Mesenchymal stem cells (MSCs) are promising candidates for treating diverse inflammatory disorders due to their capacity to be immunosuppressive. This phenotype is not present at baseline but develops in response to instructive cues. To date, clinical trials use cells grown in basic culture conditions, anticipating the cells will acquire a useful phenotype in response to in vivo cues. This strategy has failed to produce any FDA approved therapies, based on inconsistent efficacy. This thesis explores whether priming MSCs prior to administration can lead to a more uniformly therapeutic phenotype, and it details the design of an optimal in vitro priming regimen. Because interferon gamma (IFN-γ) is known to induce an anti-inflammatory state in MSCs, hypoxia can confer survival benefits, and both cues coexist in known situations of immune tolerance, we hypothesized dual IFN-γ/hypoxia priming would yield a superior immunosuppressive MSC therapy. We show that priming MSCs with hypoxia or IFN-γ alone improves their ability to inhibit T-cells in vitro, but combining these cues results in additive improvements. We next characterize the proteomic and metabolomic changes MSCs undergo when exposed to single or dual IFN-γ/hypoxia priming. While IFN-γ induces MSCs to suppress inflammation and fibrosis, hypoxia leads to cell adaptations to low oxygen, including upregulation of proteins involved in anaerobic metabolism, autophagy, angiogenesis, and cell migration. Dual priming results in additive effects, with many instances of synergy. Finally, we show initial evidence that dual primed MSCs are better able to inhibit disease progression in a mouse model of acute graft-vs-host disease (GvHD).

A osteoartrose (OA) de joelho é uma das doenças osteomioarticulares mais comuns no mundo, afetando 2693 em cada 100.000 mulheres e 1770 em cada 100.000 homens. Embora o treinamento de força (TF) seja amplamente recomendado para a melhoria das debilidades físicas encontradas em pacientes com OA, o uso de cargas entre 70-85% da força dinâmica máxima (FDM) pode ser limitado não somente pela dor, mas também pela própria etiologia da doença, representando uma limitação para esta prática. O treinamento de força associado à oclusão vascular (TFOV) baseia-se na execução do TF em intensidades entre 20 e 50% da FDM, combinado à oclusão do fluxo sanguíneo através do uso de torniquetes. Estudos têm mostrado que a magnitude das mudanças na força e massa musculares após um período de treinamento com esta técnica é similar as causadas pelo TF de alta intensidade (70-85% FDM) sem adição da oclusão vascular. O objetivo do presente trabalho foi investigar a eficácia da associação da oclusão vascular ao TF como modelo de intervenção não farmacológica para melhoria da dor, força muscular, funcionalidade e qualidade de vida em pacientes diagnosticadas com OA de joelho. Diante disso, 48 participantes mulheres foram randomicamente distribuídas em três grupos: treinamento de força de baixa intensidade (TFB), treinamento de força de alta intensidade (TFA) ou treinamento de força de baixa intensidade associado à oclusão vascular (TFOV) e receberam treinamento duas vezes por semana durante doze semanas. No período basal e após a intervenção, as pacientes passaram por avaliações físicas (testes de funcionalidade e força), responderam questionários de qualidade de vida e de dor (índice WOMAC \"Western Ontario and McMaster Universities Osteoarthritis Index\" e SF-36 \"The Short Form 36 Health Survey\") e exame de imagem da área da secção transversa (AST) do músculo quadríceps por meio de tomografia computadorizada. Durante o período de treinamento, quatro pacientes do grupo TFA foram excluídas do protocolo por dor no joelho. Após a intervenção, no WOMAC, apenas os grupos TFOV e TFB apresentaram diminuições significativas na dor (p=0,0358 e p=0,0044, respectivamente), nos demais domínios, o único grupo que apresentou diminuições significativas de escore foi o TFOV (rigidez: p=0,0167 e funcionalidade p=0,0358). Nos testes de funcionalidade, os grupos TFOV e TFA apresentaram aumentos significativos no desempenho do \"Timed-stands test\" (p=0,0251 e p=0,003), no \"Timed-up-and-go\" não foram encontradas melhoras significantes nos grupos. Com relação a força, apenas os grupos TFOV e TFA aumentaram significativamente os valores no leg-press (p<0,0001) e na extensão de joelhos (p<0,0001). Comportamento similar foi encontrado no aumento da AST, grupos TFOV e TFA apresentaram aumentos significativos (p<0,0001). A melhora de qualidade de vida foi significante nos três grupos quando analisamos a somatória dos domínios do WOMAC (TFOV: p=0,0173; TFA: p=0,0438; TFB: p=0,0301), porém o SF-36 não foi capaz encontrar melhoras significativas nos grupos. Dessa forma, concluímos que o TFOV apresenta-se como uma estratégia relevante e segura de intervenção não farmacológica para mulheres acometidas por OA sintomática de joelhos, constituindo um modelo de tratamento capaz de induzir adaptações funcionais e morfológicas de grande interesse para esta população / Osteoarthritis (OA) of the knee is one of the most common articular disease worldwide, affecting 100,000 women in 2693 and 1770 in every 100,000 men. Although strength training (ST) is widely recommended for improving the physical weaknesses found in patients with OA, using loads between 70-85% of maximal dynamic strength (MDS) can be limited not only by pain, but also by the own etiology of the disease, representing a limitation of this practice. Strength training associated with vascular occlusion (STVO) is based on the execution of the ST at intensities between 20 and 50% of MDS, combined with the occlusion of blood flow through the use of tourniquets. Studies have shown that the magnitude of changes in strength and muscle mass after a period of training this technique is similar to those caused by high-intensity ST (MDS 70-85%) without the addition of the vascular occlusion. The objective of this study was to investigate the efficacy of the combination of vascular occlusion to the ST as non pharmacologic intervention model for improving pain, muscle strength, functionality and quality of life in patients diagnosed with knee OA. Thus, 48 women participants were randomly divided into three groups: strength training low intensity (STL), strength training, high intensity (STH) or low-intensity strength training associated with vascular occlusion (STVO) and trained two times per week for twelve weeks. At baseline and after the intervention, the patients underwent physical assessments (tests of functionality and strength), answered questionnaires on quality of life and pain (WOMAC index \"Western Ontario and McMaster Universities Osteoarthritis Index\" and SF-36 \"The Short form 36 Health Survey \") and the cross section area (CSA) of the quadriceps muscle was assessed using computed tomography. During the training period the STH group, four patients were excluded from the protocol due to knee pain. After the intervention, the WOMAC, only the STVO and STL groups showed significant decreases in pain (p=0.0358 and p=0.0044, respectively), in other domains, the only group that showed significant decreases in score was the STVO (stiffness: p= 0.0167 and p = 0.0358 functionality). In functionality testing, the STVO and STH groups showed significant increases in performance \"Timed-stands test\" (p=0.0251 and p=0.003), the \"Timed-up-and-go\" were not significant improvements found in groups. Regarding strength, only the STVO and STH groups significantly increased values in leg press (p<0.0001) and knee extension (p<0.0001). Similar behavior was found in increased AST and STH STVO groups showed significant increases (p<0.0001). The improvement of quality of life was significant in all three groups when analyzing the sum of the domains of WOMAC (STVO: p=0.0173; STH: p=0.0438; STL: p=0.0301), but the SF-36 was not able to find significant improvements in groups. Thus, we conclude that the STVO presents itself as a relevant and safe strategy of non-pharmacological intervention for women suffering from symptomatic knee OA, constituting a model of treatment capable of inducing functional and morphological adaptations of great interest to this population

