponding Author shall obtain written informed consent for publication from people who might recognize themselves or be identified by others (e.g. from case reports or photographs).
\n\n3.4 The Corresponding Author and any Co-Author shall respect confidentiality rights during and after the termination of this Agreement. The information contained in all correspondence and documents as part of the publishing activity between IntechOpen and the Corresponding Author and any Co-Author are confidential and are intended only for the recipient. The contents may not be disclosed publicly and are not intended for unauthorized use or distribution. Any use, disclosure, copying, or distribution is prohibited and may be unlawful.
\n\n4. CORRESPONDING AUTHOR'S WARRANTY
\n\n4.1 The Corresponding Author represents and warrants that the Chapter does not and will not breach any applicable law or the rights of any third party and, specifically, that the Chapter contains no matter that is defamatory or that infringes any literary or proprietary rights, intellectual property rights, or any rights of privacy. The Corresponding Author warrants and represents that: (i) the Chapter is the original work of themselves and any Co-Author and is not copied wholly or substantially from any other work or material or any other source; (ii) the Chapter has not been formally published in any other peer-reviewed journal or in a book or edited collection, and is not under consideration for any such publication; (iii) they themselves and any Co-Author are qualifying persons under section 154 of the Copyright, Designs and Patents Act 1988; (iv) they themselves and any Co-Author have not assigned and will not during the term of this Publication Agreement purport to assign any of the rights granted to IntechOpen under this Publication Agreement; and (v) the rights granted by this Publication Agreement are free from any security interest, option, mortgage, charge or lien.
\n\nThe Corresponding Author also warrants and represents that: (i) they have the full power to enter into this Publication Agreement on their own behalf and on behalf of each Co-Author; and (ii) they have the necessary rights and/or title in and to the Chapter to grant IntechOpen, on behalf of themselves and any Co-Author, the rights and licenses expressed to be granted in this Publication Agreement. If the Chapter was prepared jointly by the Corresponding Author and any Co-Author, the Corresponding Author warrants and represents that: (i) each Co-Author agrees to the submission, license and publication of the Chapter on the terms of this Publication Agreement; and (ii) they have the authority to enter into this Publication Agreement on behalf of and bind each Co-Author. The Corresponding Author shall: (i) ensure each Co-Author complies with all relevant provisions of this Publication Agreement, including those relating to confidentiality, performance and standards, as if a party to this Publication Agreement; and (ii) remain primarily liable for all acts and/or omissions of each such Co-Author.
\n\nThe Corresponding Author agrees to indemnify and hold IntechOpen harmless against all liabilities, costs, expenses, damages and losses and all reasonable legal costs and expenses suffered or incurred by IntechOpen arising out of or in connection with any breach of the aforementioned representations and warranties. This indemnity shall not cover IntechOpen to the extent that a claim under it results from IntechOpen's negligence or willful misconduct.
\n\n4.2 Nothing in this Publication Agreement shall have the effect of excluding or limiting any liability for death or personal injury caused by negligence or any other liability that cannot be excluded or limited by applicable law.
\n\n5. TERMINATION
\n\n5.1 IntechOpen has a right to terminate this Publication Agreement for quality, program, technical or other reasons with immediate effect, including without limitation (i) if the Corresponding Author or any Co-Author commits a material breach of this Publication Agreement; (ii) if the Corresponding Author or any Co-Author (being an individual) is the subject of a bankruptcy petition, application or order; or (iii) if the Corresponding Author or any Co-Author (being a company) commences negotiations with all or any class of its creditors with a view to rescheduling any of its debts, or makes a proposal for or enters into any compromise or arrangement with any of its creditors.
\n\nIn case of termination, IntechOpen will notify the Corresponding Author, in writing, of the decision.
\n\n6. INTECHOPEN’S DUTIES AND RIGHTS
\n\n6.1 Unless prevented from doing so by events outside its reasonable control, IntechOpen, in its discretion, agrees to publish the Chapter attributing it to the Corresponding Author and any Co-Author.
\n\n6.2 IntechOpen has the right to use the Corresponding Author’s and any Co-Author’s names and likeness in connection with scientific dissemination, retrieval, archiving, web hosting and promotion and marketing of the Chapter and has the right to contact the Corresponding Author and any Co-Author until the Chapter is publicly available on any platform owned and/or operated by IntechOpen.
\n\n6.3 IntechOpen is granted the authority to enforce the rights from this Publication Agreement, on behalf of the Corresponding Author and any Co-Author, against third parties (for example in cases of plagiarism or copyright infringements). In respect of any such infringement or suspected infringement of the copyright in the Chapter, IntechOpen shall have absolute discretion in addressing any such infringement which is likely to affect IntechOpen's rights under this Publication Agreement, including issuing and conducting proceedings against the suspected infringer.
\n\n7. MISCELLANEOUS
\n\n7.1 Further Assurance: The Corresponding Author shall and will ensure that any relevant third party (including any Co-Author) shall, execute and deliver whatever further documents or deeds and perform such acts as IntechOpen reasonably requires from time to time for the purpose of giving IntechOpen the full benefit of the provisions of this Publication Agreement.
\n\n7.2 Third Party Rights: A person who is not a party to this Publication Agreement may not enforce any of its provisions under the Contracts (Rights of Third Parties) Act 1999.
