\r\n\tThe development of the interpersonal model and the Kleinian school in the second half of the last century allowed the emergence of an original understanding of the unconscious mind. Within the intersubjective paradigm, the psychoanalytic situation is conceptualized as an interpersonal field to which both the analyst and the patient contribute substantially. We have shown elsewhere how the failure to give a full account of such an intersubjective dimension in both psychoanalytic theory and practice amounts to a core liability in contemporary psychoanalytic discourse.
\r\n
\r\n\tThe present book will focus on a few areas where the insufficient development of our discipline is currently apparent: five wounds that mark the body of the psychoanalytic enterprise.
\r\n
\r\n\tNew contributions are particularly needed in the following areas: Current conceptualization of the unconscious mind is mechanistic and not suited to incorporate the full network of interpersonal exchanges which unfolds in the analytic room; Furthermore, the development of interpersonal psychoanalysis and the theory of the object relations warrants a greater appreciation of the impact of extratranference relations (e.g., couple, family, peers) on the patient's inner life both within and without the psychoanalytic situation.
\r\n
\r\n\tAn integration of theories and models from other psychological paradigms is clearly in order here; the book will also focus on Barangers’ theory of the bi-personal field that makes traditional unipersonal models of the psychoanalytic process untenable. Also, it will help in the understanding of the reciprocal interactions of the two partners in the psychoanalytic dyad in most psychoanalytic institutes the training format relies naively on models from the academic or the professional domains. This fosters rigidity, conformism, and a hierarchical organizational style in the institutional life; e) all over the long span of his creative life Freud showed consistent interest in the application of psychoanalysis to literature, the arts, religion, and politics. Contemporary psychoanalysis is getting more and shyer and is pressed at the margins of social and political debate. The psychoanalytic theory includes unique lore of knowledge about the conscious and unconscious mind. Without it, a comprehensive understanding of human reality will stay out of the reach of contemporary culture.
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1. Introduction
Lung cancer is the leading cause of cancer deaths in both men and women, with more than 1 million deaths worldwide each year [1]. Unfortunately, this difficult therapeutic area has shown the highest failure rate in clinical trials over the last 30 years [2] and hitherto, there is no effective treatment for patients with lung cancer. The reasons for this drug attrition are multiple, but one major explanation is considered to be the lack of relevant preclinical models to appropriately validate potential drug targets and rank novel therapeutic agents before engaging in clinical trials [3].
Genetically engineered mice, ectopic and orthotopic xenotransplantation of tumors into immunodeficient mice are common models used as surrogate of patients to evaluate drug candidates before clinical testing. Although animal models can recapitulate important facets of human responses, their limitations as preclinical cancer models have now been widely documented [4-7]. Fundamental differences in transcriptional regulation [8], telomerase activity [9], neoplastic transformation mechanisms [10], cytokines production [11] as well as matrix metalloproteinases biology [12] are but a few features which compromise the design of efficient cancer therapies. Even the patient derived xenograft model (PDX), which better recapitulate the phenotypic features of the human tumor, displays a number of inherent limitations [13]. In this system, the tumorgraft established from primary tumor fragments has to be maintained through serial transplantations into mice, which will lead to the loss of the human stroma environment after 2-3 passages [14]. Clearly, such a replacement of the original tumor microenvironment by murine host components has significant consequences on growth features as well as response to therapies. Indeed, a number of oncogenic mouse ligands fail to cross-activate their related human receptors [15, 16] while stromal mediators have been identified as a critical source triggering tumour cells resistance to treatment [17, 18]. These observations highlight the importance of considering tumor-extracellular matrix interactions in the design of in vitro cancer models. Modern tumor biology has moved away from the traditionalist view conveyed for years by 2D cultures saturated with growth factors by revealing that the many varieties of cells that compose a tumor don’t just grow on their own but constantly integrate and react to signals coming from the extracellular matrix components. Solid tumors are now regarded as complex organs able to instruct the surrounding tissue to promote their own growth and progression, but also dependent on both molecular and mechanical signals coming from the adjacent healthy environment [19, 20]. Therefore, experimental models recapitulating true human cancer biology are mandatory for the validation of therapeutic agents in order to keep away from the risk of studying no more than adaptive cancer biology in a wrong environment that will eventually result in translation failure.
Figure 1.
(A) Cancer deaths anticipated in 2014. Estimated leading cancer sites mortality in European Union for the year 2014 expressed as percent of total cancer deaths. Column diagram highlights the mortality rate within the population specifically affected by lung cancer. (B) Age-standardized (world population) EU male (blue) and female (red) lung cancer death rates per 100,000 from 1982 to 2014. The graph shows the unfavourable trend for female lung cancer with a regular increase in case numbers over the last 30 years. Source: Malvezzi et al, Annals of oncology, 2014; 25(8):1650-6. and Bosetti et al, Annals of oncology, 19: 631–640, 2008.
In this article we report the significant efforts ongoing within academia and industry to developing in vitro novel complex human tumor models that should improve the identification and selection of efficient lung cancer therapies. We first focus on the human lung cancer cell lines currently available, their contribution to lung cancer biology and their use in research. Then, we will move from cell monolayers to three-dimensional (3D) cultures, exploring at first the function of natural and synthetic extracellular matrix before to document some recent advances in the field, including spheroids, bioscaffolds, decellularized lung matrix models and precision-cut lung tumor slices. Finally, we will present the bioengineering of a new generation of lung cancer models: OncoCilAir™. These 100% human models, which combine in vitro both tumor nodules surrounded by a functional airway epithelium and stroma, open new ways to test simultaneously drug efficacy, side toxicity and tumor recurrence in a single, integrated and accessible 3D model.
2. Lung cancer, current treatments and perspectives
Lung cancer (LC) is one of the major health concerns in the western world. LC is the most frequently diagnosed cancer in men and women and represents the most common cause of cancer-related deaths, both in the United States and in Europe, with a significant rate of 27% and 21% of total cancer deaths, respectively [21] (Fig.1A). The LC epidemic has been clearly linked to tobacco smoking [22], and while mortality rate in men has regularly fallen over the last decades (53 lung cancer deaths for every 100,000 European males in the late 1980s to 41.1/100,000 in 2009) thanks to strong measures for smoking control and prevention in middle-aged men, female LC rates are predicted to rise 8% in 2015 [1] (Fig.1B). There are two main types of lung cancer: non-small lung cancer (NSCLC) which account for about 85% of all lung cancers and small cell lung cancer (SCLC, 15%). SCLC is the most aggressive form of LC, with fast growing cells leading to large tumors. Histologically, NSCLC includes 3 subgroups: adenocarcinoma, squamous cell and large cell carcinoma [23]. As in many other forms of cancer, LC does not display too many symptoms, develops slowly over a period of several years, and only manifests itself in advanced stages (III or IV), where five-year survival rates are less than 10% because of high degree of metastasis. The overall median survival in stage IV is only about 8-10 months [24]. Platinum and taxane based chemotherapies (cisplatin, paclitaxel) has remained for years the treatments of choice, but more recently LC patients have been selected based on their tumor mutation profile. In most cases, oncogene driver mutations are exclusives (EGFR, ALK/EML4, KRAS, PTEN, etc...) and importantly, they divided patient populations into molecular subsets that do not show the same sensitivity to different treatments [25]. This patient stratification has enabled the introduction of targeted therapies directed against specific signaling pathways whose tumors are dependent on. Indeed, humanized recombinant antibodies directed against the vascular endothelial growth factor (VEGF) (bevacizumab) or small molecule inhibitors of EGFR-TK (erlotinib, gefitinib and afatinib), ALK and MET (crizotinib, ceritinib) have recently been used as a promising new line of therapies to treat lung cancer. Unfortunately, this drug portfolio extends survival only by a few months (Table 1) since most of the patients develop resistance to treatments, leading invariably to the recurrence of the disease [26-32]. Huge efforts are now undertaken to understand and circumvent drug resistance mechanisms. First observations have pointed out two main mechanisms for drug resistance acquisition: the selection of another pre-existing oncogene mutation or the activation of a bypass track, i.e. the deregulation of an alternative growth signaling pathway to maintain cell proliferation and tumor progression [33]. In EGFR-mutant lung cancer treated with the EGFR-TK inhibitors erlotinib and gefitinib, resistance is generally mediated by the T790M EGFR second-site mutation (~50% of the cases) [34] or phosphatidylinositol 3-kinase PI3K–AKT pathway activation via focal amplification of MET as second signaling pathway (~5%) [35]. For ALK-mutant lung cancer treated with crizotinib, mechanisms of resistance include the gatekeeper mutation L1196M (~30% of the cases) and KIT and EGFR signaling pathways activation as bypass track (~45%) [36]. Overall, these findings argue for the use of combined therapies in a manageable way. But recent data based on the analysis of tumor specimens at the time of acquired resistance suggest a much more complex landscape. In fact, multiple mechanisms, as diverse as epigenetic changes [37], epithelial to mesenchyme transition (EMT) or conversion from a LC histological type to another (EGFR-dependent NSCLC to SCLC), are induced under the selective pressure of targeted therapies [38, 39]. These observations imply the arrival of a personalized medicine, where a careful profiling of patient tumor’s mutation status (germline and somatic) and epigenetic signature will be mandatory all along the therapy to identify and adapt the correct treatment strategy. However, such scheduled combinatorial regimen would require the development of multiple-generation inhibitors to overcome specific subsets of resistance mutations and induce durable remissions. But the ability to escape multiple types of treatment could well be a hallmark of cancer cells. With this in mind, alternative strategies are worth considering. Instead of constraining tumor cells signalling through exogenous therapies making them overreact, a different approach might be to restrict tumor progression through its own microenvironment. The original 1975’s experiment of teratocarcinoma injection into blastocysts from Mintz and Illmensee was the first example of tumor repression by the microenvironment [40]. Today, tumor reprogramming through stroma instruction is emerging as a new treatment paradigm [41-43]. From this perspective, advanced human three-dimensional (3D) cell culture approaches modelling tumor-stroma communication could be key to accelerate the development of new lung cancer therapeutics.
