A disease has consequences not only for the afflicted person but also for those who interact with him or her. A low-grade glioma is a brain tumour whose regarding its psychosocial implications for adult patients and their next of kin has received little attention in the literature. In the light of this the overall aim of the present thesis was to provide increased knowledge about how patients with low-grade glioma and their next of kin experience and deal with everyday life. The methods of the studies were mainly qualitative. Thirty-nine patients and 28 next of kin were interviewed and all except one next of kin completed a quality of life questionnaire. The onset of low-grade glioma was described from the patients’ perspective as a process, either rapid (up to a few months) or prolonged over several years. This phase of low-grade glioma encompassed repeated visits to physicians and care institutions. The onset of low-grade glioma was accompanied by stress, anxiety and uncertainty in the case of both the patients and those nearest. The symptoms and problems the patients experienced covered a broad range of consequences, physical, psychological and social. The patients presented a wide range of ways to cope with illness-related problems. The next of kin were often deeply involved in the patients’ situation and many of them experienced extremely stressful emotions mainly in the early period of the illness. They had experience of positive encounters in health care but more often they had had a sense both of powerlessness and of being invisible and neglected. Relations and roles changed in ways that mostly were experienced as negative. Enabling strength in everyday life had to do with alleviation of strain and having a positive outlook upon life. By means of the questionnaire Subjective estimation of Quality of Life (SQoL) the patients and those nearest estimated their quality of life as being comparatively high. Only one variable, among the patients the absence of work/meaningful occupation and among the next of kin the absence of own children, being estimated at below 60% of the maximum score.

Mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) both belong to the group of mature B-cell malignancies. However, MCL is typically clinically aggressive while the clinical course of CLL varies. CLL can be divided into prognostic subgroups based on IGHV mutational status and into multiple subsets based on closely homologous (stereotyped) B-cell receptors. In paper I we investigated 31 MCL cases using high-density 250K single-nucleotide polymorphism arrays and gene expression arrays. Although most copy-number aberrations (CNAs) were previously reported in MCL, a novel deletion was identified at 20q (16%) containing the candidate tumor suppressor gene ZFP64. A high proliferation gene expression signature was associated with poor prognosis, large CNAs, 7p gains and 9q losses. Losses at 1p/8p/13q/17p were associated with increased genomic complexity. In paper II we sequenced exons 4 to 8 of the TP53 gene in 119 MCL cases. 17p copy-number status was known from previous studies or determined by real-time quantitative polymerase chain reaction. TP53 mutations were detected in 14% of cases and were strongly associated with poor survival while 17p deletions were more common (32%) but did not predict survival. In papers III and IV we applied high-resolution genomic 27K methylation arrays to 20 MCL and 39 CLL samples. In paper III MCL displayed a homogenous methylation profile without correlation with the proliferation signature whereas MCL was clearly separated from CLL. Gene ontology analysis revealed enrichment of developmental genes, in particular homeobox transcription factor genes, among targets methylated in MCL. In paper IV we compared three different stereotyped CLL subsets: #1 (IGHV unmutated), #2 (IGHV3-21) and #4 (IGHV mutated). Many genes were differentially methylated between each two subsets and immune response genes (e.g. CD80 and CD86) were enriched among genes methylated in subset #1 but not in subsets #2/#4. In summary, CNAs were frequent and not random in MCL. Specific CNAs correlated with a high proliferation gene expression signature or genomic complexity. TP53 mutations predicted short survival whereas 17p deletions did not. A high proliferation signature was not associated with differential DNA methylation in MCL, which demonstrated a homogeneous methylation pattern. In contrast, genomic methylation patterns differed between MCL and CLL and between stereotyped CLL subsets.