Pulmonary fibrosis is a common consequence of lung inflammation, leading to organ dysfunction and significant morbidity and mortality. Macrophages, through their diverse functions associated with polarisation status, play a role in lung homeostasis and alternatively activated (M2) macrophages have been associated with lung fibrosis. Prolyl hydroxylases (PHDs) are the main oxygen sensors and regulators of hypoxia inducible factors (HIFs). The PHD/HIF pathway is known to play a role in tissue inflammation and fibrosis, but their role in macrophage polarisation is not fully understood. Aim To study the role of the PHD/HIF pathway in macrophage polarisation and lung fibrosis, and specifically in Idiopathic Pulmonary Fibrosis (IPF). Hypothesis It was hypothesised that pan hydroxylase inhibition alters macrophage polarisation and modulates lung inflammation and fibrosis. Methods A combination of pharmacological (pan hydroxylase inhibitors DMOG and FG41) and genetic (HIF and PHD-null) tools were used to manipulate the PHD/HIF pathway. The bleomycin induced lung fibrosis model was used to define the effect of pan hydroxylase inhibition during the early, inflammatory or the late, fibrotic phase of this model. Murine bone marrow derived macrophages (BMDM), human monocyte derived macrophages and alveolar macrophages obtained from patients with lung fibrosis were used to study the effect of pan hydroxylase inhibition on macrophage polarisation. Bronchoalveolar lavage fluid (BALF) from patients was used to define the association between lung CCL18, an M2 associated chemokine, and disease progression in IPF. Results DMOG therapy during the early phase of the bleomycin model significantly reduced lung fibrosis at day 24. In contrast, late phase pan hydroxylase inhibition enhanced lung fibrosis at day 24. In both instances there was evidence of enhanced alveolar macrophage M2-like polarisation following pan hydroxylase inhibition. Reduced fibrosis after early pan hydroxylase inhibition was not a consequence of reduced acute lung inflammation or direct inhibition of collagen synthesis. In BMDM, pan hydroxylase inhibition resulted in an ‘augmented M2-like’ macrophage. Using LysM-Cre HIF-1α, HIF-2α and PHD-3 KO mice as well as chetomin, a potent inhibitor of HIF-1α and HIF-2α mediated gene expression, the HIF-dependent and HIF-independent polarisation markers were defined. PHD-3 deficiency was not sufficient to enhance M2 skewing. In contrast to murine BMDM, in human monocyte derived macrophages and alveolar macrophages from healthy volunteers and patients with interstitial lung disease including IPF, pan hydroxylase inhibition did not augment M2 polarisation and indeed significantly inhibited macrophage CCL18 expression. CCL18 studies in clinical BALF samples confirmed that CCL18 was elevated in the lungs of patients with IPF and other ILDs compared to controls. However, baseline BALF CCL18 concentrations did not correlate with disease severity or with disease progression, suggesting this is not a useful biomarker in IPF. Further, a unique study of serial BAL in IPF patients showed no association between 12-month change in CCL18 and disease progression over the same period. Indeed CCL18 concentrations mostly fell over 12 months in patients that did progress, strongly suggesting that CCL18 does not play a major pathogenic role in IPF. Concluding, it was shown that in both BMDM and murine lung pan hydroxylase inhibition promoted an ‘augmented M2-like’ polarisation. Pharmacological pan hydroxylase inhibition during the late fibrotic phase of injury enhanced fibrosis but it is not known if there was a causal association between M2 macrophages and lung fibrosis. Similarly, the functional relevance of finding enhanced M2 polarisation observed during early DMOG therapy, which subsequently resulted in attenuated fibrosis, is not known. In human macrophages, pan hydroxylase inhibition unexpectedly attenuated CCL18 production, a chemokine associated with an M2-like phenotype in man whilst other M2 markers were unchanged. However, there was no evidence to support a pathogenic role for CCL18 in IPF, and therefore there is little potential for using pan hydroxylase inhibition to target CCL18 and treat IPF.