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Table 1.
Targeted therapies approved by the Food and Drug Administration in the treatment of lung cancer. The median time to progression on targeted therapy (Progression Free Survival - PFS) is given in months. rHMAb: recombinant human monoclonal antibody. Source: National Cancer Institute database, 2014.
3. Lung cancer cell lines
Cell lines derived from human tumors provide an unlimited, self-replicating source of malignant cells that can be studied by investigators throughout the world. Therefore, even if cell lines represent only a highly selected fraction of the original tumor, their ease of access has resulted in the production of a very large body of literature. Indeed, it is acknowledged that most of our understanding of the molecular mechanisms involved in LC comes from studies done on mouse or human cell lines [44].
3.1. Lung cancer cell lines collection
To date, more than 250 LC cell lines have been established, mainly from Western population. Currently, the American Type Culture Collection (ATCC, Manassas, VA) catalog lists 121 human lung tumor cell lines. Among this panel, SCLC is less represented, first due to the lower frequency of the disease, and second because SCLC tumors are rarely surgically resected. Indeed, only small tissue samples from biopsy examinations, malignant aspirates, and rare malignant effusions are available for research use. Moreover, the fact that SCLC tumor cells lack the ability to adhere to culture dishes and required to be grown in vitro as floating cell aggregates or spheroids has precluded for a long time their expansion as cell lines. SCLC was first successfully cultured in Japan in 1971 [45].
Regarding NSCLC, primary and metastatic tumor materials are more easily accessible, even through routine bronchoscopy [38, 39]. However, although cells from metastatic tumors, especially from malignant effusions, are relatively easy to culture, cell cultures from primary solid tumor are not obvious to establish, with success rates ranging from 2.6 to 5% [46, 47]. Various protocols are in use, but on the whole, tumor tissues minced in small pieces are either directly cultured as fragments in a matrix (e.g. Matrigel®) or subjected to enzymatic dissociation (collagenase/hyaluronidase) and then suspended in culture medium. Clearly, positive results are higher when starting from material corresponding to advanced stages as MHC III and IV [46]. The culture medium composition is also critical and the development of serum-free chemically defined media (e.g. ACL4) has significantly improved success rates [48].
The resulting current LC cell lines depository represent therefore a unique resource that can be extremely valuable in term of genetic manipulation, high-throughput screening and development of more complex co-culture models.
3.2. Lung cancer cell lines as in vitro model
LC cell lines have been used for decades in functional studies with the aim to identify new oncogene drivers or tumor suppressors. Thus, LC cell lines compared to normal human bronchial epithelial cells were instrumental to generate list of differentially expressed genes that could account for tumorigenicity and represent therefore new therapeutic targets. As an example, this strategy lead to the detection of ERBB3, a gene associated with the EGF signaling pathway, among the genes over-expressed in LC cell lines [49]. Interestingly, this result was validated later on by another study that identified the activation of ERBB3 signaling as a mechanism of resistance to gefitinib [35]. Since these initial findings, ERBB3 has been recognized as a key node of LC progression and several humanized anti-ErbB3 antibodies are currently in pre-clinical development [50].
However, cell lines limitations have emerged as our knowledge about the disease increased. As an example, several studies have shown that differences in genetic background are important in defining cancer biology as well as in drug sensitivity [51]. Thus, a potential shortcoming of the current LC collection may reside in its under-representation of some populations, like the East Asian population, possibly introducing bias in research and drug discovery. Indeed, epidemiologic surveys have revealed that in the US, 10% of patients with NSCLC have tumour associated with EGFR mutations, while this number increases to 35% in East Asia, suggesting different selection mechanisms or sensitivity for lung cancer subtypes among different ethnic groups.
Accordingly, the recent classification of lung cancers into genetic subsets based on mutations in driver oncogenes (see previous section) prompted the community to accurately characterize the LC cell lines collection at the genomic and genetic levels. In this perspective, the Sanger Cancer Institute has initiated the genetic characterization of a panel of cancer cell lines (The Cancer Genome Project, http://cancer.sanger.ac.uk/cancergenome/projects/cell_lines/). Using current high throughput techniques this program provides information on mutations, copy number variations, single nucleotide polymorphisms (SNPs) and microsatellite instability of 136 cell lines representative of the different type of lung cancers (adenocarcinoma, small cell carcinoma, etc...) with the aim to define a genetic profile predictive of drug sensitivity. Such a signature should contribute to stratify patient population and to identify efficient targeted therapies.
Another emerging use of LC cell line is related to the identification of resistance mechanisms. As documented in section 2, so far all the approaches used in the treatment of lung cancer have resulted in the acquisition of resistance by the patients. One successful approach to discovering resistance mechanisms has been to culture sensitive cell lines with increasing concentrations of the drug until resistance emerges. The resistant cell line can subsequently be analysed to identify the resistance mechanisms, leading to the identification of resistance biomarkers and new strategies to overcome resistance [36].
Undoubtedly, cell lines have proven to be useful in elucidating important aspect of lung cancer biology. However, thanks to modern deep-sequencing technologies, we now know that lung tumors are composed of population of cells with distinct molecular and phenotypic properties [52] and consequently, that cell lines do not fully recapitulate human tumor biology. Clearly, the scientific community has taken into account these limitations, as shown by the growing interest for the establishment of complex in vitro cell models intended to bridge the gap between animal models and human studies.
4. Biocompatible matrices for 3D cell culture
In this section, we will try to briefly resume different ways and techniques used to culture the cells in 3D. Maintaining a 3D structure is critical to reproduce the tumour-stroma environment, communication between tumour cells, and the interaction with other surrounding cell types such as epithelial cells or fibroblasts [19]. Advances in materials chemistry and processing technologies, as well as developmental biology have led to the design of 3D cell culture matrices and bioscaffolds that better represent the geometry, chemistry, and signaling environment of natural extracellular matrix.
To obtain 3D cell cultures, cells are generally seeded onto/into biocompatible scaffolds or matrices. The 3D differentiation of cells depends on various parameters but it is generally accepted that best results are obtained when the natural environment is closely imitated [53]. Natural extracellular matrices are mainly composed of fibrous network made of collagen, elastic fibers, water and other materials like glycosaminoglycans, proteoglycans and glycoproteins [54]. To mimic the natural extracellular environment of the cells important parameters have to be taken into account [55]:
The matrix composition (collagen, fibrin, alginate, etc.)
The structure (pore size, pore distribution, pore geometry, etc.)
The manufacturing method (electrospinning, 3D printing, inverted colloidal crystal, spontaneous polymerization, etc.)
The biocompatibility
As the fate of a cell is largely determined by its environment, the elaboration of the right extracellular context is critical to promote the correct differentiation of a cell population.
For example, it is well known that epithelial cells have to be cultured at the air-liquid interface to differentiate. This could be easily obtained by seeding cells onto micro porous supports or scaffolds allowing nutrients to come from the back. The apical side of the cells remains generally exposed to the air [56]. This basic principle of air-liquid interface cultivation can be transposed to most of epithelial cells such as airway, vaginal, buccal, intestinal, etc. However, this approach is no more suitable when the cells are not from epithelial origin [57, 58].
It is typically the case for fibroblasts that are not able to survive when directly exposed to the air. To culture fibroblasts in 3D, a different type of environment is required. Cells can be embedded into a biocompatible matrix based on various components [53]. Among the most used we find collagen and fibrin. Collagen is the major component of connective tissues, it is naturally produced by fibroblasts and can be easily isolated from many type of tissues such as dermis, bone, tendon, etc...
Whereas collagen is easily obtained, applications for human therapies and 3D cell culture remain limited because of contamination risks between animals and humans (e.g. Creutzfeldt-Jakob). Moreover, the gel polymerization can be difficult to control thus reducing the field of applicability. Another drawback is the variation between batches to batches. These weaknesses have led to the development of new generations of synthetic matrices and scaffolds where polymerization as well as intrinsic properties of materials (elasticity, porosity, permeability, hydration, etc.) can be more easily controlled. The matrices used today for 3D cell culture can be divided into 3 groups:
Mixed compounds, made of natural compounds synthetically modified. These included peptide-coupled alginates, chitosan, hyluranan, tyrosine-derived polymers, etc...
Each material has its own strengths and weaknesses and therefore it is fundamental to select matrix components in function of the needs. In the context of lung tumor cell model, it is pertinent to determine the final end-point studied. For example, if cells invasion has to be studied, it is necessary to use matrix components where cells are able to adhere, migrate and proliferate [59]. Moreover, some cells have the ability to digest and transform the scaffold and elaborate a new environment adapted to their needs. In that case it will be valuable to select a natural matrix component like collagen or fibrin. If the goal of the experiment is to obtain a 3D scaffold for human therapy, the best choice will be the use of synthetic matrices where all components can be defined and controlled [60]. If the sensitivity of a cancer cell to a drug has to be studied, a relevant choice could be the use of cell spheroids embedded into a non degradable matrix component, like alginate [61]. In that case, cells are immobilized and their drug susceptibility can be determined using a simple viability test. Alginate scaffold has been optimized for 3D tumor modeling using H460, A549 and H1650 NSCLC cell lines [62]. In this study the anticancer effects of various chemotherapeutic agents were studied and compared with conventional 2D cell culture models. Results have shown that cells grown in 3D demonstrated a more realistic drug response with higher resistance to chemotherapy [62].
