La Dystrophie Facio-Scapulo-Humérale (FSHD) est la plus fréquente maladie neuromusculaire chez l'adulte (7 cas pour 100 000 naissances). D'origine autosomique dominante, elle apparait en général durant la seconde décade et se caractérise principalement par une perte de force et une fonte asymétrique des muscles du visage, des épaules et des bras puis progresse au reste du corps. L'analyse du motif répété D4Z4, dont le nombre de répétitions est diminué chez les patients, a mis en évidence la présence d'une phase ouverte de lecture codant pour un facteur de transcription appelé DUX4. Son expression a été retrouvée spécifiquement chez les patients FSHD. Mes travaux de thèse se sont concentrés sur l'étude de DUX4 et son implication dans la physiopathologie de la FSHD. Parce que la FSHD est une maladie progressive, nos analyses ont été faites sur des cultures de cellules et des biopsies f¿tales FSHD1 et Contrôles. Nous avons ainsi mis en évidence pour la première fois que DUX4 est exprimé dès le stade f¿tal et que les dérégulations géniques identifiées chez l'adulte sont également présentent à ce stade. De plus nos expériences semblent montrer que le niveau d'expression de DUX4 entre le f¿tus et l'adulte FSHD est similaire. Enfin, le niveau d'expression de DUX4 est extrêmement restreint (environs 0,5 à 10% des noyaux), mais son effet sur un grand nombre de gènes cibles est lui très fortement prononcé et quantifiable. Des essais de co-cultures ont ainsi mis en évidence que in vivo, DUX4 est à même de diffuser d'un noyau à l'autre au sein d'une fibre musculaire et de diffuser le phénomène pathologique. Nos travaux ouvrent ainsi un nouveau champ d'étude pour la compréhension des mécanismes physiopathologiques conduisant à la FSHD. / Facioscapulohumeral muscular dystrophy (FSHD) is inherited in an autosomal dominant pattern and is one of the most common muscular dystrophies (7/100 000). FSHD usually manifests in the second decade of life and includes an asymmetric wasting and weakness of facial, shoulder and arm muscles and is affecting the distal muscles in later stages of the disease. D4Z4 repetitions, which are known to be decreased in FSHD patients, comprise an open reading frame encoding a transcription factor called DUX4 that is only expressed in patients affected by FSHD. My PhD thesis project is aiming to better understanding of the role played by DUX4 in human skeletal muscle in order to elucidate its involvement in the pathophysiology of FSHD. As FSHD is a progressive disease, I studied DUX4 mRNA expression in both primary human fetal muscle cells and in fetal muscle tissue of control subjects and FSHD1 patients. For the first time, we were able to demonstrate DUX4 expression at the fetal stage, and in addition, we showed abnormal expression of various genes that has been reported to be altered in adult FSHD patients. Also, our experiments on fetus and adult FSHD patient cells suggested an equal expression of DUX4 protein. Although DUX4 protein is expressed at a very low level in patients (about 0.5 to 10% of the nuclei), it leads to a strong misexpression of a large number of DUX4 target genes. By performing co-cultures between C2C12 mouse myoblasts and control or FSHD human myoblasts, we demonstrated that expression of toxic DUX4 protein occurs only in a limited number of nuclei in FSHD patient cells. Interestingly, we revealed that the expressed DUX4 protein is able to spread from one nucleus into nearby nuclei within the myotubes and hence transmitting the molecular pathological abnormalities. Our research project will give us new insights into the pathophysiological mechanisms underlying FSHD.