Poor oxygenation of solid tumours has been linked with resistance to chemo- and radio-therapy and poor patient outcomes. Measuring the functional status of the tumour vasculature, including blood flow fluctuations and changes in oxygenation is important in cancer staging and therapy monitoring. A robust method is needed for clinical non-invasive measurement of the oxygen supply and demand in tumours. Current clinically approved imaging modalities suffer high cost, long procedure times and limited spatio-temporal resolution. Optoacoustic tomography (OT) is an emerging clinical imaging modality that can provide static images of endogenous haemoglobin concentration and oxygenation. In this work, an integrated framework for quantitative analysis of functional imaging using OT is developed and applied in vivo with preclinical cancer models. Oxygen Enhanced (OE)-OT is established here to provide insight into tumour vascular function and oxygen availability in the tissue. Tracking oxygenation dynamics using OE-OT reveals significant differences between two prostate cancer models in nude mice with markedly different vascular function (PC3 & LNCaP), which appear identical in static OT. OE-OT metrics are shown to be highly repeatable and correlate directly on a per-tumour basis to tumour vascular maturity, hypoxia and necrosis, assessed ex vivo. Dynamic Contrast Enhanced (DCE) OT demonstrates the relationship between OE-OT response and tumour perfusion in vivo. Finally, the possibility of using OT data acquired at longer wavelengths to report on tumour water and lipid content is investigated, with a view to future providing intrinsically co-registered imaging of tumour oxygenation and cellular necrosis. These findings indicate that OE-OT holds potential for application in prostate cancer patients, to improve delineation of aggressive and indolent disease, while combined with DCE-OT, it may offer significant advantage for localised imaging of tumour response to vascular targeted therapies. Further work is needed to establish whether OT can provide a new method to detect tumour necrosis in vivo.

Hypoxia (low tissue oxygenation) is an important signalling cue for many cell types. The study of its effects has direct relevance to surgery since hypoxic gradients are generated with every cut. On a cellular level, changes in molecular oxygen are sensed by the Hypoxia-Inducible Factors (HIFs). The HIFs are a family of transcription factors that are master regulators of over 100 genes and can effect changes in multiple cellular processes including migration, survival and differentiation. The broad nature of the response to hypoxia means that study of the HIF system is also important in cancer; where many tumour cells have found ways of subverting the HIF response to ensure their continued growth and survival. This thesis explores the role of hypoxia and the HIF system in the regulation of migration, survival and differentiation in both cancer and stem cells. The first experimental chapter examines the role of hypoxia and the HIF system in the regulation of migration and three-dimensional organisation in several cancer cell lines. Using biochemical and functional assays, the HIF system is shown to exert a pleiotropic effect across a panel of cancer cell lines. In particular, HIF 1α is shown to activate proliferation in a prostate cancer cell line in findings that may be useful to inform future clinical strategies for the management of this disease. In the second experimental chapter, the first epigenetic mechanism involving histone modification for the specific regulation of HIF 2α expression is characterised. Here the family with sequence similarity 60, member A (Fam60a) protein is shown to repress expression of the HIF 2α gene through its association with the class 1 Sin3-HDAC co-repressor complex, achieving specificity by co-operation with the SP1 transcription factor. This novel mechanism is demonstrated to be important in the regulation of the basal expression of HIF 2α. Modification of HIF 2α expression through this mechanism is shown to alter cell migration, three dimensional organisation and angiogenesis in vitro. The clinical importance of these findings is demonstrated in a series of 45 patients suffering from colorectal cancer of known stage. In this cohort, the reciprocal relationship between Fam60a and HIF 2α is maintained, and both are identified as potential novel biomarkers for the development of this disease. In the final experimental chapter, the role of hypoxia in the regulation of differentiation is explored. These effects are documented in mesenchymal progenitors primarily derived from human fat. Here, hypoxia is shown to regulate differentiation in a context-dependent manner, promoting osteogenic and retarding adipose and neural differentiation in-vitro. The roles of Fam60a and HIF 2α are explored in this system. These data may be useful in optimising future surgical engraftment of these cells for regenerative purposes.