Colorectal cancer (CRC) remains a significant burden in contemporary society due to an aging population, unhealthy dietary choices and an increasingly sedentary lifestyle. While the underlying defects for many hereditary forms of CRC have been determined, many genetic and epigenetic changes promoting common sporadic CRCs have yet to be identified. The Mutated in Colorectal Cancer (MCC) gene, identified in 1991, was initially thought to be responsible for the hereditary form of CRC, familial adenomatous polyposis, before the discovery of the susceptibility gene Adenomatous Polyposis Coli (APC), which then became the focus of intense research. Recent data, however, suggests that MCC may also be important in the development of CRC. I have investigated the mechanism of MCC gene silencing, the putative structure, and multiple functions of MCC. MCC was frequently silenced by promoter hypermethylation in CRC cell lines and primary tumours. MCC methylation showed strong molecular and clinicopathological associations with hallmarks of the serrated neoplasia pathway. Furthermore, MCC methylation was more frequent in serrated precursor lesions compared with adenomas, thus occurring early during carcinogenesis. MCC is highly conserved in complex multicellular organisms. Re-introduction of MCC in CRC cell lines resulted in partial G1 to S phase, and G2/M phase cell cycle blocks, potentially by upregulating cell cycle inhibitor gene transcription and interfering with the process of mitotic checkpoints and division, respectively. Changes in MCC levels also modulated NF?B pathway signalling, the pathway required for maintaining cell viability and proliferation in colonic epithelial cells. In particular, MCC overexpression suppressed both TNF? and LPS-induced NF?B activation, decreasing both the magnitude and rate of cellular responses. Overexpression also resulted in downregulation of proteins involved in canonical NF?B pathway signalling, while increasing the transcription of non-canonical NF?B genes. Therefore, MCC may direct activation of this pathway to a specific subset of NF?B-regulated genes. These data provide a molecular basis for the role of MCC as a tumour suppressor gene in CRC. MCC may have multiple functions, regulating cell cycle progression and modulating NF?B pathway signalling, either through direct involvement in pathway signalling cascades, or by providing a scaffold on which signalling events can occur.

Infectious diseases are increasingly being recognised as significant threatening processes in conservation biology. Developing strategies to effectively manage infectious diseases in wildlife is, therefore, of the utmost importance to the maintenance of global biodiversity. The effective management of infectious diseases relies on understanding the ecology of the host, the epidemiological characteristics of the pathogen and the impacts of the pathogen on the host population. However, for most wildlife-disease systems this information remains poorly understood. This is particularly true for endangered species threatened by novel infectious agents as opportunities to observe and assess disease impacts and host-pathogen dynamics in the wild are limited. The Tasmanian devil (Sarcophilus harrisii), the world’s largest carnivorous marsupial, is threatened with extinction as a result of an epidemic of an emerging disease, a fatal infectious cancer known as Devil Facial Tumour Disease (DFTD). In this thesis I capitalised on a unique dataset from a population of Tasmanian devils where disease arrived part-way through an intensive longitudinal study, and utilised existing genetic samples collected prior to DFTD outbreak, to determine the impact of DFTD on the demography, population dynamics, genetic diversity and population genetic structure of wild Tasmanian devils. I then used this knowledge of the impacts of DFTD impacts in an unmanaged population to evaluate the effectiveness of a disease management trial involving the selective culling of infected individuals. I employed mark-recapture models to investigate the impact of DFTD on age-specific and sex-specific apparent survival rates, to examine the pattern of variation in infection rates (force of infection), and to investigate the impact of DFTD on population growth rate. I investigated demography, life-history traits and morphometric parameters of infected and uninfected individuals to determine the impacts of DFTD on age-structure and sex-structure, female fecundity and individual growth rates. I used this information to assess the population’s ability to respond to low population densities and to compensate for the detrimental impacts of DFTD. To determine the genetic consequences of disease-induced population decline I used microsatellite DNA to compare genetic diversity, population genetic structure and dispersal patterns in three Tasmanian devil populations prior to and following DFTD outbreaks. Capture-mark-recapture analyses revealed that the arrival of DFTD triggered an immediate decline in apparent survival rates of devils, the rate of which was predicted well by the increase in disease prevalence in the population over time. Transition rates of healthy individuals to the diseased class (the force of infection) increased in relation to disease prevalence, while the arrival of DFTD coincided with a marked and ongoing decline in the population growth rate. There was a significant change to the age structure following the arrival of DFTD. This shift to a younger population was caused by the loss of older individuals as a direct consequence of DFTD-driven declines in adult survival rates. Evidence of reproductive compensation in response to these disease impacts was observed via a reduction in the age of sexual maturity of females over time. However, widespread