The CD-ROM disc containing supplementary material is kept in the cardboard box in the Systems Office. / The heterogeneity of breast cancer (disease stage and phenotype) makes it challenging to differentiate between each subtype; luminal A, luminal B, HER2, basal-like and claudin-low, on the basis of a single gene or protein. Therefore, a collection of markers is required that can serve as a signature for diagnosing different types of breast cancer. New developments in proteomics have provided the opportunity to look at phenotype-specific breast cancer cell lines and stage-specific liquid biopsies (nipple aspirate fluid [NAF], plasma samples) to identify disease and phenotype specific signature. An 8-plex iTRAQ quantification strategy was employed to compare proteomic profiles of a range of breast cancer and ‘normal-like’ cell lines with primary breast epithelial cells. From this, 2467 proteins were identified on Orbitrap Fusion and Ultraflex II, of which 1430 were common. Matched pairs of NAF samples from four patients with different stages of breast cancer, were analysed by SCX-LC-MS and a total of 1990 unique gene products were identified. More than double the number of proteins previously published data, were detected in NAF, including 300 not detected in plasma. The NAF from the diseased patients have 138 potential phenotype biomarkers that were significantly changed compared to the healthy volunteer (7 for luminal A, 9 for luminal B, 11 for HER2, 14 for basal-like and 52 for claudin-low type). The average coefficient of variation for triplicate analyses by multiple reaction monitoring mass spectrometry (MRM-MS), was 9% in cell lines, 17 % in tissue biopsies, 22% in serum samples and 24% in NAF samples. Overall, the results provide a strong paradigm to develop a clinical assay based on proteomic changes in NAF samples for the early detection of breast cancer supplementary to established mammography programmes. / The supplementary material submitted with the thesis is not available online.

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La transplantation pulmonaire pour les patients avec une maladie pulmonaire en phase terminale est leur seul espoir de survie. Malheureusement, certains greffés du poumon rencontrent des difficultés après la transplantation du poumon, dont l'un est le rejet chronique du greffon pulmonaire également connu histologiquement comme la bronchiolite oblitérante et cliniquement comme syndrome de bronchiolite oblitérante. L'étiologie exacte de la BO reste mal comprise. Certaines hypothèses suggèrent l'implication des cellules épithéliales dans le processus de remodelage des voies respiratoires, conduisant à l'obstruction des voies aériennes. Un des mécanismes proposés est un processus de transition, connue sous le nom de transition épithéliale-mésenchymateuse (TEM). Lors de ce processus, les cellules perdent leurs propriétés épithéliales, acquièrent un phénotype mésenchymateux et deviennent plus mobiles et envahissantes. Cette transformation leur permet de participer activement au processus de remodelage bronchique dans la bronchiolite oblitérante. L’induction de la TEM peut être due à certains facteurs tels que l'inflammation et l'apoptose. Le principal objectif de ce travail de maîtrise est de détecter in vivo la présence de la TEM dans des biopsies transbronchiques obtenues chez des greffés et de l’associer à leurs conditions cliniques. Le deuxième objectif est d'induire la TEM in vitro dans les cellules épithéliales des petites voies aériennes à l'aide de milieux conditionnés apoptotiques et non apoptotiques produits par les cellules endothéliales microvasculaires humaines du poumon. D’autre part, nous avons évalué si des médiateurs connus pour participer au processus de TEM tels que le facteur de croissance du tissu conjonctif (CTGF)et le facteur de croissance transformant bêta (TGF-beta) ainsi que le perlecan sont présents dans les milieux conditionnés utilisés. / For patients with end-stage lung disease, lung transplantation is their only hope for survival. Unfortunately, some of the lung transplant recipients (LTRs) might face obstacles following lung transplantation, one of which is chronic lung transplant rejection also known as bronchiolitis obliterans (BO) histologically and bronchiolitis obliterans syndrome (BOS) clinically. The exact etiology behind BO development remains poorly understood. Speculations have suggested the involvement of epithelial cells in the airway remodeling process leading to airway obstruction. One of the proposed mechanisms is a transitional process, known as epithelial-mesenchymal-transition (EMT). In this process epithelial cells lose their properties and acquire mesenchymal ones causing them to be more mobile and invasive which allow them to take part of the airway remodeling process in BO. Induction of EMT can be due to several factors such as inflammation, apoptosis. In our study we try to detect in vivo the presence of EMT in transbronchial biopsies (TBB) obtained from LTRs and associates it with their clinical conditions. We also try to manipulate and induce EMT in vitro in small airway epithelial cells (SAEC) using conditioned apoptotic (SSC4h) and non apoptotic (ZVAD) media produced from human microvascular endothelial cells (HMVEC) from lung. In addition, we worked on detecting possible mediators such as connective-tissue growth factor (CTGF), transforming growth factor-beta (TGF-β), and perlecan in produced media.