Chiu, Wui Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 107-128). / Abstracts in English and Chinese. / Abstract --- p.i / 中文摘要 --- p.iv / Acknowledgements --- p.vi / Publications --- p.viii / Table of Contents --- p.ix / List of Tables --- p.xiv / List of Figures --- p.xv / List of Abbreviations --- p.xviii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Hypoxic-ischemic encephalopathy in human infants --- p.1 / Chapter 1.1.1 --- Incidence --- p.1 / Chapter 1.1.2 --- Biphasic development of HI brain damage --- p.2 / Chapter 1.1.2.1 --- Initiating mechanism: energy failure in immature brain --- p.3 / Chapter 1.1.2.2 --- Biochemical cascades --- p.4 / Chapter 1.1.2.2.1 --- Excitatory amino acid receptor activation by glutamate --- p.4 / Chapter 1.1.2.2.2 --- Intracellular calcium accumulation --- p.4 / Chapter 1.1.2.2.3 --- Formation of free radicals --- p.5 / Chapter 1.1.2.2.3.1 --- Reactive oxygen species (ROS) --- p.5 / Chapter 1.1.2.2.3.2 --- Nitric oxide (NO) --- p.6 / Chapter 1.1.2.3 --- Release of inflammatory mediators --- p.6 / Chapter 1.1.2.4 --- Mitochondrial dysfunction --- p.7 / Chapter 1.1.2.5 --- Final path to death: necrosis or apoptosis --- p.8 / Chapter 1.1.2.6 --- Ways to change: neuronal survival and proliferation signaling --- p.8 / Chapter 1.1.3 --- Interventions for neonatal hypoxia-ischemia --- p.9 / Chapter 1.2 --- Animal models mimicking hypoxia-ischemia brain injury --- p.12 / Chapter 1.2.1 --- Comparisons of animal models of hypoxia-ischemia --- p.12 / Chapter 1.2.2 --- Development of neonatal rat model with hypoxic-ischemic damage --- p.14 / Chapter 1.3 --- Neural stem/progenitor cells --- p.15 / Chapter 1.3.1 --- Effect of hypoxic-ischemia on neural stem/progenitor cells --- p.17 / Chapter 1.4 --- Thrombopoietin --- p.18 / Chapter Chapter 2 --- Objectives --- p.23 / Chapter Chapter 3 --- Materials and Methodology --- p.24 / Chapter 3.1 --- Establishment of neonatal rat model of HI brain damage and effects of TPO on neural protection --- p.24 / Chapter 3.1.1 --- Animal protocols --- p.24 / Chapter 3.1.2 --- Induction of HI brain damage in neonatal rats --- p.24 / Chapter 3.1.3 --- Treatment with TPO --- p.25 / Chapter 3.1.4 --- Sacrifice of rats --- p.25 / Chapter 3.1.5 --- Read-out measurements --- p.26 / Chapter 3.1.5.1 --- Brain weight --- p.26 / Chapter 3.1.5.2 --- Gross injury assessment of the right hemisphere --- p.26 / Chapter 3.1.5.3 --- Histology --- p.27 / Chapter 3.1.5.4 --- Blood cell count --- p.27 / Chapter 3.1.5.6 --- Functional assessments --- p.28 / Chapter 3.1.5.6.1 --- Grip traction test --- p.28 / Chapter 3.1.5.6.2 --- Elevated body swing test --- p.28 / Chapter 3.1.5.7 --- Statistical analysis --- p.28 / Chapter 3.2 --- Establishment of in vitro model of primary mouse NSPs and the effect of TPO on their proliferation --- p.29 / Chapter 3.2.1 --- Mouse embryo dissection for the extraction of NSP --- p.29 / Chapter 3.2.2 --- Culturing of NSP --- p.30 / Chapter 3.2.3 --- Immunofluorescence staining for stem cell markers --- p.31 / Chapter 3.2.4 --- Neurosphere assay with different combinations of mitogens --- p.31 / Chapter 3.2.5 --- Neurosphere assay with different concentrations of TPO --- p.32 / Chapter 3.2.6 --- Neurosphere assay under hypoxia --- p.32 / Chapter 3.2.7 --- Statistical analysis --- p.33 / Chapter Chapter 4 --- Effects of thrombopoietin on neonatal rat models of hypoxia-ischemia brain damage --- p.39 / Chapter 4.1 --- Summary of experimental settings --- p.39 / Chapter 4.2 --- Results --- p.39 / Chapter 4.2.1 --- Mortality --- p.39 / Chapter 4.2.2 --- Effects of TPO on p7 mild damage model 1 week post-surgery --- p.40 / Chapter 4.2.2.1 --- Body and brain