Approximately 22% of the United States population suffers from a chronic orofacial pain condition. One such condition is known as trigeminal neuropathic pain frequently reported as continuous aching and burning pain, often accompanied by intermittent electrical shock-like sensations. Dental procedures or trauma are known causes of peripheral trigeminal nerve injury and inflammation. Patients who have this type of facial pain also suffer from emotional distress. For these reasons, trigeminal neuropathic pain needs to be studied in more detail to improve the understanding of the etiology and maintenance of this condition, as well as to develop effective treatment strategies. The first experiment was focused on characterizing the behavioral aspects of the Trigeminal Inflammatory Compression (TIC) mouse model. The findings determined that the TIC injury model induced mechanical and cold hypersensitivity that persist at least 21 weeks. This orofacial, neuropathic pain condition was accompanied by anxiety- and depressive-like behaviors at week 8 post injury. The TIC injury mouse model’s chronicity and development of psychosocial impairments demonstrated its usefulness as a facial pain model. The second experiment used the mouse TIC injury model to test the ability of pioglitazone (PIO), a PPARγ agonist used clinically for treatment of diabetes, on alleviating trigeminal pain. A single low dose of PIO had no effect, but a higher dose attenuated facial pain. The third experiment determined that combining ineffective low doses of PIO and D-cycloserine (DCS) produced a potentiated anti-allodynic response of these drugs and attenuated the anxiety associated with the TIC injury. Ex vivo studies revealed that cortical mitochondrial dysfunction occurred after the TIC injury but could be reversed by the combination of DCS/PIO which improves mitochondrial function. Overall, the present studies determined that the novel mouse TIC injury model is a clinically relevant facial neuropathic pain model. The results suggest that PPARγ and brain mitochondria may represent new molecular targets for the treatment of trigeminal neuropathic pain. These studies support the future “repurposing” of PIO and DCS as well as the combination of the two drugs for this new use in patients with trigeminal neuropathic pain.

Ovarian cancer is thought to be derived from the ovarian surface epithelium (OSE), but it is often diagnosed during the late stages and therefore the events that contribute to the initiation and progression of ovarian cancer are poorly defined. Epidemiological studies have indicated an association between the female reproductive hormones and ovarian cancer etiology, but the direct effects of 17β-estradiol (E2), progesterone (P4), luteinizing hormone (LH) and follicle stimulating hormone (FSH) on disease pathophysiology are not well understood. A novel transgenic mouse model of ovarian cancer was generated that utilized the Cre/loxP system to inducibly express the oncogene SV40 large and small T-Antigen in the OSE. The tgCAG-LS-TAg mice developed poorly differentiated ovarian tumours with metastasis and ascites throughout the peritoneal space. Although P4 had no effect; E2 significantly accelerated disease progression in tgCAG-LS-TAg mice. The early onset of ovarian cancer was likely mediated by E2’s ability to increase the areas of putative preneoplastic lesions in the OSE. E2 also significantly decreased survival time in ovarian cancer cell xenografts. Microarray analysis of the tumours revealed that E2 mainly affects genes involved in angiogenesis and cellular differentiation, proliferation, and migration. These results suggest that E2 acts on the tumour microenvironment in addition to its direct effects on OSE and ovarian cancer cells. In order to examine the role of the gonadotropins in ovarian cancer progression, the tgCAG-LS-TAg mice were treated with 4-vinylcyclohexene-diepoxide (VCD) to induce menopause. Menopause slowed the progression of ovarian cancer due to a change in the histological subtype from poorly differentiated tumours to Sertoli tumours. Using a transgenic mouse model, it was shown that E2 accelerated ovarian cancer progression, while P4 had little effect on the disease. Menopause (elevated levels of LH and FSH) altered the histological subtype of the ovarian tumours in the tgCAG-LS-TAg mouse model. These results emphasize the importance of generating animal models to accurately recapitulate human disease and utilizing these models to develop novel prevention and treatment strategies for women with ovarian cancer.