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Background and Objective In stereotactic brain tumor biopsies, the combination of real-time fluorescence spectroscopy with the detection of microvascular perfusion using laser Doppler flowmetry provides an improved localization of the brain tumor while decreasing the risk of intra-cranial hemorrhage. The surgeon using the measurement probe is required to view signal values on a screen or usually, when her or his visual focus is directed at the patient, the verbal feedback of a biomedical engineer who is monitoring the measurement signals is needed. In this process possible important information can be overlooked and time is lost. The aim of the thesis was the development a visual and auditory user interface (UI) for use in stereotactic brain tumor biopsies. Materials and Methods The system translates the fluorescence intensity of protoporphyrin IX (PpIX) into sound and visual indicators that are easy and fast to recognize and transmits warning signals in case of signal error or the detection of microvascular perfusion. The increasing and de-creasing fluorescence values at tumor margins were reproduced to improve the precision of de-tecting varying fluorescence intensities when entering tumor tissue with color gradient models. The algorithm produced five signal values when specific fluorescence intensities were measured and compared at different wavelengths.For the development of the UI, a user-centered design was implemented. The user-, operating room- and safety requirements were gathered by communicating with the biomedical engineers and neurosurgeons who had experience in working with fluorescence guided brain tumor biop-sies. The requirements were considered when designing the UI’s features in LabVIEW and the auditory feedback was generated using OSC (Open Sound Control). The user interface intended to deliver measurement data to the user that triggered a high response accuracy by being easy to understand while inducing high user acceptance. The user interaction and function response accuracy of the visual and auditory interface were evaluated in statistical tests where operating room situations were mimicked. The user acceptance of the UI was evaluated. Results Signals for no, low (increasing and decreasing) and high fluorescence indicators, as well as two warning indicators for a blocked signal and vessel occurrence were represented visually and auditorially by the user interface. An intensity/time graph and intensity/wavelength graph, along with the option of recording measurement files and opening saved files allowed the inspec-tion of detailed measurement values. The user study exhibited auditory response accuracy of 95 ± 3% in the intuition test and 91±16% in a memory test. The testing of the response accuracy of the individual signal values displayed accurate responses in 84% to 100% of times a signal was played back. The user acceptance rating of the auditory and visual interface showed no negative results. Conclusion A UI was developed to visually and auditorially represent measurement values to a neurosurgeon performing a stereotactic brain tumor biopsy procedure and biomedical engineers monitoring the measurement signals. The visual display was successful in representing data in a way that was easy to understand. The auditory interface showed high response accuracies for the individual tones representing measurement values. The majority of the test subjects per-ceived the signals to be intuitive, easy to understand and easy to remember. The auditory and visual UI showed high user acceptance ratings, indicating that the user interface was useful and satisfactory in its application.