weights --- p.40 / Chapter 4.2.2.2 --- Gross injury score --- p.41 / Chapter 4.2.2.3 --- Cortex and hippocampus area --- p.41 / Chapter 4.2.2.4 --- Blood cell counts --- p.42 / Chapter 4.2.3 --- Effects of TPO on p7 severe damage model 1 week post-surgery --- p.43 / Chapter 4.2.3.1 --- Body and brain weights --- p.43 / Chapter 4.2.3.2 --- Gross injury score --- p.43 / Chapter 4.2.3.3 --- Cortex area --- p.44 / Chapter 4.2.3.4 --- Blood cell counts --- p.44 / Chapter 4.2.4 --- Effects of TPO on p7 severe damage model 3 week post-surgery --- p.45 / Chapter 4.2.4.1 --- Body and brain weights --- p.45 / Chapter 4.2.4.2 --- Gross injury score --- p.46 / Chapter 4.2.4.3 --- Blood cell counts --- p.46 / Chapter 4.2.4.4 --- Functional outcomes --- p.46 / Chapter 4.2.5 --- Effects of TPO on pl4 severe damage model 1 week post-surgery --- p.47 / Chapter 4.2.5.1 --- Body and brain weights --- p.47 / Chapter 4.2.5.2 --- Gross injury score --- p.48 / Chapter 4.2.5.3 --- Cortex area --- p.48 / Chapter 4.2.5.4 --- Blood cell counts --- p.49 / Chapter 4.3 --- Discussion --- p.49 / Chapter Chapter 5 --- Effects of thrombopoietin on the proliferation of primary mouse neural stem/ progenitor cells in culture --- p.83 / Chapter 5.1 --- Summary of experimental settings --- p.83 / Chapter 5.2 --- Results --- p.83 / Chapter 5.2.1 --- Effect of EGF or bFGF withdrawal on NSP proliferation --- p.84 / Chapter 5.2.2 --- Dose effect of TPO treatment on NSP proliferation --- p.85 / Chapter 5.2.3 --- Effect of hypoxia --- p.85 / Chapter 5.2.4 --- Effect of TPO treatment in combination with hypoxia --- p.86 / Chapter 5.2.5 --- Detection of neural progenitor cell marker --- p.87 / Chapter 5.3 --- Discussion --- p.88 / Chapter Chapter 6 --- General discussion --- p.101 / Bibliography --- p.106

Ascent to altitude presents a significant challenge to the human body. Specifically, it is associated with an increased ventilation and pulmonary vasoconstriction. In healthy subjects these are related such that a high ventilatory drive is associated with blunted pulmonary vasoconstriction. Adults born prematurely and given supplemental oxygen at birth have a blunted ventilatory response to hypoxia. We hypothesized that the hypoxic ventilatory and pulmonary vasoconstrictor responses would be unrelated following perinatal supplemental oxygen exposure. To test our hypothesis, we used a well-established rat model of 80% O2 (80%) exposure for 14 days post-natally, with 21% O2 exposure as a control (21%). We assessed the ventilatory response to graded hypoxia using barometric plethysmography 6-9 months post hyperoxia exposure. The left and right ventricles were catheterized to evaluate the hemodynamic response to 10 minutes of 12% O2 (hypoxia). To our surprise we found that 80% animals did not demonstrate a depressed ventilatory response to hypoxia. However, these animals experienced increased right ventricular systolic pressure in response to 12% O2. An increase in cardiac output was the primary driving force behind the increase in right ventricular end systolic pressure, not an increase in vascular resistance. We found no relationship between the hypoxic ventilatory drive and right ventricular pressure. In 21% animals exposed to hypoxia, the increase in right ventricular pressure was driven primarily by vasoconstriction and, as previous studies have shown, there was a relationship between the ventilatory and pressure responses. These data suggest that neonatal supplemental oxygen alters the hemodynamic response to hypoxia, possibly through enhanced sympathetic drive. The relationship between ventilation and pulmonary pressure may not translate to individuals born prematurely.