In the era of personalized cancer treatment, adoptive T cell therapy (ACT) shows promise for the treatment of solid cancers. However, partial or mixed responses remain common clinical outcomes due to the heterogeneity of tumours. Indeed, in many patients it is typical to see a response to ACT in one tumour nodule, while others show little or no response. Thus, defining the tumour features that distinguish those that respond to ACT from those that do not would be a significant advance, allowing clinicians to identify patients that might benefit from this treatment approach. The first chapter of this thesis provides the necessary background to understand the principals behind and components of ACT. This chapter also offers selected historical advances contributing to the current state of the field. The second chapter introduces a novel murine model of breast cancer developed to investigate the tumour-specific mechanisms associated with immune evasion in an ACT setting. The third chapter describes the in vivo characterization of mammary tumour cell lines derived from our mouse model that reliably showed complete, partial or no response to ACT. Using these cell lines, we were able to characterize in vivo tumour-specific differences in cytotoxic T cell trafficking, infiltration, activation, and proliferation associated with response to ACT. In the fourth chapter, we used bioinformatics approaches to develop a preliminary predictive gene signature associated with response to ACT in our mammary tumour model. We used this signature to predict outcome and then test a number of murine mammary tumours in vivo, with promising results, wherein 50% of tumours responded to ACT as predicted based upon gene expression. Thus, using an innovative model for breast cancer, these results suggest that there are tumour-specific features that can be used a priori to predict how a tumour will respond to adoptive T cell therapy. Importantly, these findings might facilitate the design of immunotherapy trials for human breast cancer. / Graduate

SYNGAP1 haploinsufficiency is one of the most common monogenic causes of nonsyndromic moderate to severe intellectual disability (NSID) and autism (Hamdan et al., 2009; Pinto et al., 2010). De novo truncating or frameshift mutations in the SYNGAP1 gene lead to the loss of the encoded protein Synaptic GTPase activating protein (SynGAP), one of the most abundant of postsynaptic proteins (Hamdan et al., 2011). SynGAP, present at excitatory and inhibitory synapses (Kim et al., 1998), acts as a key regulator of highly conserved signaling pathways linked to AMPA- and NMDA-receptor dependent plasticity at the post synaptic density (Krapivisky et al., 2004; Vazquez et al., 2004). The Syngap mouse model has been extensively used to understand the pathophysiology underlying abnormal SynGAP-mediated signaling. Syngap heterozygous (het) mice demonstrate a range of physiological and behavioural abnormalities from development to adulthood (Komiyama et al., 2002; Muhia et al., 2010). However, recent advances in techniques for genome manipulation have allowed for the generation of rat models of neurodevelopmental disorders, including Syngap; enabling phenotypes to be validated across species and to address cognitive and social dysfunction, using paradigms that are more difficult to assess in mice. In this study, we examined the pathophysiology associated with a heterozygous deletion of the C2 and catalytic GAP domain of the protein, in Long-Evans rats (het). In contrast with het mice, het rats do not present with hyperactivity and can be habituated to an open field environment. To examine associative recognition memory, we tested the rats in five spontaneous exploration tasks for short-term and long-term memory, object-recognition (OR), object-location (OL), object-place (OP), object-context (OC) and object-place-context (OPC). Both groups were able to perform short-term memory tasks, but only wild type rats performed above chance in OL with a 24hour delay, suggesting deficits in long- term spatial memory. We also tested if partial loss of the GAP domain in SynGAP affects social behaviour in rats and we found that het rats exhibited impaired short- term social memory, with no signs of social isolation. These findings do not fully recapitulate previous abnormalities reported in the mouse model of SYNGAP1 haploinsufficiency, suggesting that some key behavioural