precocial breeding in devils was precluded by physiological and ecological constraints that limited the ability of one year olds to breed. Using temporally-replicated spatial genetic data, I found evidence of increased inbreeding following DFTD arrival and greater population genetic differentiation in post-disease populations. These changes appeared to be driven by a combination of selection and altered dispersal patterns of females in DFTD-affected populations. Comparison of demographic and epidemiological parameters indicative of disease progression and impact between the managed and unmanaged populations revealed that selective culling of infected individuals neither slowed the rate of disease progression nor reduced the population level impacts of this debilitating disease; with culling mortality simply compensating for disease mortality. This thesis provides one of the few direct empirical evaluations of the impact of an emerging wildlife disease epidemic on a wild population. This thesis revealed that infectious diseases can result in major demographic and genetic changes in host populations over relatively few generations and short time-scales. Results showing dramatic and ongoing population declines and very limited population compensation in DFTD-affected populations indicate that DFTD poses a significant extinction risk for wild devil populations. Hence, this study confirms that host-specific pathogens can pose a significant extinction risk for wild species, even in the absence of alternate reservoir hosts, a finding critical to our understanding of host-pathogen dynamics. My thesis also highlights the potential negative interplay between disease susceptibility and host genetic variability, which is of utmost importance to the management of novel wildlife epizootics and the conservation of threatened wildlife in general. The thorough understanding of the ecology and impacts of DFTD in the wild obtained in this study has provided a solid base from which to both rigorously assess the outcome of management strategies and also formulate recommendations for the management of this disease in the wild. The lack of evidence for successful control of the DFTD epidemic in a wild population during the first phase of a selective culling experimental adaptive management approach, points to the need to implement a multi-faceted disease management program when attempting to control a novel infectious disease in the wild. By drawing on the lessons learnt in this case study I show that it is possible to establish a set of general guidelines for the future management of infectious diseases in threatened wildlife.

Name: Anna Jadwiga Kielkowska Dissertation title: The role of PTEN as a PI(3,4)P2 lipid phosphatase in Class I phosphoinositide 3-kinase signalling Abstract Class I phosphoinositide 3-kinases (Class I PI3Ks) are essential players involved in the signalling events in the cell and are critical promoters of cellular growth, survival and metabolism. Once activated by environmental stimuli such as growth factors, cytokines or antigens, they exert their catalytic activity by phosphorylating phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2) to yield a second messenger - PI(3,4,5)P3. Unrestrained PI(3,4,5)P3 signalling has been classically associated with hyperactivation of the Class I PI3K/AKT pathway and has been shown to be a molecular trigger of many pathophysiologies in humans, including autoimmune disorders, respiratory diseases and cancer. To date, two classes of lipid phosphatases SHIP1/2 and PTEN have been reported, which dephosphorylate PI(3,4,5)P3 on positions 5’ and 3’ of the inositol ring to generate PI(3,4)P2 and PI(4,5)P2 respectively, and thus quench Class I PI3K signalling. Moreover, PI(3,4)P2 levels in the cell are regulated by two important lipid 4-phosphatases - INPP4A/B. While the role of PTEN as a tumour suppressor is well established, functions of SHIP1/2 and INPP4A/B are just starting to emerge. A major barrier to progress in this field has been the lack of high quality measurements of PI(3,4)P2, to assess the impact it may have on shaping cellular behaviour. This dissertation summarises the work performed to develop a novel, HPLC-ESI MS/MS based method, in order to measure the product of PI(3,4,5)P3 5-dephosphorylation, PI(3,4)P2, separated from its more abundant regioisomer in cells - PI(4,5)P2. This and an existing HPLC-ESI MS/MS method for measuring PI(3,4,5)P3, have enabled us to describe the fluxes through Class I PI3K-controlled PI(3,4,5)P3 generation and its subsequent 3- and 5- dephosphorylation pathways in human mammary epithelial cells (Mcf10a) stimulated with epidermal growth factor (EGF). By means of genetic suppression of PTEN and INPP4B, we revealed an unexpectedly high level of PI(3,4)P2 that accumulates in EGF-stimulated PTEN-INPP4B-KO Mcf10a cells. Further, an in vitro biochemical assay suggested a novel role for PTEN as a direct PI(3,4)P2 3-phosphatase in Mcf10a cells. This important observation was supported by in sillico phosphatidylinositol lipid modelling of the relevant pathways. In an effort to understand its potential physiological significance, we demonstrated that PI(3,4)P2 accumulation correlates with the ability of genetically modified Mcf10a cells to form gelatin-degrading invadopodia. Finally, we used a mouse prostate cancer model to show PTEN’s importance in controlling PI(3,4)P2 levels in vivo, pointing to a potential role for PI(3,4)P2 in PTEN-dependent tumourigenesis. I hope that the work described in this dissertation will contribute to the current knowledge of phosphatidylinositol lipid biology in the context of Class I PI3K signalling and will simulate future efforts to gain an in-depth understanding of the roles of PTEN and PI(3,4)P2 in cellular physiology.