Clearly, there is a tremendous flexibility to reconstitute a scaffold and the choice of synthesis should be guided by the type of cells, the application and the desired physical properties [53]. In addition, new perspectives are offered by bioprinting technologies. The possibility to organize extracellular matrix into precise geometries should help engineering 3D complex tumor tissues for in vitro assays [63].
5. Tumor spheroid models for lung cancer research
Numerous anchorage-independent assays have been developed for drug discovery. The most popular is the spheroid model because it allows both 3D self-organization of tumor cells and drug screening in high-throughput format. Many normal and malignant cell types can be grown as sphere-shaped cell colonies, so called spheroids. Cells that don’t form spheroids spontaneously can be induced to do so by co-culturing with spheroid-forming non-clonogenic feeder cells [64]. As spheroid environment can be controlled, effects on tumor cell viability can be carefully examined. This model is particularly adapted to high throughput screening in 96-well plate assays, and numerous solutions are commercially available.
Phenotypic and functional differences between lung tumor cells grown as 2D monolayer cultures, versus cells grown as 3D spheroids have been observed. Indeed, the 3D spheroid culture changed the cellular response to drugs and growth factors suggesting to be more accurately mimicking the natural tumor microenvironment than classical culture of lung cell line [65]. Multi-cell type tumor spheroids are a valuable model to reproduce cellular heterogeneity and provide more comprehensive assessment of tumor response to therapeutic strategies. 3D co-culture model using NSCLC cell lines in combination with lung fibroblasts can be prepared [66]. To date, co-cultures involving up to three different cell types in a single spheroid (tumor cells, fibroblasts and endothelial cells) have been established, but without any proof of micro-capillary functionality [67]. Recent studies report that NSCLC can acquire epithelial-mesenchymal transition and cancer stem-like phenotypes within chitosan-hyaluronan membrane-derived 3D tumor spheroids [68].
6. Microfluidic chip-based 3D co-cultures
In the continuity of the pioneering work of Donald Ingber (organ on chip), a series of 3D lung-on-a-chip microfluidic devices have been developed. Briefly, lung-on-a-chip is a biomimetic microsystem that reconstitutes the critical functional alveolar-capillary interface of the human lung, with periodic mechanical stretching and flow of the medium carrying immune cells. Using this micro-fluidic device, the authors were able to replicating the immune responses against bacterial infections in vitro [69]. Afterwards, devices were optimized as a drug screening platform to select individualized treatment for lung cancer. In these systems, lung cancer and stromal cell lines were co-cultured as 3D spheroids under continuous media supplementation, mimicking the circulation of nutrients and metabolic waste out of the cultures [70]. Another similar model has been developed for chemoresistive testing of pleural mesothelioma cancer spheroids. Interestingly, growth inhibitory concentration of cisplatin showed higher concentration in perfused tumor spheroids compared with spheroids cultured under static conditions [71]. These systems represent therefore valuable tools to get information about the efficacy of chemotherapeutic drugs in a dynamic microenvironment which recapitulate the actual in vivo situation, but they do not address side-toxicity on normal lung physiology. The challenge will be to improve the model so that it incorporates normal and functional tissues. That could be achieved by connecting such devices with other microphysiological organotypic chips, representative of healthy lung tissues.
7. Ex vivo 3D lung cancer model based on decellularized matrix
As it is not obvious to identify the ideal matrix components and conditions suitable for the development of various lung tumor types, an alternative strategy is to take advantage of existing natural matrices. This methodology relies on the initial work of Ott and colleagues that first succeed in regenerating a bioartificial organ from a rat cadaveric lung [72]. Briefly, in this model the organ of interest is perfused with a detergent in order to remove donor cells and leave the components of the extracellular matrix. The resulting decellularized matrix is then reloaded with human lung adenocarcinoma tumor cells. In addition to their well-adapted composition, decellularized matrices also display specific elasticity which has been pointed out as critical for tumor cell growth. To date, rat decellularized lung matrix [73] and porcine decellularized intestinal submucosa [17] have been used as scaffold. Interestingly, tumor cell lines (A549, H460 and H1299) engrafted in this microenvironment formed 3D lung tumor nodules and displayed histological features reminiscent of the original primary tumor [74]. They also recovered functionalities (e.g. MMP-9 production) that were lost in 2D culture [73]. These ex vivo 3D models can be kept in culture for up to 28 days and exhibit sensitivity to treatment comparable to what is observed in clinic [17]. Although relevant for fundamental research, current ex vivo 3D lung models clearly show limitations. First, they are difficult to produce, the cultivation of the cells must take place in a special incubator, and consequently they cannot be used for high-throughput screening. Second, they do not recapitulate the human – human interactions between tumor and stroma. Indeed, epithelial and mesenchymal cells have been removed from epithelial space by the decellularization process. Third, they necessitate large amount of tumor cells (~25 millions) in order to colonize the matrix, precluding personalized medicine. And finally, they required the sacrifice of animals for matrix supply. However, ex vivo 3D lung models must be seen as the gold standard to be reached by 3D bioprinting technologies combined to synthetic matrices.
8. Precision-cut lung tumor slices
As previously mentioned, the tumor microenvironment provides essential signaling necessary for establishing and maintaining tumor specific morphogenic programs. Precision-cut lung slices (PCLS) obtained from freshly isolated human lung cancer tissues maintain both the original cancer microenvironment and preserve the complexity of the tumor-stroma interaction [75, 76]. Usually thin tissue slices (~200 µM) are prepared with a vibratome and cultured submerged into medium for several days. PCLS constitute a valid tool for the in vitro evaluation of tumor morphology, proliferation, viability and resistance to therapy [75]. Moreover, a major advantage of this model is the preparation of multiple experimental replicates from a single tumor, allowing performing drug efficacy studies. Indeed, dose-response experiments with the PIK3 inhibitor LY294002 have shown that PCLS cultures from lung cancer may be used to predict tumor sensitivity to drugs in a patient-specific manner [75]. In a different study, tumor PCLS were used to investigate nanoparticles delivery of antisense as lung cancer treatment. The model was instrumental to demonstrate that nanoparticles could penetrate into tumor tissue and target telomerase activity, without disturbing adjacent tissue architecture or inducing significant side-toxicity [76]. PCLS established from human lung tumor tissue represent therefore a useful in vitro tumor model that has the potential to enhance preclinical drug evaluation studies. However, an obvious limitation of PCLS is their short lifespan, ~5 days, which prevents long-term exposure, and therefore chronic treatment evaluation.
9. Engineered 3D lung tumor tissues: The OncoCilAir™ model
Tissue engineering is an innovative technology designed at first to produce artificial functional tissues to repair or replace portion of injured tissues. While initially seen as unrealistic, this field has made tremendous progress over the past decade, and regenerative medicine will soon become a routine technique [77]. Today, it is possible by combining human cells with suitable bioscaffolds to produce in vitro tissue equivalents from many sources (e.g. corneal, cartilage, intestinal, muscle, respiratory, skin, etc...). More recently, this promising technology has been applied to the field of oncology with some attempts to develop engineered tumor tissues for pre-clinical research (e.g. human melanoma model) [78]. Here we took advantage of our tissue engineering know-how in the respiratory field [79] to develop a complex, but accessible, 3D lung cancer model: OncoCilAir™ [80, 81]. To this purpose, human primary bronchial cells, lung fibroblasts and lung adenocarcinoma cell lines were co-cultured at the air-liquid interface in a transwell insert (Fig.2). After appropriate differentiation, the system closely reproduces malignant pulmonary nodules invading a human functional airway epithelium (Fig.3).
Figure 2.
Schematic representation of the OncoCilAir™ lung cancer model. OncoCilAir™ is a complex cellular model based on the co-culture at the air-liquid-interface of three different human components: bronchial cells, lung fibroblasts and NSCLC cell lines. After 30 days, the cells differentiate into a functional respiratory epithelium which comprises ciliated cells (pink), goblet cells (blue) secreting mucus (light blue), basal cells (yellow), fibroblasts (brown) and tumor nodules (green).
Figure 3.
Cultured at the air-liquid interface in a convenient 24-wells format (A), the OncoCilAir™ model mimics the in vivo lung tissue of a patient with characteristic tumour lung nodules (B & magnification in C).
Several properties contribute to make OncoCilAir™ a relevant pre-clinical in vitro alternative: First, it is a 100% human three-dimensional model which summarizes human tumour-stroma interactions to assess therapies targeting host-tumor interactions (Fig.4). Second, it is a flexible system: depending on the cell line used to build its tumour component, OncoCilAir™ offers the possibility to recapitulate distinct molecular subsets of lung cancers (EGFR, KRAS, etc...) and thus to simulate patient stratification. Third, it is a bi-competency model: the fact that it includes both compromised and healthy tissues brings the possibility to experiment simultaneously drug efficacy and drug side-effect within a single culture. Lastly, its long lifespan (>3 months) allows to test chronic treatments and recurrence while reducing animal testing.
Figure 4.
