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Les médulloblastomes (MB) sont les tumeurs cérébrales les plus fréquentes en pédiatrie. Ils apparaissent le plus souvent au niveau du cervelet. Ils peuvent être stratifiés en quatre groupes : les groupes WNT et SHH, où ces voies de signalisation sont altérées, et les groupes 3 et 4, présentant des anomalies chromosomiques et amplifications multiples, dont c-Myc (groupe 3) et N-Myc (groupe 4). L’une des altérations génétiques les plus retrouvées dans les MB est la surexpression du facteur de transcription OTX2. Ce facteur est exprimé dans les précurseurs des cellules granulaires (GCP) du cervelet, cellules d’origine de la majorité des MB. Pendant la période périnatale, les GCP subissent une phase de prolifération très intense en réponse au mitogène Sonic Hedgehog (SHH), ce qui les rendrait particulièrement sensibles à la tumorigenèse. Au cours de cette thèse, nous nous sommes intéressé à la fonction d’Otx2 dans ces GCP. Nous avons montré que l’ablation conditionnelle d’Otx2 conduit à un défaut de prolifération des ces cellules. L’analyse approfondie de ce phénotype a permis de révéler qu’Otx2 stimule la prolifération des GCP parallèlement à la voie de signalisation Shh. Par ailleurs, l’ablation d’Otx2 dans un modèle murin mimant la formation de MB Shh-dépendants a montré qu’Otx2 s’avère indispensable pour leur maintien à long terme. En parallèle, nous avons tenté de créer un nouveau modèle murin mimant la formation de MB de groupe 3 en induisant l’expression, pendant la période postnatale, d’un dominant actif de c-Myc dans les cellules exprimant Otx2. Cette approche a donné des résultats inattendus : des carcinomes de plexus choroïdes, et non des MB, ont été obtenus. / Medulloblastomas (MB) are the most common brain tumors in paediatrics. They appear during development in the posterior part of the brain, mainly in cerebellum. MB can be stratified in four groups: the WNT and SHH groups, where these signalling pathways are aberrantly activated, and the groups 3 and 4, which display chromosomal abnormalities and multiple amplifications, including c-Myc (group 3) and N-Myc (group 4). One of the most frequent genetic alterations in MB is the overexpression of the Otx2 transcription factor (in 75% of cases). This factor, which is essential for central nervous system development, is expressed in granule cell precursors (GCP) of the cerebellum, which represent the cell of origin of the majority of MB. During the perinatal period, GCPs undergo intense proliferation in response to Sonic Hedgegog (SHH), making them particularly susceptible to tumorigenesis. During this thesis, we were interested in examining the function of Otx2 in GCPs. We have shown that conditional ablation of Otx2 leads to a GCP proliferation defect and that Otx2 stimulates the proliferation of these cells independently of the Shh signaling pathway. Moreover, ablation of Otx2 in a mice model of Shh-dependent medulloblastomas yielded very interesting results: while Otx2 does not seem to be required for the initiation of these tumors, it is essential for their long-term maintenance. In parallel, we tried to create a new murine model for the MB group 3 by inducing the expression, during the postnatal period, of an active dominant of c-Myc in cells expressing Otx2. This approach yielded unexpected results: choroid plexus carcinomas, instead of MB, were obtained.

Mouse strains are frequently used to model human disease states, to test the efficiency of drugs and therapeutic principles. However, the direct translation of murine experimental data to human pathological events often fails due to sufficient differences in the organization of the immune system of both species. Here we give a short overview of the principle differences between mice and humans in defense strategies against pathogens and mechanisms involved in response to pathogenic microorganisms and other activators of the immune system. While in human blood mechanisms of immune resistance are highly prevailed, tolerance mechanisms dominate for the defense against pathogenic microorganisms in mouse blood. Further on, species-related differences of immune cells mainly involved in innate immune response as well as differences to maintain oxidative homeostasis are also considered. A number of disease scenarios in mice are critically reflected for their suitability to serve as a model for human pathologies. Due to setbacks in these studies, novel mouse models were created to bridge the immune system of both species: humanized mice. Accordingly, a special section of this review is devoted to new results applying humanized mouse models taking limitations and prospects into account.

To understand the pathophysiology of hearing loss, it is necessary to identify genes responsible for embryonic development of neurosensory cells in the inner ear. The aim of this work is to clarify the role of LIM-homeodomain transcription factor ISL1 in the development of these cells. Using Cre-loxP recombination strategy, we generated a mouse line with time and site- specific deletion of Isl1 gene in NEUROD1-Cre expressing cells (Isl1 CKO). Although the early development of stato-acoustic ganglion was not affected by Isl1 deletion, at E14,5, we observed abnormalities in neuronal migration, formation of spiral ganglion and axon guidance in the Isl1 CKO cochlea. The length of the cochlear sensory epithelium was shortened by 20% as a consequence of lower proliferation activity of sensory precursor cells. Our results suggest that ISL1 is necessary for spiral ganglion formation and innervation of the Organ of Corti. Key words: transcription factor ISL1, neurons, Cre-loxP system, mouse model