Early Effects of Castration Therapy in Non-malignant and Malignant Prostate Tissue BACKGROUND. Androgen ablation, the standard treatment for advanced prostate cancer, results in increased apoptosis, decreased cell proliferation and subsequent involution of the prostate gland. The mechanisms behind these responses are largely unknown, but effects in the prostatic epithelium are believed to be mediated by primary changes in the stroma. The purpose of this thesis was to investigate short-term cellular effects of castration-induced prostate tissue involution in mice and humans. METHODS. Prostate tissue factors affected by castration were investigated using cDNA-arrays, micro-dissection, RT-PCR, immunohistochemistry and Western blot analysis. The effects of local insulin-like growth factor-1 (IGF-1) administration were investigated in intact and castrated mice. Non-malignant and malignant epithelial and stromal cells were micro-dissected from human prostate biopsies taken before and within two weeks after castration treatment from patients with advanced prostate cancer. These tissue compartments were analyzed by RT-PCR and/or immunohistochemistry for IGF-1, IGF-1 receptor, androgen receptor (AR) and prostate specific antigen (PSA) expression. Treatment-induced changes in these factors were related to apoptosis and proliferation as well as to clinical data and cancer specific survival. RESULTS. Similar to our observations in mouse ventral prostate (VP), non-malignant and malignant human prostate tissues responded with increased epithelial cell apoptosis and decreased proliferation after androgen withdrawal. Also, the PSA mRNA levels were reduced within the first days after therapy both in non-malignant and malignant human prostate epithelial cells. However, neither of these changes was related to subsequent nadir serum PSA or to survival. Locally injected IGF-1 increased epithelial cell proliferation and vascular volume in intact but not in castrated mice. IGF-1 was found to be mostly, but not exclusively, expressed in the stroma, and it decreased rapidly after castration in both humans and mice. This decrease was, however, largely absent in prostate tumor stroma, and tumor stroma cells showed lower pre-treatment levels of AR than stroma surrounding normal epithelial glands. Furthermore, decreased levels of IGF-1 mRNA in the non-malignant and tumor stroma cells, and in tumor epithelial cells in response to castration, were associated with high levels of apoptosis in epithelial cells after therapy. CONCLUSIONS. In the prostate, IGF-1 may be an important mediator of stroma-epithelial cell interaction that is involved in castration-induced epithelial and vascular involution. Moreover, reduced AR in the tumor stroma may play an important role in prostate cancer progression towards androgen-independency, resulting in inadequate IGF-1 reduction and apoptosis induction in response to castration. Most primary tumors initially respond to castration with markedly decreased PSA synthesis and cell proliferation, and moderately increased apoptosis. Death due to metastatic disease is, however, still common, despite primary tumor regression. This may suggest that tumor cells in metastases respond differently to treatment than primary tumor cells, probably influenced by a different and possibly androgen-independent stroma. Further studies should test the hypothesis that the effect of castration therapy can be enhanced by simultaneous blocking of IGF-1 signaling.

Background: Tobacco smoking is the main cause of chronic obstructive pulmonary disease, COPD, but the mechanisms by which cigarette smoke induces COPD are still elusive. T lymphocytes have been implicated in the pathogenesis of the disease, but their role in the airway inflammation in COPD is not fully understood. The aim of this thesis was therefore to address T lymphocyte subsets and their activation in the airways of subjects with COPD, in comparison to smokers with normal lung function (S) and never smokers (NS). Methods: Subjects with moderate to severe COPD were recruited along with controls. They were all non-atopic and clinically stable, without any exacerbation during at least three months prior to inclusion. Only medication with short-acting β2-agonists and/or anti-cholinergic drugs was permitted. All subjects underwent bronchoscopy with endobronchial mucosal biopsy sampling as well as bronchial wash, BW, and bronchoalveolar lavage, BAL, collection. Biopsies were immunohistochemically stained for inflammatory cells and markers. BW and BAL fluids were prepared for differential cell counts. Soluble markers were measured in BW and lymphocyte subsets were determined in BAL using flow cytometry. Results: In biopsies, an increase in epithelial CD3+ and CD8+ cells was found in COPD, compared