A tumor nodule expanding within the OncoCilAir™ human airway epithelium. Adenocarcima cells GFP+ (green) and human bronchial epithelial cells nuclei DAPI+ (blue) were visualized by confocal laser scanning microscopy. Scale bar represents 100 µm.
Accordingly, a dose response efficacy study with the investigational MEK inhibitors selumetinib and trametinib demonstrated that OncoCilAir™ showed responsiveness to anticancer drugs in agreement with previously reported data, and therefore can be used as a predictive tool for anticancer drug evaluation [82].
10. Future perspective
Hanahans and Weinberg highlighted six cancer hallmarks which provide us with a framework to understand this complex disease. These hallmarks include (i) sustaining proliferative signaling, (ii) evading growth suppressors, (iii) resisting cell death (iv) enabling replicative immortality, (v) inducing angiogenesis, and (vi) activating invasion and metastasis [83, 84]. However, in essence, cancer is a genetic disease with germ or autosomal mutations affecting genes implicated in cell division and/or in tissue integrity. These genetic alterations lead to unrestricted cell division and formation of a clone of cells which undergo further genetic changes. Some of these mutations promote features that endow cells with a selective advantage over normal cells, thus creating a more aggressive subclone with an even higher mutation rate, eventually leading to tumor formation [85]. The clonal theory has been corroborated by several decades of cancer researches: we now know that mutation in some specific genes, so-called oncogenes and tumor supressors, are primary cause for cancers. For lung cancers, the components of EGF signaling, such as EGF receptor and its downstream effectors (KRAS, BRAF, ALK, etc...) are the main drivers [33]. With the advance in biotechnology, it is now possible to rapidly identify the underlying mutations of the lung cancers for diagnostics and for personalized treatment.
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Table 2.
Current and future in vitro lung cancer models sorted according to cancer hallmarks.
However, a genetic change is only one side of the same coin. It is generally recognized now that the microenvironment surrounding the cancer cells plays also a crucial role in cancer development [19, 83, 84]. Indeed, tumor cells have to overcome at least six barriers in order to become invasive [86]. The extracellular matrix, stroma cells, immune cells, etc... form an integral part of the tumor, therefore should be taken into account. In fact, not all the cancer cells can grow in standard cell culture conditions: out of 160 tumors, only 8 Chinese NSCLC cell lines have been established in culture [47].
Therefore, for tissue engineering, the most important, as well as the most challenging task is to recreate the in vivo-like tumor micro-environment.
Another important issue is to maintain the heterogeneity of the tumor populations. Despite of huge progresses made in cancer research, the toll cancer claims in both human lives and funds spent on health care has been only marginally reduced, and in some cases even increases [87, 88]. One of the reasons for this situation is the drug resistance. The underlying cause is the heterogeneity of the tumor cells: within a tumor several clones with different mutations may co-exist. Furthermore, another process termed the community effect may be involved. Studies have suggested that the ability of a cell to respond to a signal may be enhanced by, or even dependent on, other neighboring cells reacting in the same way at the same time [89]. This effect helps to explain the formation of blocks of tissue from sheets of cells, and could be of widespread occurrence and significance in various morphogenesis processes, including tumor development. The underlying mechanism of the community effect could be the autocrine or paracrine positive feedback loops, which have also been suggested and identified during tumor formation. Several studies have outlined the importance of autocrine IL-6 signaling in lung and breast cancers. For example, one group found a positive correlation between persistently activated tyrosine-phosphorylated STAT3, found in 50% of lung adenocarcinomas, and IL-6. Further investigation revealed that mutant EGFR could activate the oncogenic STAT3 pathway via upregulated IL-6 autocrine signaling [90].
The fact that most of the cancer cells, even the aggressive ones, cannot grow in culture once dissociated strongly supports this notion. In other words, all the cancer hallmarks are the hallmarks of the tumor as a whole, not that of individual cancer cells. This has to be taken into account during the development of in vitro cancer models.
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Table 3.
Strengths and weaknesses of the different human in vitro models for lung cancer.
Ideally, in vitro lung cancer models should recapture all the hallmarks of human lung cancer. Each model has its own strength and weaknesses (Table 3). But depending on the application, simple models may be more relevant and sufficient. Models can therefore be sorted by complexity:
Cell lines
As simplest 3D model, the tumor spheroids represent already a progress with regarding to monolayer culture of tumor cell lines or primary tumor cells: the cell-cell interaction is restored. Stroma cells and or matrix could also be added to better mimic the in vivo situation.
Since the lung tumor cells are located at air-liquid interface, co-culture with the normal airway epithelial cells and fibroblast cells at air-liquid interface, illustrated by OncoCilAir™, is another realistic scenario for modeling the lung tumors.
In vitro PnP (Plug and Play) models with primary tumor derived from the patient.
In addition, Table 2 summarizes how the current, as well as the future in vitro cancer models can replicate some or all the cancer hallmarks, their use and limitations for drug development.
Finally, we would like to propose an ideal in vitro lung cancer model based on the above considerations, so-called in vitro PnP (Plug and Play) model (Fig.5).
Figure 5.
Schematic representation of an ideal in vitro PnP (Plug and Play) lung cancer model. A primary tumor derived from the patient is incorporated into a fully differentiated and healthy airway epithelium and cultured at air-liquid interface in a setting similar to OncoCilair™ (Mas et al., 2015); Then this co-culture model is plugged into a micro-fluidic device with artificial blood/or lymphatic vessels (pink color) containing circulating immune cells (blue color); liver cells (hepatocytes as spheroids, brown color) can also be integrated into the circuit through the plug number 2, providing metabolic capacity of drugs. If needed, other cells/organs can be further inter-connected in similar way. An input/output plug allows the addition of drug or the uptake of medium for analysis. The lung tissue culture remains accessible to apical exposure during all the experiment.
A primary tumor derived from the patient is incorporated into a fully differentiated and healthy airway epithelium and cultured at air-liquid interface, a setting similar to OncoCilair™ [82]. Then this co-culture model is plugged into a micro-fluidic device with artificial blood/or lymphatic vessels containing circulating immune cells; liver cells (hepatocytes as spheroids) can also be plugged into the circuit, providing metabolic capacity of drugs. If needed, other cells/organs can be further inter-connected in a similar way.
We are convinced that, with the development of new technologies such as microfluidic devices and 3D bio-printing, such models should quickly emerge and strengthen in vitro pre-clinical cancer research.
\n',keywords:null,chapterPDFUrl:"https://cdn.intechopen.com/pdfs/48483.pdf",chapterXML:"https://mts.intechopen.com/source/xml/48483.xml",downloadPdfUrl:"/chapter/pdf-download/48483",previewPdfUrl:"/chapter/pdf-preview/48483",totalDownloads:2643,totalViews:610,totalCrossrefCites:2,totalDimensionsCites:5,totalAltmetricsMentions:0,impactScore:4,impactScorePercentile:88,impactScoreQuartile:4,hasAltmetrics:0,dateSubmitted:"May 28th 2014",dateReviewed:"April 15th 2015",datePrePublished:null,datePublished:"June 3rd 2015",dateFinished:"May 28th 2015",readingETA:"0",abstract:null,reviewType:"peer-reviewed",bibtexUrl:"/chapter/bibtex/48483",risUrl:"/chapter/ris/48483",book:{id:"4539",slug:"drug-discovery-and-development-from-molecules-to-medicine"},signatures:"Samuel Constant, Song Huang, Ludovic Wisniewski and Christophe\nMas",authors:[{id:"72832",title:"Dr",name:"Samuel",middleName:null,surname:"Constant",fullName:"Samuel Constant",slug:"samuel-constant",email:"samuel.constant@epithelix.com",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",institution:null},{id:"166624",title:"Dr.",name:"Christophe",middleName:null,surname:"Mas",fullName:"Christophe Mas",slug:"christophe-mas",email:"christophe.mas@oncotheis.com",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",institution:null}],sections:[{id:"sec_1",title:"1. Introduction",level:"1"},{id:"sec_2",title:"2. Lung cancer, current treatments and perspectives",level:"1"},{id:"sec_3",title:"3. Lung cancer cell lines",level:"1"},{id:"sec_3_2",title:"3.1. Lung cancer cell lines collection",level:"2"},{id:"sec_4_2",title:"3.2. Lung cancer cell lines as in vitro model",level:"2"},{id:"sec_6",title:"4. Biocompatible matrices for 3D cell culture",level:"1"},{id:"sec_7",title:"5. Tumor spheroid models for lung cancer research",level:"1"},{id:"sec_8",title:"6. Microfluidic chip-based 3D co-cultures",level:"1"},{id:"sec_9",title:"7. Ex vivo 3D lung cancer model based on decellularized matrix",level:"1"},{id:"sec_10",title:"8. Precision-cut lung tumor slices",level:"1"},{id:"sec_11",title:"9. Engineered 3D lung tumor tissues: The OncoCilAir™ model",level:"1"},{id:"sec_12",title:"10. Future perspective",level:"1"}],chapterReferences:[{id:"B1",body:'Malvezzi, M., et al., European cancer mortality predictions for the year 2013. 