Parasitic protists of genera Trypanosoma and Leishmania are members of Trypanosomatidae family. In our studies, we investigated genetic influence on infections caused by these parasites in a mouse model. These diseases are on genetic level controlled by quantitative trait loci (QTLs), when the resulting phenotype is controlled by set of genes with small individual effect. As a mouse model for mapping of QTLs controlling these infections, we used recombinant congenic strains (RCS). Each RCS carry unique set of 12.5% of the genome from donor parental strain on genetic background of other parental strain. For mapping of QTLs controlling infections caused by Trypanosoma brucei brucei (T. b. brucei) and Leishmania tropica (L. tropica) and eosinophil infiltration into inguinal lymph nodes after Leishmania major (L. major) infection, we used RCS from CcS/Dem series, where STS is donor strain and BALB/cHeA is strain of genetic background. First, it was necessary to find suitable model strains for mapping. In all three studies, we selected RCS, which exceeded range of monitored phenotype parameters in comparison with any other tested RCS or parental strains. Mice of RCS CcS-11 showed shorter survival after T. b. brucei infection and strain CcS-9 exhibited higher eosinophil infiltration after L. major infection. For...

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1 Abstract Diabetes mellitus is characterized by the dysfunction and reduction of insulin-producing cells, resulting in hyperglycemia, which in long term harms the organism. For future therapy, it is crucial to understand the function of various factors participating in the differentiation and maturation of endocrine pancreatic cells. The aim of this study was to unravel the functional role of ISL1 during the development of the pancreas. ISL1 is expressed in all endocrine cells of the islets of Langerhansbut its function remains unclear, especially during early pancreatogenesis. As the global deletion of this gene is embryonically lethal, we used the tissue specific deletion of Isl1 in Neurod1 possitive cells using the Cre-loxP system. In this work we studied the effect of this deletion on the structure of islets of Langerhans, the formation of endocrine cell types and relative expression of genes during early pancreatic development. A defective achitecture of islets together with postnatal absence of α-cells was found in the Isl1 deletion mutant. Also, the expression of genes important for the specification of α-cell lineage and their subsequent function was decreased. The secondary outcome was the optimalization of a protocol for effective sorting of endocrine cells using fluorescent flow cytometry, which...

Gastric cancer is the second leading cause of cancer related deaths and the fifth most common malignancy worldwide. The prognosis of gastric cancer is often poor. Frequently, the lack of clinical signs lead to a delayed diagnosis with three quarters of patients presenting with non-curable advanced disease. The only curative option is surgery, supported in recent years by perioperative chemotherapy. However, known molecular alterations represent possibilities for targeted therapies to improve overall survival. Nevertheless, biomarkers to predict therapy response are missing, resulting in several failed clinical trials for targeted drugs. Organoids are a recently developed three-dimensional culture system derived from different sources, i.e. adult tissue stem cells, embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC). While in ESC or iPSC derived organoids a functional niche is present that maintains stem cells, this niche is missing in adult stem cell derived organoids and needs to be replaced by a definite medium containing the relevant growth factors. Organoids have the ability of proliferation, self-renewal and self-organization. They show a comparable functionality of the organs they are derived from. In sum, organoids are valuable tools to study diseases on a patient level. In this work, we focused on the characterization of gastric cancer by using human and mouse cancer organoids. Firstly, a human gastric cancer organoid biobank was established. The patient derived organoid lines were characterized concerning their molecular profile, treated with classical chemotherapeutics and mutation specific targeting was performed. The generated human cancer organoids showed a high similarity to the tissue they were derived from and allowed a detailed analysis of observed alterations for each individual patient. However, the high number of mutations effected targeted therapies and needed to be interpreted in the whole mutation spectrum of each specific organoid line. In order to establish organoids with defined mutations for in depth analysis of pathway interference, we decided to combine inducible alleles of frequently altered signaling pathways in gastric cancer in mice and derived organoids of the stomach. These organoid lines were further analyzed by their morphology, functionality and drug response. Successful interference with activated pathways demonstrated their potential usefulness as living biomarkers for therapy response testing. In order to analyze gastric cancer in vivo a stomach specific mouse model was established. Intensive literature and database research resulted in the identification of Annexin10 (Anxa10) as potential stomach specific gene which at the same time is expressed in all different cell types of the stomach epithelium. We therefore generated an inducible Cre recombinase mouse line under the Anxa10 promotor. The Anxa10 CreERT2 line showed only stomach specific recombination events and no restriction to a specific cell type. Nevertheless, activation of Cre resulted in a patchy recombination pattern throughout the whole gland and not a uniform recombination in all cells. Due to this patchy expression, the mouse line is an optimal tool for cancer models, where a complete transformation of an organ is not desired. On the other side it is not useful, if a complete knock-out of a certain floxed allele is needed. This new stomach specific mouse line was then used to model gastric cancer subtypes in vivo. Frequently altered pathways and hotspot mutations of each gastric cancer subtype were defined based on the TCGA database. Alterations were mainly found in the following pathways: RTK/RAS, PI3K/AKT, WNT, TGF β, cell adhesion and chromatin remodelling. We generated and analyzed three different mouse models: one for the chromosomal instability (CIN) subtype and two for the genomically stable (GS) subtype. The different models mimicked very closely the histology of known human gastric cancer subtypes. The intestinal CIN model with mutations in Kras, Smad4 and Tp53 developed tumors with glandular and tubular structures showing morphologies to human intestinal type gastric cancer. The first GS model with alterations in Kras, Cdh1 and Smad4 showed cancers with a diffuse tumor cell morphology with the presence of typical signet ring cells. The second GS model with Kras, Cdh1 and Apc alterations showed similarities to the adenomatous tooth like gastric cancer subtype. Taken together, this study demonstrates that gastric cancer organoids might serve as living biomarkers to predict therapy response and resistance in individual patients. Additionally, the generated gastric cancer mouse model is to our knowledge the first model initiating tumor formation exclusively in the stomach with similar characteristics as described for human gastric cancer. This mouse represents a prime tool for further gastric cancer research.