This study aimed to investigate the pharmacology of selected pyrrolobenzodiazepine (PBD) compounds shown to have cytotoxic activity with predicted DNA sequence selectivity. Research focused upon the PBD dimer, SJG-136, selected for clinical trials, and the novel PBD monomer compounds D709119, MMY-SJG and SJG-303. SJG-136, a novel sequence-selective DNA minor groove cross-linking agent, was shown to have potent tumour cell type selective cytotoxicity in in vitro assays. Pharmacokinetic studies in mice via both the i.p. and i.v. route (dosed at the maximum tolerated dose (MTD)) showed that SJG-136 reaches concentrations in plasma well in excess of the in vitro IC50 values for 1 h exposure, and was detected in tumour and brain samples also above the in vitro IC50 values. Furthermore, SJG-136 showed linear pharmacokinetics over a 3-fold drug dose range. Metabolism studies showed SJG-136 is readily metabolised in vitro by hepatic microsomes, predominantly to a monodemethylated metabolite; this metabolite could be detected in vivo. Analytical method development work was also conducted for the imminent Phase I clinical trial of SJG-136 resulting in a sensitive and selective bio-analytical detection protocol. Comet analysis showed that SJG-136 dosed at the MTD and ⅓MTD causes significant interstrand DNA cross-linking in lymphocytes in vivo. In vitro studies demonstrated that SJG-136 localises within the cell nucleus, and acts to disrupt cell division via a G2/M block in the cell cycle at realistic concentrations and exposure times that are achievable in vivo. In vivo pharmacokinetic studies of D709119 showed the compound is easily detectable in mouse plasma following i.p. dosing at the MTD, but could not be detected in either tumour or brain samples. In vitro cytotoxicity studies revealed D709119 to have potent activity across a selection of tumour cell lines. SJG-136, D709119, MMY-SJG, SJG-303 and DC-81 demonstrated a non-enzyme-catalysed reactivity with the biologically relevant thiol, reduced glutathione (GSH). Studies demonstrated that reactivity of the PBD compounds toward GSH was dependent on GSH concentrations. At levels of GSH found in plasma, the PBD compounds showed considerably lower reactivity with GSH than at intracellular GSH levels. SJG-136 and D709119 also showed favourable pharmacokinetic profiles in mice, and warrant further study for anti-tumour activity in vivo and progression to use in patients.

<b>Background:</b> Systemic chemotherapy and radiotherapy play an important role in the treatment of colorectal cancer. Tumour perfusion and oxygenation is known to influence radiosensitivity and chemosensitivity. In this thesis, I propose that the evaluation of changes in tumour perfusion using perfusion CT (pCT) and dynamic contrast-enhanced (Dce) MRI can guide the rational sequencing of drugs and radiation. <b>Methods:</b> Dce-MRI and pCT scans were incorporated into a clinical trial of hypofractionated pelvic radiotherapy and nelfinavir in 10 patients with rectal cancer. Toxicity and tissue biomarkers (tumour cell density, microvessel density, CAIX, HIF1-alpha, phospho-Akt and phospho-PRAS40) were evaluated. pCT liver scans were incorporated into an imaging study in patients with colorectal liver metastases randomised to receive either oxaliplatin/ 5FU chemotherapy or oxaliplatin/ 5FU chemotherapy plus selective internal radiotherapy. <b>Results:</b> After 7 days of nelfinavir concurrent with hypo-fractionated pelvic radiotherapy, there was a mean 42&percnt; increase in median K^trans (P=0.03, paired t test) on Dce-MRI and a median 30&percnt; increase in mean blood flow on pCT (P=0.028, Wilcoxon Rank Sum), although no statistically significant changes in perfusion parameters were demonstrated after 7 days of nelfinavir prior to radiotherapy. The feasibility of evaluating tumour cell density in rectal biopsies before and after radiotherapy and a radiosensitising drug as an early endpoint of response was demonstrated. In patients with colorectal liver metastases who received oxaliplatin and modified de Gramont chemotherapy alone, after 4 cycles of chemotherapy, a 28&percnt; decrease in the mean hepatic arterial fraction was observed (P=0.018, paired t test). Between pCT scans 2 days before SIRT and 39-47 days following SIRT and continued 2-weekly chemotherapy, there was a mean 62&percnt; (P=0.009) reduction in Blood Flow and 61&percnt; (P=0.006) reduction in Blood Volume (paired t test). <b>Conclusions</b> This research does not support the hypothesis that nelfinavir before radiotherapy improves blood flow to human rectal cancer. Increases in rectal tumour perfusion during radiotherapy and concurrent nelfinavir are likely to be primarily explained by the acute biological effects of radiation. Four or more cycles of oxaliplatin and modified de Gramont chemotherapy may result in changes in tumour perfusion of colorectal liver metastases which would be detrimental to subsequent radiotherapy. Selective internal radiotherapy resulted in substantial reductions in tumour perfusion 39-47 days after the treatment. Perfusion imaging can be used to detect changes in tumour perfusion in response to radiotherapy and systemic therapy which have implications for the sequencing of therapies.