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Biomaterials, 2013. 34(16): p. 4109-17.'},{id:"B71",body:'Ruppen, J., et al., A microfluidic platform for chemoresistive testing of multicellular pleural cancer spheroids. Lab Chip, 2014. 14(6): p. 1198-205.'},{id:"B72",body:'Ott, H.C., et al., Regeneration and orthotopic transplantation of a bioartificial lung. Nat Med, 2010. 16(8): p. 927-33.'},{id:"B73",body:'Mishra, D.K., et al., Human lung cancer cells grown on acellular rat lung matrix create perfusable tumor nodules. Ann Thorac Surg, 2012. 93(4): p. 1075-81.'},{id:"B74",body:'Mishra, D.K., et al., Human lung cancer cells grown in an ex vivo 3D lung model produce matrix metalloproteinases not produced in 2D culture. PLoS One, 2012. 7(9): p. e45308.'},{id:"B75",body:'Vaira, V., et al., Preclinical model of organotypic culture for pharmacodynamic profiling of human tumors. 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I-4-204.'},{id:"B81",body:'Mas C, Huang S, Wiszniewski L, Constant S., In vitro Human Airway Tissue Model For Lung Cancer Research. Am J Respir Crit Care Med 2014. 189: p. A3455.'},{id:"B82",body:'Mas C, Boda B, CaulFuty M, Huang S, Wiszniewski L and Constant S., Antitumour efficacy of the selumetinib and trametinib MEK inhibitors in a combined human airway-tumour-stroma lung cancer model. J Biotechnol., 2015. in press.'},{id:"B83",body:'Hanahan, D. and R.A. Weinberg, The hallmarks of cancer. Cell, 2000. 100(1): p. 57-70.'},{id:"B84",body:'Hanahan, D. and R.A. Weinberg, Hallmarks of cancer: the next generation. Cell, 2011. 144(5): p. 646-74.'},{id:"B85",body:'Nowell, P.C., The clonal evolution of tumor cell populations. Science, 1976. 194(4260): p. 23-8.'},{id:"B86",body:'Gatenby, R.A. and R.J. Gillies, A microenvironmental model of carcinogenesis. 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'}],corrections:null},book:{id:"4539",type:"book",title:"Drug Discovery and Development",subtitle:"From Molecules to Medicine",fullTitle:"Drug Discovery and Development - From Molecules to Medicine",slug:"drug-discovery-and-development-from-molecules-to-medicine",publishedDate:"June 3rd 2015",bookSignature:"Omboon Vallisuta and Suleiman Olimat",coverURL:"https://cdn.intechopen.com/books/images_new/4539.jpg",licenceType:"CC BY 3.0",editedByType:"Edited by",isbn:null,printIsbn:"978-953-51-2128-2",pdfIsbn:"978-953-51-4222-5",reviewType:"peer-reviewed",numberOfWosCitations:63,isAvailableForWebshopOrdering:!0,editors:[{id:"73943",title:"Prof.",name:"Omboon",middleName:null,surname:"Vallisuta",slug:"omboon-vallisuta",fullName:"Omboon Vallisuta"}],equalEditorOne:null,equalEditorTwo:null,equalEditorThree:null,coeditorOne:{id:"252327",title:"Dr.",name:"Suleiman",middleName:"M.",surname:"Olimat",slug:"suleiman-olimat",fullName:"Suleiman Olimat"},coeditorTwo:null,coeditorThree:null,coeditorFour:null,coeditorFive:null,topics:[{id:"1184"}],productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"},chapters:[{id:"47844",type:"chapter",title:"The Evolving Role of Natural Products from the Tropical Rainforests as a Replenishable Source of New Drug Leads",slug:"the-evolving-role-of-natural-products-from-the-tropical-rainforests-as-a-replenishable-source-of-new",totalDownloads:2964,totalCrossrefCites:3,signatures:"Ibrahim Jantan, Syed Nasir Abbas Bukhari, Mohamed Ali Seyed\nMohamed, Lam Kok Wai and Mohammed Ahmed Mesaik",reviewType:"peer-reviewed",authors:[{id:"172418",title:"Prof.",name:"Ibrahim",middleName:null,surname:"Jantan",fullName:"Ibrahim Jantan",slug:"ibrahim-jantan"}]},{id:"48182",type:"chapter",title:"Edible and Medicinal Mushrooms as Promising Agents in Cancer",slug:"edible-and-medicinal-mushrooms-as-promising-agents-in-cancer",totalDownloads:3461,totalCrossrefCites:4,signatures:"Ken Yasukawa",reviewType:"peer-reviewed",authors:[{id:"100634",title:"Prof.",name:"Ken",middleName:null,surname:"Yasukawa",fullName:"Ken Yasukawa",slug:"ken-yasukawa"}]},{id:"48125",type:"chapter",title:"Challenges of Patient Selection for Phase I Oncology Trials",slug:"challenges-of-patient-selection-for-phase-i-oncology-trials",totalDownloads:4727,totalCrossrefCites:1,signatures:"Mark Voskoboynik and Hendrik-Tobias Arkenau",reviewType:"peer-reviewed",authors:[{id:"68479",title:"Prof.",name:"Hendrik-Tobias",middleName:null,surname:"Arkenau",fullName:"Hendrik-Tobias Arkenau",slug:"hendrik-tobias-arkenau"},{id:"172261",title:"Prof.",name:"Hendrik-Tobias",middleName:null,surname:"Arkenau",fullName:"Hendrik-Tobias Arkenau",slug:"hendrik-tobias-arkenau"},{id:"172343",title:"Dr.",name:"Mark",middleName:null,surname:"Voskoboynik",fullName:"Mark Voskoboynik",slug:"mark-voskoboynik"}]},{id:"48483",type:"chapter",title:"Advanced Human In vitro Models for the Discovery and Development of Lung Cancer Therapies",slug:"advanced-human-in-vitro-models-for-the-discovery-and-development-of-lung-cancer-therapies",totalDownloads:2643,totalCrossrefCites:2,signatures:"Samuel Constant, Song Huang, Ludovic Wisniewski and Christophe\nMas",reviewType:"peer-reviewed",authors:[{id:"72832",title:"Dr",name:"Samuel",middleName:null,surname:"Constant",fullName:"Samuel Constant",slug:"samuel-constant"},{id:"166624",title:"Dr.",name:"Christophe",middleName:null,surname:"Mas",fullName:"Christophe Mas",slug:"christophe-mas"}]},{id:"48066",type:"chapter",title:"Nuclear Receptor Modulators — Current Approaches and Future Perspectives",slug:"nuclear-receptor-modulators-current-approaches-and-future-perspectives",totalDownloads:2282,totalCrossrefCites:2,signatures:"Thales Kronenberger, Oliver Keminer, Carsten Wrenger and Björn\nWindshügel",reviewType:"peer-reviewed",authors:[{id:"172265",title:"Dr.",name:"Carsten",middleName:null,surname:"Wrenger",fullName:"Carsten Wrenger",slug:"carsten-wrenger"},{id:"175203",title:"Dr.",name:"Bjoern",middleName:null,surname:"Windshuegel",fullName:"Bjoern Windshuegel",slug:"bjoern-windshuegel"},{id:"175204",title:"Dr.",name:"Thales",middleName:null,surname:"Kronenberger",fullName:"Thales Kronenberger",slug:"thales-kronenberger"},{id:"175205",title:"Dr.",name:"Oliver",middleName:null,surname:"Keminer",fullName:"Oliver Keminer",slug:"oliver-keminer"}]},{id:"48274",type:"chapter",title:"Lipids and Liposomes in the Enhancement of Health and Treatment of Disease",slug:"lipids-and-liposomes-in-the-enhancement-of-health-and-treatment-of-disease",totalDownloads:2395,totalCrossrefCites:0,signatures:"Simon A. 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1. Introduction
Radon is a colorless, odorless, and tasteless inert gas. It can only be detected or measured with the help of special detectors. It can travel through cracks of the bedrock, soil, and through groundwater. In underground mines or underground structures, high concentrations of radon may be detected in the absence of adequate ventilation. In underground mines with uranium-bearing mineralization, radium 226 (radium’s most stable isotope) is a natural source of radiation. Other isotopes of radon, such as radon 220 and radon 219, also exist naturally; however, because of the small amount and short lifetime, other isotopes are of less concern. Radium 226 decays into radon 222, which in turn decays into its short-lived radioactive daughters in the mine atmosphere. The uranium decay chain can be summarized as shown in Figure 1.
Figure 1.
Uranium decay chain.
Until the late 1970s, radon and its daughter products were of concern only at uranium mines. A study conducted by Daniels and Schubauer in 2017 shows that the radon exposure varied widely among several working populations, most of whom were employed in industries unrelated to the uranium fuel cycle. With the recent advancement of scientific knowledge, there has been more interest and attention to the hazards in non-uranium mines, underground structures, and residential buildings. In the absence of control measures, occupational exposures outside the uranium fuel cycle (e.g., tourist cave workers, waterworks employees) can exceed those found in most uranium workers [1].
Dehnret [2] reported high radon concentrations in old underground workings in Germany and protective steps taken for miners’ safety. Sahu et al. [3] reported the sources of radon, its emanation rate, and measurement techniques, particularly for underground uranium mines. Hu et al. [4] highlighted radon and radon progeny problems in Chinese uranium mines. In the United States, radon has been listed as the second major cause of lung cancer after tobacco [5]. A study of underground miners shows that 40% of lung cancer deaths may be due to radon progeny exposure [6]. MSHA has regulations for radon concentration in underground mines and sampling procedures depending on the concentration.
Considering the short half-life and the high radiation dose of radon gas and its daughter products, its mitigation in the underground environment becomes very important. In the absence of mitigation techniques, both the uranium and non-uranium mines (with uranium mineralization in the orebody) pose a serious threat to the personnel working in the underground environment.