Idiopathic Pulmonary Fibrosis (IPF) is an interstitial lung disease with no known cause or cure. IPF has an incidence of 75/1,000,000 of the population, predominately in men over the age of 60. This relatively rare disease develops in a chronic and progressive way, ultimately leading to death within two to five years of diagnosis. Our use of translatable methodologies in the bleomycin mouse model of IPF led to the novel identification of the similarities between the average percent loss of lung function in previous human clinical trials to that of our mouse model data. There is no treatment for IPF outside of lung transplantation, therefore our goal is to develop a protein therapy to halt the progression of IPF. B6_BP_dslf is a small, 93.36 kDa minibinder protein with a nanomolar affinity to αvβ6, an integrin of therapeutic potential for IPF when inhibited by halting αvβ6/TGF-β signaling. Our hypothesis is that B6_BP_dslf will halt the progression of pulmonary fibrosis induced in a mouse model of IPF. To test this hypothesis, a de novo design method was used resulting in the B6_BP_dslf minibinder having high β unit selectivity and nanomolar affinity for αvβ6, and maintenance of its secondary structure after aerosolization. These attributes led to testing in the bleomycin mouse model for IPF as an inhaled therapy. We found that B6_BP_dslf inhalation by mice with induced pulmonary fibrosis had reduced pathogenesis through the quantification of biomarkers for αvβ6/TGF-β mediated fibrosis, lowered histopathological scores, and improved lung function. These positive results from standard biochemical analysis and clinically translatable methods show that BP_B6_dslf has clinical potential as an inhaled therapy for IPF. Additionally, we tested the use of lung function tests in an animal model of chronic obstructive pulmonary disease (COPD), using secondhand smoke exposure to induce the disease and to identify inflammatory pathways. We found that smoke exposure increased inflammatory signaling through receptors for advanced glycation end-products, and inhibition of these receptors using a novel therapy of semi-synthetic glycosaminoglycan ethers (SAGEs) reduced inflammation and improved lung function. Together, the data from two different lung disease models supports the use of lung function as a preclinical efficacy variable for experimental drugs. The combination of biochemical and functional assessments of B6_BP_dslf and SAGEs gives weight to their therapeutic potential.

<p>The PRLs are a subfamily of dual specificity phosphatases that appear to play important roles in oncogenesis. Much of the current understanding of PRL function has been either correlative, and deduced from observed PRL overexpression in pathological conditions, or from in vitro analysis of signaling pathways following PRL deletion or overexpression. Such studies, necessitated by the general lack of synthetic inhibitors or compounds to probe the substrate specificity and biological interactions of the PRLs, are nonetheless now providing critical insight into potential biological substrates and roles of the PRL phosphatases. The recent identification of PTEN as a substrate for PRL2 provided the foundation for studies to further define the role of PRL2 in oncogenesis and, by analogy, the normal physiological function of PRL2. In the studies described herein, a novel PRL2 conditional knock-out animal was generated and used to validate the PRL2/PTEN interaction in a leukemic phenotype, and further demonstrated that PRL2 inhibition can restore dysregulated PTEN/AKT pathways to significantly attenuate disease progression. Inhibition of PRL2 therefore represents a novel potential therapeutic strategy in the management and treatment of AML. This thesis project also sought to further examine the role of the PRLs in oncogenesis through their regulation and interaction of targets within the TME. Functional analyses revealed that PRL3 was the only PRL to have a prominent role in host response to TME development, and that previously proposed roles for PRL3 in angiogenesis and immune cell recruitment is dependent upon PRL3 expression and activity in cells external to the TME. The study also revealed a previously unrecognized synergism between VEGF and PRL3 in the host in promoting TME angiogenesis. The studies of PRL3 in the TME suggest the potential physiological role of PRL3 in wound healing.</p>