Les gliomes sont les principales tumeurs primitives du cerveau affectant environ 4000 nouveaux patients par an en France. La moitié des gliomes est détectée au stade avancé de glioblastome (grade IV) tandis que 15% des tumeurs sont diagnostiquées au stade II de gliomes diffus dit de bas grade. Ces tumeurs affectent des patients jeunes et présentent des mutations caractéristiques, notamment une mutation pour l’enzyme IDH1 communément retrouvée dans les glioblastomes secondaires. Ces tumeurs de bas grade sont traitées par une chirurgie, idéalement en condition éveillée mais du fait de leur nature diffuse, la partie résiduelle progressera inexorablement vers un stade III ou IV avec une survie globale entre 5 ans et 15 ans après diagnostique. La progression tumorale est hautement variable et non prédictible d’un patient à l’autre. Des foyers de progression tumorale chez 20% des patients atteints de gliome diffus de bas grade ont été identifiés. Ces foyers montrent une densité cellulaire plus élevée ainsi qu’un Ki67 augmenté. Mon travail de thèse aura consisté à étudier les modifications cellulaires et moléculaires associées à ces foyers de progression tumorale. À partir du profil ARN des foyers et des territoires adjacents, j’ai pu mettre en évidence par des techniques haut-débit la baisse d’expression significative de gènes dans les foyers notamment de AGXT2L1/ETNPPL, carboxypeptidase E, EDNRB, SFRP2. J’ai émis l’hypothèse que SFRP2 et ETNPLL pourraient s’opposer à la prolifération cellulaire et que leur diminution dans les foyers ouvrirait la voie à la transformation tumorale. Une corrélation inverse entre la quantité d’ETNPPL enzyme et la survie de patients atteints d’hépatocarcinomes a été publiée. En limitant la quantité de précurseurs de phospholipides dans la cellule, ETNPPL pourrait agir comme un frein en s’opposant à la prolifération et de fait, sa diminution dans les foyers de transformation des gliomes pourrait lever cette inhibition. Mes travaux auront été innovants tant dans leur approche comparative des différents compartiments tumoraux pour chaque patient étudié et auront révélés ETNPPL comme corrélé à la gliomagenèse et potentielle cible thérapeutique. / Gliomas are the main primary brain tumours affecting around 4000 new patients in France each year. Half of gliomas are detected in the advanced stage of glioblastoma (grade IV) while 15% of tumours are diagnosed in stage II (diffuse low-grade gliomas-DLGG). These tumors affect young patients and bear characteristic mutations, including a mutation for the enzyme IDH1 commonly found in secondary glioblastomas. These low-grade tumours are treated by surgery, ideally in awake condition but due to their diffuse nature, the residual part will progress inexorably to stage III or IV with overall survival between 5 and 15 years after diagnosis. Tumor progression is highly variable and unpredictable from one patient to another. Foci of tumor progression have been identified in 20% of patients with DLGG. These foci show a higher cell density and an increased Ki67. My thesis work consisted in studying the cellular and molecular changes associated with tumor progression. From the RNA profile of the foci and adjacent territories, I was able to highlight through high-throughput techniques significant decrease in gene expression in the foci, particularly of AGXT2L1/ETNPPL, carboxypeptidase E, EDNRB, SFRP2. I hypothesized that SFRP2 and ETNPLL could oppose cell proliferation and that their decrease would pave the way for tumor transformation. An inverse correlation between the amount of ETNPPL and the survival of patients with hepatocarcinoma has been published. By limiting the amount of phospholipid precursors in the cell, ETNPPL could act as a brake against proliferation and indeed, its decrease in glioma transformation foci could remove this inhibition. My PhD work will have been innovative in the comparative approach of the different tumors’ compartments for each patient studied and will have revealed ETNPPL as correlated to gliomagenesis and as potential therapeutic target.