Ventilation plays a significant role by supplying fresh air and removing the contaminated air from the working areas, thereby minimizing the radon concentrations in the mine environment. In addition, an appropriate mining method and well-designed mining sequence can also help control radon gas in the mine atmosphere [4]. In this chapter, the different radon mitigation methods that are specific to the underground mining operations are discussed.
2. Sources of radon
Radium 226 decays into radon 222, which in turn decays into its short-lived radioactive daughters in the mine atmosphere. Common sources of radon emissions in underground mines are summarized in Table 1.
Sources
Remarks
Mine walls
In low/medium ore grades, porosity and micro-fractures are dominant factors affecting the rate of radon gas emanation.
Broken ore
Fragmented ore provides a source of higher radon emanation due to the increased exposed surface area.
Backfill tailings
Radon emanation rate increases with increasing water content up to a certain saturation level, and beyond the saturation level, it decreases with the increase in water content.
Mine water
Mine water carries radon from the mineralized rocks to mine openings and transports it to a considerable distance in the mine galleries.
Table 1.
Common sources of radon in underground uranium mines [3].
3. Radon monitoring
The concentration of radon gas is measured in units of picocuries per liter (pCi/L) or becquerels per cubic meter (Bq/m3) of ambient air. Due to difficulties in measuring radon gas concentration, potential alpha particles per liter of air are usually measured. The ratio of all the short-lived radon daughters’ activity to the parent radon gas activity is called the equilibrium factor. The equilibrium factor is 1 when both are equal. Radon daughter activities are usually less than the radon activity, and hence, the equilibrium factor is generally less than 1. In artificially ventilated scenarios such as underground mines, the equilibrium factor is in the range of 0.4 to 0.5.
In the United States, radioactivity for radon decay products is measured in terms of Working Level (WL). A WL is defined as the concentration of short-lived radon daughters, representing 1.3 × 105 MeV of potential alpha particle energy while decaying to the stable Pb-210. The worker’s prolonged exposure to radon daughters is expressed in Working Level Months (WLM). One WLM is equivalent to 1 WL exposure for 170 hours.
In underground mines as per the Mine Safety and Health Administration (MSHA) regulations, personnel shall not be exposed to air containing concentrations of radon daughters exceeding 1.0 WL. No person shall be permitted to receive exposure over 4 WLM (Working Level Months) in any calendar year. In all mines, at least one sample must be taken in exhaust mine air by a competent person to determine whether concentrations of radon daughters are present [7]. Table 2 provides the radon sampling frequency for uranium and non-uranium mines and households. Gamma radiation surveys shall be conducted annually in all underground mines where radioactive ores are mined. Gamma radiation dosimeters shall be provided to all personnel working in the area where gamma radiation exceeds 2.0 milliroentgens; annual individual gamma radiation exposure shall not exceed 5 Roentgen Equivalent Man [7].
Type of mine
Radon daughter concentration level (a)
Frequency of monitoring
Uranium mine
a > 0.1 WL
Radon daughter concentration shall be determined at least every 2 weeks at random times in all working areas.
a > 0.3 WL
Radon daughter concentration shall be determined weekly in that area until the concentration reaches 0.3 WL or less for 5 consecutive weeks.
a < 0.1 WL (exhaust mine air sample)
Radon daughter concentration shall be determined by taking at least one sample in the exhaust mine air monthly.
Non-uranium mine
0.1 WL < a < 0.3 WL
Radon daughter concentration shall be determined at least every 3 months at random times until the concentration is below 0.1 WL in that area and annually thereafter.
a > 0.3 WL
Radon daughter concentration shall be determined at least weekly in that area until the concentration drops to 0.3 WL or less for 5 consecutive weeks.
a < 0.1 WL (exhaust mine air sample)
No further exhaust mine air sampling is required.
Houses
a > 0.04 WL (equilibrium factor of 1)
The EPA (Environmental Protection Agency) guidelines recommend the installation of radon mitigation systems.
The measurement techniques for radon can be classified based on a) whether the technique measures radon gasR222nor its daughter products, b) time resolution, and c) radioactive detection of the type of emission resulting from radioactive decay—alpha, beta, gamma radiation. The commonly used methods for measuring radon and its daughter products are shown in Figure 2.
Figure 2.
Radon gas and daughter (progeny) product measurement methods [8].
Active methods require electric power for measurements, whereas passive methods require no power. Measurements can be performed at specified intervals and data can be stored and read directly with active methods. In contrast, in the case of passive methods, integrated exposure concentrations can be measured, and data analysis requires special equipment. Time resolution techniques can be classified into three types, as shown in Figure 3.
Figure 3.
Time resolution techniques for measuring radon and its daughter products [8].
Grab sample technique: This technique involves measurement of R222n in a discrete sample of air collected over a very short period of time (compared with the mean life of R222n at a single point). Radon measuring instruments such as RAD7 can be used to measure R222n and its daughters. When it is used in “sniffer” mode, in which radon is typically present with minimal growth of its progeny, a large number of measurements can be taken in a relatively less period of time [8].
Grab sampling technique for radon progeny involves drawing a known air volume through a filter and counting the alpha activity during or following the sampling. Usually, a known volume of air is drawn through a filter using an air sampling pump for very short sampling periods usually 5 minutes. Filters are counted for alpha particle emissions during mathematically determined periods after the sample is collected. There are three main methods available for counting these particles, namely the Kusnetz method, where the filter is counted once, and the modified Tsivoglou method, where the filter is counted three times to measure the decay. Another method, named the Rolle method, is quite popular in Canadian mines. It is similar to Kusnetz method but is more rapid, and the procedure differs only in the timing of filter counting after sample collection. Figure 4 shows one of the MSHA recommended instruments for sampling radon progeny that works based on the Kusnetz method.
Figure 4.
Ludlum 2000 with accessories.
Continuous technique: This technique provides time series concentrations of R222nin air samples; in this method, sampling and counting are performed simultaneously. Most of the continuous monitors are portable, and nearly all of those are designed to detect alpha radiation (by an ionization chamber, gross alpha counting, or alpha spectrometry). Specific ionization and scintillation chambers are shown in Figures 5 and 6, respectively.
Figure 5.
Schematic diagram of an ionization chamber [9].
Figure 6.
A typical scintillation detector [10].
Integrating technique: This technique provides the integrated concentration over a certain period of time. Such measurements are used to determine monthly or averageR222n. The passive detectors, which are expensive, are examples of integrating techniques [8].
Apart from measuring alpha particles during the decay ofR222n, radon concentrations are determined by measuring the beta activity during the decay of P214b and B214i by assuming secular equilibrium between R222n and its progeny in the air. The beta activity is assayed with plastic scintillators mounted on photomultiplier tubes or the filter paper can be counted in a beta counter with the appropriate use of absorber film. Gamma spectrometry can also be used to determine radon concentrations by measuring the gamma activity during the decay of P214b andB214i.
5. Prediction techniques
There are a few traditional approaches for predicting radon flux such as uninterrupted short-term monitoring to represent radon concentration over an extended period and laboratory investigations. These methods do not apply to all cases. Recently, Kayode et al. [11] developed an approach for predicting radon flux from fractured rocks, a discrete fracture network (DFN) model that can predict radon transport through fractures considering diffusion, advection, and radon generation with radon decay.
6. Mitigation methods
Some of the important techniques to mitigate radon gas in underground mines are discussed below.
6.1 Sealant coating
The major sources of radon gas in non-uranium underground mines (with uranium mineralization) are the drift walls, floor, and roof. Shotcreting or applying radon sealants to the walls and roof effectively minimizes radon gas emissions into the mine atmosphere. The effectiveness of sealant coating in controlling the radon gas depends on the size of the capillary in which the acrylics (contained in the sealants) form barriers to prevent the escape of radon gas [12].
6.2 Bulkhead
Isolation of mined-out areas using bulkheads is one of the popular methods of controlling radon gas emissions into the active mine workings. Bulkheads prevent the contaminated air of mined-out regions containing high radon gas concentrations from mixing with the fresh air. Loring and others [13] reported that styrofoam and shotcrete/concrete bulkheads are used in a panel cave mine for a temporary and permanent sealing purpose, respectively. These bulkheads are installed at a 60-degree layback angle of the planned cave area to minimize damage to the bulkheads during the caving process. As the bulkheads are not leak proof, bleeder pipes creating a negative pressure inside the bulkhead area and connected to the main exhaust ventilation system can also be an effective measure [13]. Figure 7 shows the typical designs of bulkheads.
Figure 7.
Typical designs of bulkheads [14].
6.3 Mine pressurization by mechanical ventilation
Mine pressurization can also play an important role in controlling radon gas emissions in underground workings, especially near-working faces. In a forced ventilation system, which is considered quite effective for control of radon gas in the mine environment, fresh air is pumped into underground workings with the help of fans; this follows the path of least resistance taking the contaminants along with it and out of the mine. In the forced ventilation system, the direction of seepage is toward the rock surface, causing less radon to be released.
Studies [13] have shown that a successful blend of positive and negative pressure systems in a panel cave mine effectively reduces the radon gas concentrations at the production level. Negative pressure on the cave top minimizes the escape of radon gas from the broken ore to the production levels. Positive pressurization in the undercut levels also reduces the escape of radon gas into the working areas. Mine pressurization greatly depends on the porosity and permeability of the broken rock/ore for its effectiveness in controlling radon concentrations in the underground environment. Figure 8 shows a typical cave ventilation system in a block/panel cave mine.
Figure 8.
A typical cave ventilation system in a panel/block cave mine.