to NS. In BAL fluid, CD8+ cells were enhanced, whereas CD4+ cells were reduced in subjects with COPD and S, compared to NS. Furthermore, CD4+ and CD8+ cells were more activated both in COPD and S, in terms of increased expression of CD25, CD69 and HLA-DR. NKG2D-expressing CD8+ T cells in BAL fluid were enhanced in both COPD and S. CD4+CD25bright cells were upregulated in COPD and S, suggesting the presence of regulatory T cells. Further analyses of T cell subsets with the more specific markers for regulatory T cells, FoxP3 and CD127, indicated a smoking-induced expansion of non-regulatory T cells, which tended to normalize after smoking cessation in COPD. Currently smoking subjects with COPD still expressed high proportions of activated non-regulatory CD4+ T cells. The data on FoxP3 expression further indicated that the increase in CD25 expression in COPD and S was not only associated with the expansion of regulatory T cells. As CD127 expression is reported to be inversely associated with FoxP3, the data indicate the expansion of a non-regulatory CD25+ population in smokers and patients with stable COPD. The immunohistochemical staining for the NKG2D ligands MICA and MICB on epithelial cells was unchanged. Conclusion: The results of this thesis suggest a role for CD4+ and CD8+ T-cells in clinically stable COPD, indicating that T-cells are of importance in the long-term inflammatory response in COPD. Regardless of current smoking habits, activated CD8+ T lymphocytes were found to be increased in BAL fluid from subjects with COPD, suggesting that changes in CD8+ T cells are associated with a persistent immune response and, thus, of importance in COPD pathogenesis. In contrast, the expansion of non-regulatory CD25+CD4+ cells in BAL fluid seemed to be preferentially smoke-related. In summary, the data indicate that, among airway T cells, changes in CD8+ cells seem to be highly associated with COPD pathogenesis, whereas changes in CD4+ cells appear to be related to cigarette smoke-induced responses. Further, a non regulatory population of helper T cells was identified in BAL fluid of COPD patients, which may contribute to the persistent cytotoxic T cell responses.

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Ce travail avait pour objectif i) d’analyser les caractéristiques physiologiques et musculaires(déterminées d’après des biopsies) de rameurs poids légers entraînés, ii) de proposer une méthode de153calcul pour estimer de façon non-invasive la quantité de lactate accumulé dans l’organisme (QTLS) au cours d’un exercice épuisant sur ergomètre aviron d’après la modélisation de la cinétique lactique pendant la récupération et iii) d’explorer l’influence des caractéristiques musculaires, et de l’aptitude à échanger et à éliminer le lactate sur la capacité anaérobie des rameurs appréciée par la mesure du déficit maximal d’O2 cumulé (DMOC).Premièrement, les rameurs étudiés possédaient un rapport masse musculaire - masse corporelle élevé et leurs paramètres physiologiques et musculaires étaient caractéristiques des athlètes spécialisées en endurance.Dans un deuxième temps, nous avons démontré que QTLS était corrélé positivement à DMOC.Cette relation supporte notre hypothèse et confirme la cohérence de la méthode proposée pour calculer QTLS.Dans une dernière étude, les résultats ont démontré que DMOC était corrélée positivement à l’aptitude à éliminer le lactate. Cette dernière était également significativement corrélée à la densité capillaire et au contenu musculaire en MCT4, une protéine impliquée dans le cotransport lactate-proton à travers le sarcolemme. / The aim of this work was i) to analyse physiological and muscle characteristics (determinedfrom muscle biopsies) in trained lightweight oarsmen, ii) to propose a non-invasive method to estimatelactate accumulation in the organism (QTLS) using the blood lactate recovery kinetics in response to anall-out exercise on rowing ergometer and iii) to explore the influence of muscle characteristics andlactate exchange and removal abilities on the anaerobic capacity of our subjects determined from themeasurement of the maximal accumulated oxygen deficit (MAOD).Firstly, the studied oarsmen displayed an elevated muscle - body mass ratio and their muscleand physiological characteristics were typical of those of elite endurance athletes.Secondly, we showed that QTLS was positively correlated with MAOD. This relationshipsupports our hypothesis and reinforces the interest of our method to estimate QTLS.Finally, the results demonstrated that MAOD was positively correlated with the lactate removalability. This latter was also positively correlated with the capillary density and the muscle content ofMCT4, a protein involved in the cotransport of lactate and proton across the sarcolemma