Largely as a consequence of the ongoing obesity epidemic, research into adipose tissue biology has increased substantially in recent years. Worldwide, the number of people classed as overweight or obese is growing, and this represents a major public health concern. Adipose tissue is broadly divided into two types; white and brown. Whilst white adipose tissue (WAT) functions to store and mobilise triglycerides, brown adipose tissue burns chemical energy to generate heat. WAT is further divided into visceral “bad” fat and subcutaneous “good” fat depots, and it is an increase in the former that is linked to obesity-associated diseases. As well as adipocytes, several other cell types including haematopoietic and endothelial are found within adipose tissue, and comprise the stromal vascular fraction (SVF). Adipocyte precursor cells (APCs) also reside within the SVF and are essential for the maintenance and expansion of adipose tissue. The protein encoded by the Wilms’ tumour 1 (Wt1) gene is predominantly known to function as a transcription factor, but also has a role in post-transcriptional processing. Deletion of Wt1 in adult mice results in a considerable loss of fat tissue. Moreover, recent work has revealed that a proportion of the APCs from all visceral WAT depots express Wt1, therefore revealing heterogeneity within the APC population. Additionally, visceral WAT depots are encapsulated by a WT1 expressing mesothelial layer, which has its origins in the lateral plate mesoderm (LPM), and can give rise to mature adipocytes. Lineage tracing has demonstrated that a significant proportion of the mature adipocytes in all adult visceral WAT depots (but not subcutaneous) are derived from cells that express Wt1 in late gestation. These findings uncovered key ontogenetic differences between visceral and subcutaneous WAT and led us to ask whether Wt1 functions in visceral adipose tissue biology. Preliminary work has shown that adipocytes derived from Wt1 expressing (Wt1+) precursor cells have fewer, larger lipid droplets than those derived from non-Wt1 expressing (Wt1-) precursors. In this thesis, this heterogeneity is explored further using a Wt1GFP/+ knock-in mouse. When Wt1+ and Wt1- APCs are cultured separately, the Wt1+ population differentiate into adipocytes more readily. Moreover, the Wt1+ APCs are more proliferative than the Wt1-. Preliminary results also suggest that the Wt1+ APCs may secrete a factor(s) that causes the Wt1- APCs to exhibit improved adipogenic differentiation, a result that is supported by data from comparative transcriptomic analysis. Finally, the percentage of APCs decreases when mice are fed a high fat diet. Interestingly, this decrease is more pronounced for the Wt1+ population. Therefore, it appears that as well as exhibiting differing behaviours in vitro, the Wt1+ and Wt1- populations respond differently to physiologically relevant conditions in vivo. Whilst the LPM is a major source of visceral WAT, the origin of subcutaneous WAT is currently unknown. Here, the Prx1-Cre and Prx1-CreERT2 mouse lines are used to investigate this. It is shown that the majority of subcutaneous WAT adipocytes and APCs are labelled by Prx1-Cre, however this is not the case for most of the visceral WAT depots. The exception to this is the pericardial (heart fat) depot, in which approximately 70% of the adipocytes and 40% of the APCs are labelled. Moreover, a proportion of the Prx1-Cre labelled pericardial APCs also express Wt1, therefore suggesting additional heterogeneity. Preliminary results show that this heterogeneity may have functional consequences, at least in vitro. Additionally, lineage tracing studies suggest that the somatic LPM may be one source of subcutaneous WAT and pericardial visceral WAT Finally, it is shown that the conditional deletion of Wt1 in the Prx1-Cre lineage results in abnormal diaphragm development. Congenital diaphragmatic hernia (CDH) is severe birth defect, the etiology of which is not well understood. Here, a new model of CDH has been developed, and the cellular and molecular mechanisms responsible for the defect in this model are investigated.