Computational fluid dynamic (CFD) simulation studies by Kayode et al. [15] showed the effect of an undercut ventilation system on radon gas distribution in the production drift and cave. It was observed that the air flowing through the cave transports some of the radon generated within the cave into the production drift, increasing the production drift concentration. However, in the absence of undercut ventilation, radon concentration decreases significantly within the production drift but increases inside the cave. The radon growth through the production drifts is nonlinear due to differences in the source of radon. Maintaining a negative pressure on top of the cave and undercut pressurization significantly reduces radon concentration in the production drift. However, maintaining a negative pressure on top of the cave is not very effective without undercut pressurization. An increase in air volume flow rate reduces radon concentration through the production drifts; based on the drift configuration for radon source, different empirical relationships relate airflow and working level for each drift.
The knowledge of airflow behavior and system characteristics is vital in ventilating the block cave operations and reducing radon concentrations. Using field observations and laboratory experiments (scale model studies), Pan [16] investigated the effects of porosity, material size combinations, additional fan, ventilation devices, and undercut structure on cave airflow resistance. The study found that the cave airflow resistance increases with a decrease in porosity and particle size, additional fan operation, regulator installation, and air gap reduction in the undercut drifts. An additional fan operation can contribute extra total airflow through the system, but regulators will not increase the total airflow in the system; the air gap observed in the undercut drifts might lead to less airflow through the production drifts.
Rahul et al. [17] investigated the effect of changes in the bulk porosity of the broken rock on the cave airflow resistance using the computational fluid dynamics (CFD) approach. This study reveals that porosity plays a vital role in changing the resistance offered by the broken rock to the airflow leaking into the cave. The airflow resistance increases as the porosity of the broken rock pile decreases. The resistance of the block cave mine changes dynamically with the bulk porosity of the broken rock.
Jha et al. [18] studied the utility of different fans in reducing the radon concentration within the drifts using a physical scale model and CFD simulations. It was observed that the combination of main and cave fan is optimal in minimizing the gas concentration within the drifts. Observations of the scaled model also show that a fully operational cave fan significantly reduced the gas concentrations within the drifts. The study suggests using main fan in conjunction with a cave fan to minimize the gas concentration within the drift.
Erogul et al. [19] investigated the impact of air gap geometries on cave resistance and radon emissions using the CFD approach. This study reported an interesting airflow behavior within the air gap zone; initially, the airflow resistance increases up to a certain height and drops as the air gap height increases further.
6.4 Radon adsorption on activated carbon
Radon gas can be adsorbed by activated carbon, commonly known as a charcoal bed. The capacity of a charcoal bed to adsorb radon depends on the temperature and moisture content of the incoming air. Karunakara et al. [20] demonstrated that a coconut shell-based activated charcoal system can be used for designing effective and reliable radon mitigation systems. Degassing properties of the charcoal indicate its reusability potential. Adsorption of radon by activated carbon can also significantly reduce ventilation air requirements. Figure 9 shows the experimental setup for studying radon adsorption in a charcoal bed.
Figure 9.
Experimental setup for studying radon adsorption in charcoal bed [20].
6.5 Ground freezing
Mine water is another source of radiation in underground mines. Artificial ground freezing is an excavation support method that involves the use of refrigeration to convert in situ pore water into ice [21]. Yun and others [22] reported that artificial ground freezing, to form a frozen curtain between the water-bearing sandstone and the ore body at McArthur River mining operation in Canada, helped prevent high-pressure, radon-bearing water from entering into the mine workings. Figure 10 shows a schematic of a typical ground freezing technique.
Figure 10.
Typical freeze wall insulation [22].
6.6 Choice of mining method and ventilation system
The choice of mining method and the type of mechanical ventilation significantly impact the control of radon gas emissions into the underground mine atmosphere. Table 3 provides the type(s) of effective ventilation systems to be used for various mining methods for controlling radon emissions in underground mines.
Mining methods
Mining types
Ventilation types
Cut and fill
Dry packing
Both forced and exhaust ventilation systems can be used.
Hydraulic flushing
Both forced and exhaust ventilation systems can be used. Measures should be taken to control the release of radon from the seeping water.
Open stope
Shrinkage stoping
Downward forced ventilation can be used here to prohibit the release of radon. The air inlets are installed on the upper parts of the deposits. Local fans are installed where the amount of air introduced is inadequate.
Breast stoping
Both forced, and exhaust ventilation systems can be used. However, the amount of air required at working faces increases as the number of mined-out areas increases.
Caving
Slicing
Forced ventilation and local fans should be used.
Sublevel caving
Forced ventilation should be used.
Block/panel caving
The combination of forced and exhaust ventilation systems.
It is an indirect mitigation technique. In the environments where radon daughters’ concentration exceeds 1.0 WL, miners should wear respirators approved by the National Institute for Occupational Safety and Health (NIOSH). The use of personal respiratory protection against radon daughters must be limited to temporary situations where engineering controls have not been developed or for maintenance and investigative work. For exposures up to 10 WL, proper filter-type respirators are available where concentrations of radon daughters exceed 10 WL, air devices, or face masks containing absorbent material capable of removing both radon and its daughters [7].
6.8 Dust control and miscellaneous measures
Airborne radon progeny (daughters) has an electrical charge associated with it; so, it can be attached to dust and other particles, which can be inhaled into the lungs of mineworkers who work in the dusty environment, particularly near the working faces. Some of the best practices that can help control radon levels in the mine atmosphere include implementing appropriate dust control measures by using air filters, measuring the performance of blasting practices at the end of the shift, and minimization of main/auxiliary fan shutdowns. Abd et al. [23] showed that the radon diffusion coefficient and diffusion length reduce significantly with increased water saturation of the material. This phenomenon can be used to reduce the rate of radon diffusion into the mine air.
7. Summary
Several radon mitigation techniques, particularly bulkheads and sealant coating, are being successfully used in the underground mines in the United States. Activated charcoal bed and oxidizing agents are also viable options for treating the contaminated air locally, especially at the difficult mine working faces. The feasibility of the application of these agents in the challenging mine environment needs a greater in-depth study.
Even though sealant coatings and bulkheads effectively control radon gas concentrations in the active working areas, improvements to reduce the costs and design of application of sealants and bulkheads can be performed.
Activated charcoal beds present a viable option for radon mitigation, but a pilot study in the mine environment can be more helpful to understand their applicability and effectiveness.
The use of strong oxidizing agents to remove radon from the contaminated mine air can also be a possibility. However, high humidity and temperature conditions in the mine atmosphere might limit the applicability of a corrosive oxidizing agent inside the mine.
Acknowledgments
The authors acknowledge the financial support from the National Institute for Occupational Safety and Health (NIOSH) (200-2014-59613) for conducting this research.