\n\n7.3 Entire Agreement: This Publication Agreement constitutes the entire agreement between the parties in relation to its subject matter. It replaces and extinguishes all prior agreements, draft agreements, arrangements, collateral warranties, collateral contracts, statements, assurances, representations and undertakings of any nature made by or on behalf of the parties, whether oral or written, in relation to that subject matter. Each party acknowledges that in entering into this Publication Agreement it has not relied upon any oral or written statements, collateral or other warranties, assurances, representations or undertakings which were made by or on behalf of the other party in relation to the subject matter of this Publication Agreement at any time before its signature (together "Pre-Contractual Statements"), other than those which are set out in this Publication Agreement. Each party hereby waives all rights and remedies which might otherwise be available to it in relation to such Pre-Contractual Statements. Nothing in this clause shall exclude or restrict the liability of either party arising out of its pre-contract fraudulent misrepresentation or fraudulent concealment.
\n\n7.4 Waiver: No failure or delay by a party to exercise any right or remedy provided under this Publication Agreement or by law shall constitute a waiver of that or any other right or remedy, nor shall it preclude or restrict the further exercise of that or any other right or remedy. No single or partial exercise of such right or remedy shall preclude or restrict the further exercise of that or any other right or remedy.
\n\n7.5 Variation: No variation of this Publication Agreement shall be effective unless it is in writing and signed by the parties (or their duly authorized representatives).
\n\n7.6 Severance: If any provision or part-provision of this Publication Agreement is or becomes invalid, illegal or unenforceable, it shall be deemed modified to the minimum extent necessary to make it valid, legal and enforceable. If such modification is not possible, the relevant provision or part-provision shall be deemed deleted.
\n\nAny modification to or deletion of a provision or part-provision under this clause shall not affect the validity and enforceability of the rest of this Publication Agreement.
\n\n7.7 No partnership: Nothing in this Publication Agreement is intended to, or shall be deemed to, establish or create any partnership or joint venture or the relationship of principal and agent or employer and employee between IntechOpen and the Corresponding Author or any Co-Author, nor authorize any party to make or enter into any commitments for or on behalf of any other party.
\n\n7.8 Governing law: This Publication Agreement and any dispute or claim (including non-contractual disputes or claims) arising out of or in connection with it or its subject matter or formation shall be governed by and construed in accordance with the law of England and Wales. The parties submit to the exclusive jurisdiction of the English courts to settle any dispute or claim arising out of or in connection with this Publication Agreement (including any non-contractual disputes or claims).
\n\nLast updated: 2020-11-27
\n\n\n\n
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I am also a member of the team in charge for the supervision of Ph.D. students in the fields of development of silicon based planar waveguide sensor devices, study of inelastic electron tunnelling in planar tunnelling nanostructures for sensing applications and development of organotellurium(IV) compounds for semiconductor applications. I am a specialist in data analysis techniques and nanosurface structure. 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After obtaining a Master's degree in Mechanical Engineering, he continued his PhD studies in Robotics at the Vienna University of Technology. Here he worked as a robotic researcher with the university's Intelligent Manufacturing Systems Group as well as a guest researcher at various European universities, including the Swiss Federal Institute of Technology Lausanne (EPFL). During this time he published more than 20 scientific papers, gave presentations, served as a reviewer for major robotic journals and conferences and most importantly he co-founded and built the International Journal of Advanced Robotic Systems- world's first Open Access journal in the field of robotics. Starting this journal was a pivotal point in his career, since it was a pathway to founding IntechOpen - Open Access publisher focused on addressing academic researchers needs. Alex is a personification of IntechOpen key values being trusted, open and entrepreneurial. Today his focus is on defining the growth and development strategy for the company.",institutionString:null,institution:{name:"TU Wien",country:{name:"Austria"}}},{id:"19816",title:"Prof.",name:"Alexander",middleName:null,surname:"Kokorin",slug:"alexander-kokorin",fullName:"Alexander Kokorin",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/19816/images/1607_n.jpg",biography:"Alexander I. Kokorin: born: 1947, Moscow; DSc., PhD; Principal Research Fellow (Research Professor) of Department of Kinetics and Catalysis, N. Semenov Institute of Chemical Physics, Russian Academy of Sciences, Moscow.\r\nArea of research interests: physical chemistry of complex-organized molecular and nanosized systems, including polymer-metal complexes; the surface of doped oxide semiconductors. He is an expert in structural, absorptive, catalytic and photocatalytic properties, in structural organization and dynamic features of ionic liquids, in magnetic interactions between paramagnetic centers. The author or co-author of 3 books, over 200 articles and reviews in scientific journals and books. He is an actual member of the International EPR/ESR Society, European Society on Quantum Solar Energy Conversion, Moscow House of Scientists, of the Board of Moscow Physical Society.",institutionString:null,institution:{name:"Semenov Institute of Chemical Physics",country:{name:"Russia"}}},{id:"62389",title:"PhD.",name:"Ali Demir",middleName:null,surname:"Sezer",slug:"ali-demir-sezer",fullName:"Ali Demir Sezer",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/62389/images/3413_n.jpg",biography:"Dr. Ali Demir Sezer has a Ph.D. from Pharmaceutical Biotechnology at the Faculty of Pharmacy, University of Marmara (Turkey). 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