Le travail réalisé durant cette thèse a eu pour objectif de développer un modèle théorique spécifiquement dédié au contexte du développement tumoral tel qu'il peut être observé sous une fenêtre dorsale implantée sur une souris. Le modèle développé est un modèle hybride multi-physique et multi-échelle qui couple deux modules principaux. Le premier module modélise la croissance tumorale par un automate cellulaire qui permet de différencier l'état de chaque cellule en fonction de son histoire (cycle cellulaire), et de son environnement (espace disponible pour proliférer, présence d'oxygène). Le second module modélise le réseau vasculaire et le flux sanguin et rend compte de l'angiogenèse (apparition de nouveaux vaisseaux) et de l'adaptation du diamètre des vaisseaux, en fonction de l'évolution des contraintes hémodynamiques, nettement visible sous la fenêtre dorsale. L'ensemble des processus diffusifs (diffusion de l'oxygène et des facteurs de croissance vasculaire) sont décrits par des équations aux dérivées partielles, couplées à des automates cellulaires qui permettent de localiser à chaque instant pour chaque équation les termes sources (production) et les termes puits (consommation) pour chaque entité diffusive. Les simulations numériques réalisées montrent dans quelle mesure il est possible de rendre compte des observations expérimentales sur le plan qualitatif, qui nécessite la neutralisation des biais numériques ; et sur le plan quantitatif, pour reproduire la cinétique de croissance tumorale et l'évolution de la densité vasculaire. Le modèle numérique de l'évolution tumorale sous fenêtre dorsale est ensuite utilisé pour tester les effets de deux types de molécules : cytotoxiques et anti-vasculaires. Les simulations numériques de ces deux types de traitement explorent différents protocoles, définis par le mode d'action de la molécule, la dose administrée et la fréquence d'administration. Les résultats montrent comment il est alors possible de définir un protocole optimum pour une tumeur donnée en direction d'une individualisation de la thérapie. Ce modèle intégré a permis de poser de façon satisfaisante les bases d'un clone numérique du modèle expérimental d'évolution tumorale sous fenêtre dorsale même si certains aspects nécessitent encore quelques améliorations. La validation des aspects thérapeutiques restera encore à accomplir avant de pouvoir envisager à terme le remplacement (au moins partiel) de l'animal par l'ordinateur. / The work realised during this thesis had for objective to develop a theoretical model dedicated to the context of tumour development as observed on a dorsal skinfold chamber on a mouse. The model developed is hybrid, multi-physic and multi-scale, and associate two main modules. The first module model tumour growth with a cellular automaton which permit to differentiate the state of each cell regarding its history (cell cycle), its environment (available space to proliferate, oxygen availability). The second module model vascular network and blood flow, and accounts for angiogenesis (apparition of new vessels) and diameter adaptation of vessels, regarding hemodynamical constraints evolution which is distinctly visible on dorsal chamber. The diffusive processes (oxygen diffusion and vascular growth factors) are described by partiel differential equations, coupled with cellular automata which permit to localize at each time for each equation the source terms (production) and the well terms (consumption) for each diffusive entity. The numerical simulations realised show in which regard it is possible to accounts for the experimental observations on the qualitative basis, which require numerical bias neutralisation; and on the quantitative basis, to reproduce tumour growth kinetic and evolution of vascular density. The numerical model of tumour evolution on dorsal chamber is then used to test the effects of two types of molecules: cytotoxic and anti-vascular. Numerical simulation of these two types of treatment explore different protocols, defined by the action mode of the molecule, the dose administrated, and the administration frequency. Results show how it is possible to define an optimum protocol for a given tumour in direction of therapy individualisation. This integrated model has permitted to put in place in a satisfactory way the bases of a numerical clone of the experimental model of tumour growth on dorsal chamber, even if several aspects still necessitate some improvements. The validation of these theoretical aspects has yet to be accomplished before considering in term the replacement (at least partiallly) of animals by computers.