Plusieurs travaux s’accordent sur le fait que l’analyse de l’ADN circulant (ADNcir) est un outil à fort potentiel diagnostic, pronostic, théranostic et de suivi en oncologie clinique. Cependant, le manque de standardisation des procédures pré-analytiques, des connaissances approfondies des structures des ADNcir ainsi que les facteurs influençant la dynamique de son relargage constituent des obstacles à son transfert en pratique clinique. C’est dans ce contexte que nous avons choisi d’approfondir l’étude des structures des ADNcir d’origines nucléaire et mitochondriale, évaluer leur utilité clinique en oncologie et étudier l’effet de l’hypoxie sur leur libération. A partir d’une technique de qPCR optimisée et validée cliniquement pour l’analyse des ADN circulants, nous avons quantifié les ADN extracellulaires d’origines nucléaire et mitochondriale dans des milieux de culture cellulaire et dans des plasmas humains et murins.Nos données ont révélé l’influence des procédures pré-analytiques sur la quantification de l’ADN circulant en fonction de l’origine. En effet, nous montrons que la préparation du plasma par séparation en ficoll diminue de manière significative la quantification de l’ADNcir d’origine mitochondriale sans influencer la quantification de l’ADNcir d’origine nucléaire, ce qui suggère la présence de structures de densité différente contenant de l’ADN mitochondrial dans le plasma. D’autre part, j’ai contribué à l’évaluation d’un test de dépistage du cancer basé sur l’analyse de l’ADNcir nucléaire et mitochondrial mise au point au laboratoire. Nos résultats préliminaires montrent de façon significative la capacité diagnostic de ce test sur des cohortes de patients atteints de cancers colorectal (n = 127) versus des individus sains (n = 91) (AUC = 0,8657 ; p < 0,0001).Nous montrons in vitro que l’hypoxie module de manière différente le relargage des ADN extracellulaires en fonction de leur origine, et notamment que l’ADN extracellulaire d’origine mitochondriale semble être régulé négativement en condition hypoxique. In vivo, l’hypoxie entraîne une augmentation de la libération de l’ADNcir d’origine nucléaire (p=0,002), mais pas d’origine mitochondriale, dans le plasma de souris greffées avec des cellules tumorales de cancer de poumon.Pour conclure, les travaux réalisés au cours de cette thèse mettent en lumière : (i) l’intérêt de la mise en place de procédures pré-analytiques standardisées pour la compréhension des origines et structures des ADNcir ; (ii) le fort potentiel diagnostic de l’analyse de l’ADNcir en oncologie ; (iii) et l’influence de l’hypoxie sur le relargage de l’ADN circulant. / Different studies converge on the diagnostic, theragnostic and prognostic properties of circulating DNA analysis (cirDNA) in clinical oncology. There remain various obstacles, however, to its transfer to clinical practice. These include the lack of standardization of pre-analytical procedures, and limited knowledge of cirDNA structures and of the factors influencing the dynamics of their release. In this context, we studied the structures of cirDNA of nuclear and mitochondrial origins, evaluated their diagnostic potential, and then evaluated the effect of hypoxia on their release. Using an optimized qPCR technique previously validated in clinical studies for the analysis of cirDNA, we quantified extracellular DNA of nuclear and mitochondrial origins in cell culture medium and in human and mouse plasma.Our data also revealed the influence of pre-analytical procedures on the quantification of cirDNA, depending on its origin. Indeed, we showed that plasma preparation with Ficoll separation significantly reduces the quantification of mitochondrial cirDNA without influencing the quantification of nuclear cirDNA.In addition, we evaluated the value of nuclear and mitochondrial cirDNA analysis in a cancer-screening test developed in the laboratory. Our preliminary results demonstrated a significant discrimination between colorectal cancer patients (n=127) and healthy individuals (n=91) (AUC=0.8657; p<0.0001).We demonstrated that in vitro hypoxia modulates the release of extracellular DNA in different ways, depending on its origin, and showed that extracellular DNA of mitochondrial origin is negatively regulated. By contrast, we demonstrated in vivo that hypoxia leads to a greater release of nuclear cirDNA (p=0.002), but not of mitochondrial cirDNA, as compared to normoxia. These experiments were performed using the plasma of mice grafted with lung cancer tumor cells.In conclusion, the work carried out for this thesis highlights: (i) the importance of setting up standardized pre-analytical procedures when investigating the origins and structures of cirDNA; (ii) the strong diagnostic potential of cirDNA analysis in oncology and (iii) the influence of hypoxia on the release of cirDNA.