\n',keywords:"radon mitigation, radon measurement, underground mines, ventilation system",chapterPDFUrl:"https://cdn.intechopen.com/pdfs/79630.pdf",chapterXML:"https://mts.intechopen.com/source/xml/79630.xml",downloadPdfUrl:"/chapter/pdf-download/79630",previewPdfUrl:"/chapter/pdf-preview/79630",totalDownloads:185,totalViews:0,totalCrossrefCites:2,dateSubmitted:"September 2nd 2021",dateReviewed:"October 15th 2021",datePrePublished:"December 10th 2021",datePublished:null,dateFinished:"December 10th 2021",readingETA:"0",abstract:"Radon, a radioactive noble gas, is a decay product of uranium found in varying concentrations in all soils and rocks in the earth crust. It is colorless, odorless, tasteless, and a leading cause of lung cancer death in the USA. A study of underground miners shows that 40% of lung cancer deaths may be due to radon progeny exposure. In underground mines, radon monitoring and exposure standards help in limiting miners’ exposure to radioactivity. Radon mitigation techniques play an important role in keeping its concentration levels under permissible limits. This chapter presents a review of the radon sources and monitoring standards followed for underground mines in the USA. Also, the different radon prediction and measurement techniques employed in the underground mines and potential mitigation techniques for underground mining operations are discussed.",reviewType:"peer-reviewed",bibtexUrl:"/chapter/bibtex/79630",risUrl:"/chapter/ris/79630",signatures:"Purushotham Tukkaraja, Rahul Bhargava and Srivatsan Jayaraman Sridharan",book:{id:"10951",type:"book",title:"Mining Technology",subtitle:null,fullTitle:"Mining Technology",slug:null,publishedDate:null,bookSignature:"Dr. Andrew Hammond, B.Sc. Brendan Donnelly and Associate Prof. Nanjappa Ashwath",coverURL:"https://cdn.intechopen.com/books/images_new/10951.jpg",licenceType:"CC BY 3.0",editedByType:null,isbn:"978-1-83969-762-3",printIsbn:"978-1-83969-761-6",pdfIsbn:"978-1-83969-763-0",isAvailableForWebshopOrdering:!0,editors:[{id:"259487",title:"Dr.",name:"Andrew",middleName:null,surname:"Hammond",slug:"andrew-hammond",fullName:"Andrew Hammond"}],productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"}},authors:null,sections:[{id:"sec_1",title:"1. Introduction",level:"1"},{id:"sec_2",title:"2. Sources of radon",level:"1"},{id:"sec_3",title:"3. Radon monitoring",level:"1"},{id:"sec_4",title:"4. Measurement techniques",level:"1"},{id:"sec_5",title:"5. Prediction techniques",level:"1"},{id:"sec_6",title:"6. Mitigation methods",level:"1"},{id:"sec_6_2",title:"6.1 Sealant coating",level:"2"},{id:"sec_7_2",title:"6.2 Bulkhead",level:"2"},{id:"sec_8_2",title:"6.3 Mine pressurization by mechanical ventilation",level:"2"},{id:"sec_9_2",title:"6.4 Radon adsorption on activated carbon",level:"2"},{id:"sec_10_2",title:"6.5 Ground freezing",level:"2"},{id:"sec_11_2",title:"6.6 Choice of mining method and ventilation system",level:"2"},{id:"sec_12_2",title:"6.7 Personal respiratory protection",level:"2"},{id:"sec_13_2",title:"6.8 Dust control and miscellaneous measures",level:"2"},{id:"sec_15",title:"7. Summary",level:"1"},{id:"sec_16",title:"Acknowledgments",level:"1"}],chapterReferences:[{id:"B1",body:'Daniels RD, Schubauer-Berigan MK. Radon in US workplaces: A review. Radiation Protection Dosimetry. 2017;176(3):278-286. DOI: 10.1093/rpd/ncx007'},{id:"B2",body:'Dehnert J. Radon exposures of miners at small underground construction sites in old mining. Health Physics. 2020;118(1). DOI: 10.1097/HP.0000000000001117'},{id:"B3",body:'Sahu P, Panigrahi DC, Mishra DP. Sources of radon and its measurement techniques in underground uranium mines – an overview. Journal of Sustainable Mining. 2014;13(3):11-18. DOI: 10.7424/jsm140303'},{id:"B4",body:'Hu P, Li X. Analysis of radon reduction and ventilation systems in uranium mines in China. Journal of Radiological Protection. 2012;32(3):289-300. DOI: 10.1088/0952-4746/32/3/289'},{id:"B5",body:'Miller KJ, Coffey MA. Radon and you: Promoting public awareness of radon in Montana’s air and groundwater. Montana Bureau of Mines and Geology. 1998'},{id:"B6",body:'Lubin JH et al. Lung cancer in radon-exposed miners and estimation of risk from indoor exposure. JNCI Journal of the National Cancer Institute. 1995;87(11):817-827. DOI: 10.1093/jnci/87.11.817'},{id:"B7",body:'MSHA, “Radiation—Underground Only: 30 CFR §§ 57.5037 through 57.5047.” Available from: https://www.ecfr.gov/cgi-bin/text-idx?SID=4d0ee9eb9bdc6f357eb5e74f99e7d7e1&pitd=20200722&node=sg30.1.57_15015.sg16&rgn=div7 [Accessed: October 23, 2020]'},{id:"B8",body:'Baskaran M. Radon measurement techniques. In: Radon: A Tracer for Geological, Geophysical and Geochemical Studies. Cham: Springer International Publishing; 2016. pp. 15-35'},{id:"B9",body:'Nagaraja K, Kumar KC, Pramodh B, Prasad TR, Rao TN, Ratnam MV. Temporal variation of radioactivity at NARL, Gadanki. International Journal of Advanced Research in Science and Technology. 2015;4(5):469-471'},{id:"B10",body:'Equipco, “Introduction to Radiation Detectors.” Availabe from: http://www.equipcoservices.com/support/tutorials/introduction-to-radiation-monitors/ [Accessed: October 23, 2020]'},{id:"B11",body:'Ajayi KM, Shahbazi K, Tukkaraja P, Katzenstein K. A discrete model for prediction of radon flux from fractured rocks. Journal of Rock Mechanics and Geotechnical Engineering. 2018;10(5):879-892. DOI: 10.1016/j.jrmge.2018.02.009'},{id:"B12",body:'Kown BT, Van der Mast VC and Ludwig KL, “Technical Assessment of Radon-222 Control Technology for Underground Uranium Mines. Technical Note.” 1980. [Online]. Available from: https://inis.iaea.org/search/search.aspx?orig_q=RN:12611409 [Accessed: November 07, 2020]'},{id:"B13",body:'Loring DM, Meisburger EP IV. A discussion of radon and the mitigation strategy at the Henderson Mine. In: 13th North American Mine Ventilation Symposium. 2010. pp. 73-78'},{id:"B14",body:'Harteis SP, Dolinar DR. Water and slurry bulkheads in underground coal mines: Design, monitoring, and safety concerns. Mining and Engineering. 2006;58:41 Available from: https://me.smenet.org/abstract.cfm?articleID=1328&page=41'},{id:"B15",body:'Ajayi K, Shahbazi K, Tukkaraja P, Katzenstein K. Numerical investigation of the effectiveness of radon control measures in cave mines. International Journal of Mining Science and Technology. 2019;29(3):469-475. DOI: 10.1016/j.ijmst.2018.07.006'},{id:"B16",body:'Pan Y, Tukkaraja P. Experimental Investigation of Airflow Behavior in a Block Cave Mine. Journal of Mineral and Material Science. 2020;1(4). Available from: https://www.corpuspublishers.com/assets/articles/article-pdf-139.pdf'},{id:"B17",body:'Bhargava R, Tukkaraja P, Adhikari A, Sridharan SJ, Vytla VVS. Airflow characteristic curves for a mature block cave mine. In: Mine Ventilation. London: CRC Press; 2021. pp. 56-64'},{id:"B18",body:'Jha A, Pan Y, Tukkaraja P, Sridharan SJ. Scale model investigation of ventilation parameters in a block cave mine. In: Mine Ventilation. London: CRC Press; 2021. pp. 556-562'},{id:"B19",body:'Erogul D, Ajayi K, Tukkaraja P, Shahbazi K, Katzenstein K and Loring D, Effect of Airgap Geometry on Immature Panel Cave Resistance. 2017'},{id:"B20",body:'Karunakara N et al. Evaluation of radon adsorption characteristics of a coconut shell-based activated charcoal system for radon and thoron removal applications. Journal of Environmental Radioactivity. 2015;142:87-95. DOI: 10.1016/j.jenvrad.2014.12.017'},{id:"B21",body:'Yun X, McNamara K, Murdock G. Geotechnical challenges and strategies at McArthur River operation. Procedia Engineering. 2011;26:1603-1613. DOI: 10.1016/j.proeng.2011.11.2344'},{id:"B22",body:'Yun X, Tang B, Greg M, Brian M, Brian M. Radon bearing water protection in underground uranium mining – A case study. International Journal of Mining Science and Technology. 2017;27(4):599-603. DOI: 10.1016/j.ijmst.2017.05.013'},{id:"B23",body:'Abd Ali FS, Mahdi KH, Jawad EA. Humidity effect on diffusion and length coefficient of radon in soil and building materials. Energy Procedia. 2019;157. DOI: 10.1016/j.egypro.2018.11.203'}],footnotes:[],contributors:[{corresp:"yes",contributorFullName:"Purushotham Tukkaraja",address:"pt@sdsmt.edu",affiliation:'
Department of Mining Engineering, South Dakota Mines, Rapid City, SD, USA
Department of Mining Engineering, South Dakota Mines, Rapid City, SD, USA
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He also involved in more than 30 research projects in production optimization, rock mechanics, wellbore stability, well stimulation, cement and cementing, drilling and drilling fluid, biofuel and nanotechnology application in oil & gas.",institutionString:"University of Technology Malaysia",institution:{name:"University of Technology Malaysia",institutionURL:null,country:{name:"Malaysia"}}},{id:"120520",title:"Prof.",name:"Seyed Reza",surname:"Shadizadeh",slug:"seyed-reza-shadizadeh",fullName:"Seyed Reza Shadizadeh",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:null}]},generic:{page:{slug:"open-access-funding-funders-list",title:"List of Funders by Country",intro:"
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UK Research and Innovation (former Research Councils UK (RCUK) - including AHRC, BBSRC, ESRC, EPSRC, MRC, NERC, STFC.) Processing charges for books/book chapters can be covered through RCUK block grants which are allocated to most universities in the UK, which then handle the OA publication funding requests. It is at the discretion of the university whether it will approve the request.)
UK Research and Innovation (former Research Councils UK (RCUK) - including AHRC, BBSRC, ESRC, EPSRC, MRC, NERC, STFC.) Processing charges for books/book chapters can be covered through RCUK block grants which are allocated to most universities in the UK, which then handle the OA publication funding requests. It is at the discretion of the university whether it will approve the request.)
Wellcome Trust (Funding available only to Wellcome-funded researchers/grantees)
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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. 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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. 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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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Since then, he has been working as an Adjunct Professor in the same Department at the University of Pavia. His research activity during the first years was primarily focused on the purification and structural characterization of enzymes from animal and plant sources. During this period, Prof. Iadarola familiarized himself with the conventional techniques used in column chromatography, spectrophotometry, manual Edman degradation, and electrophoresis). Since 1995, he has been working on: i) the determination in biological fluids (serum, urine, bronchoalveolar lavage, sputum) of proteolytic activities involved in the degradation processes of connective tissue matrix, and ii) on the identification of biological markers of lung diseases. 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She gained considerable experience in developing and validating new methodologies whose applications allowed her to determine both the amount of biomarkers (Desmosine and Isodesmosine) in the urine of patients affected by COPD, and the activity of proteolytic enzymes (HNE, Cathepsin G, Pseudomonas aeruginosa elastase) in the sputa of these patients. Simona Viglio was also involved in research dealing with the supplementation of amino acids in patients with brain injury and chronic heart failure. She is presently engaged in the development of 2-DE and LC-MS techniques for the study of proteomics in biological fluids. The aim of this research is the identification of potential biomarkers of lung diseases. 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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. 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