The lungs represent one of the earliest interfaces for pathogens and noxious stimuli to interact with the body. As such, careful maintenance of the permeability barrier is vital in providing homeostasis within the lung. Essential to maintaining this barrier is the tight junction, which primarily acts as a paracellular seal and regulator of ionic transport, but also contributes to establishing cell polarity, cell-to-cell integrity, and regulating cell proliferation and differentiation. The loss of these tight junctions has been documented to result in alterations in inflammation, and ultimately the development of many respiratory disorders such as COPD, Asthma, ARDS, and pulmonary fibrosis. One critical contributor that creates this permeability barrier is the tight junctional protein Claudin. While studies have begun to elucidate the various functions and roles of various Claudins, our understanding is still limited. To initially investigate these proteins, we looked at both temporal and spatial expression patterns for family members during development. A consistent pattern was demonstrated in mRNA expression for the majority of Claudin members. In general, Claudin expression underwent rapid increase during time periods that correlate with the pseudoglanduar/canalicular periods. One notable exception was Claudin 6 (Cldn6), which demonstrated decreasing levels of mRNA expression throughout gestation. We also sought to understand expression dynamics during the addition of maternal secondhand smoke (SHS) which resulted in an almost universal decrease in Claudin proteins. To more fully explore expression mechanisms that affect Claudin-6 (Cldn6), we exposed pulmonary alveolar type II (A549) cells to cigarette smoke extract (CSE) and found that it transcriptionally regulated Cldn6 expression. Using a luciferase reporter, we determined that transcription was negatively regulated at multiple promoter response elements by CSE, and transcription was equally hindered by hypoxic conditions. These findings identified Cldn6 as a potential target of SHS and other respiratory irritants such as diesel particulate matter (DPM). We next sought to assess whether an increase in Cldn6 was sufficient to provide a protective advantage against harmful exogenous exposure. To test this, we utilized a doxycycline induced Cldn6 over-expressing mouse, and subjected it to SHS for 30 days to stimulate an inflammatory state. Our findings demonstrated that Cldn6 transgenic animals have decreased inflammation as evidence by decreased total cell infiltration into the airways, decreased polymorphonuclocyte (PMNs) extravasation, total protein in bronchoalveolar lavage fluid (BALF), and decreased cytokine secretion. Anti-inflammatory advantages were also discovered during experiments involving acute exposure to DPM. In both cases, while stimulation of transgenic mice with SHS or DPM diminished Cldn6 expression, anti-inflammatory evidence emerged suggesting that genetic up-regulation of Cldn6 likely causes the recruitment of other tight junctional components during an organism's response to environmental assault.

The Louisiana (LA) shelf is chronically affected by seasonal hypoxia that has been shown to be spatially expanding and growing progressively more severe. Hypoxic conditions on the shelf have been closely linked to the large quantities of nutrients delivered to the Gulf of Mexico via the Mississippi River. Multiple geochemical proxies on a suite of sediment cores from the LA shelf provide a record of environmental change that parallels the onset of hypoxic conditions over the last century and prior to anthropogenic influences. The sedimentary record for the last century shows a shift from terrestrial to algal sources to the sediments, as well as a trend of increasingly enriched nitrogen isotopes, which is probably caused by a combination of large amounts of denitrification, either within the Mississippi watershed or on the continental shelf, increased primary productivity, compounded with relatively minor increases of enriched nitrogen source inputs. The current chronic hypoxia, especially since the early 1970's, is exacerbated by anthropogenic nutrient loading from the Mississippi River basin, but other processes must be responsible in the past. In the historic record, several episodic low-oxygen events are defined by forminiferal assemblages and shifts in the geochemical records. Geochemistry of three sediment cores from the Louisiana (LA) shelf indicates that these historic events are likely caused by increased inputs of terrestrially-derived organic matter during peaks in Mississippi River discharge. These results suggest that low-oxygen conditions may be a natural, aperiodic phenomenon on the LA shelf, and that the current seasonally severe hypoxia resulted from the excess anthropogenic nutrient input.