phenotypes may be species-specific. Furthermore, based on physiological deficits that Syngap het mice exhibit, such as alterations in mEPSC/mIPSC amplitude and frequency and evoked cortical hyperexcitability in vitro (Guo et al., 2009; Ozkan et al., 2014), we also aimed to test if in vivo neuronal activity and circuit properties are altered. Using two-photon calcium imaging in awake mice, we focused on two areas of the cortex; a primary sensory area, the binocular region of the visual cortex (V1), and an association area, the medial posterior parietal cortex (PPC). Both areas have been found to maintain activity during visual discrimination tasks but to present with divergent activity trajectories (Harvey et al., 2012; Goard et al., 2016). We found preliminary evidence that neurons in layer 2-3 of the PPC of Syngap mice are hypoactive in basal conditions when animals are still in the dark, compared to wild type controls. When we assessed whether that changes when animals are running, we found that during locomotion neurons of both genotypes increase their activity, consistent with previous findings in wild type mice (McGinley et al., 2015; Pakan et al., 2016). However, this response gain is exaggerated in Syngap het neurons of the PPC. In contrast to above findings in PPC, results in V1 show that layer 2-3 neurons are hyperactive during both behavioural states, suggesting seemingly different computations of these two cortical areas. This work provides the first evidence for a dysregulated neuronal circuit in vivo in both visual and parietal cortex of Syngap mice, two areas critical for sensory processing that has been found to be affected in individuals with NSID and autism (Joosten and Bundy, 2010). We also provide first evidence of the effect of loss of SynGAP activity in behaviour of rats, complimenting existing data in the literature in a species-specific manner and providing greater insight into sensory and cognitive dysfunction associated with dysregulation in SynGAP-mediated signaling.

L’hypertension artérielle est une maladie induite par de multiples facteurs génétiques et environnementaux. De nombreuses pathologies y sont associées. A travers ce travail, j’ai abordé trois aspects de la régulation de la pression artérielle in vivo et in vitro. Dans une première partie, j’ai étudié le rôle de l’activation du récepteur AT1 de l’angiotensine II dans le développement de la fibrose, indépendamment de l’hypertension artérielle. Un modèle animal exprimant un récepteur constitutivement actif et des modèles cellulaires (MEF, HEK293, H295) exprimant le récepteur constitutivement actif de façon inductible ont été utilisés. Contrairement aux souris sur fond mixte, les souris mutées sur fond pur C57Bl6 ne développent pas de fibrose cardiaque et rénale et ont une hypertension modérée, qui est difficile à réduire par les anti-hypertenseurs. De plus, les cellules fibroblastiques MEF ne sont pas un bon modèle pour étudier la fibrose induite par l’angiotensine II. Seule l’ostéopontine est un marqueur induit par l’expression du récepteur AT1 contitutivement actif. Ces différents modèles, étudiés extensivement, ne sont donc pas adaptés pour répondre aux questions posées. Dans la seconde partie de ma thèse, un travail collaboratif a permis de mettre en évidence le rôle majeur de Wnk1 au cours de l’hypertension et du remodelage cardiovasculaire induits par une infusion chronique d’angiotensine II. En effet, les souris Wnk1+/- (haplo-insuffisantes pour le gène Wnk1) présentent une résistance transitoire à l’hypertension induite par l’angiotensine II, particulièrement au cours de la première semaine d’infusion. Cette résistance est associée à une altération du remodelage hypertrophique cardiovasculaire mais la fonction rénale et la sécrétion d’aldostérone sont préservées. Au niveau mécanistique, nos résultats ont identifié Wnk1 comme un activateur important de la phosphorylation de Mypt1, un marqueur connu de l’activité de la voie Rho-kinase. Les aortes de souris Wnk1+/- présentent une diminution transitoire de la phosphorylation de Mypt1 après une semaine d’infusion d’angiotensine II. De façon importante, nous montrons aussi que l’infusion chronique d’angiotensine II induit une activation de l’expression du gène Wnk1 au niveau aortique, et la surexpression de Wnk1 in vitro active de façon importante et reproductible la phosphorylation de Mypt1, indépendamment de l’activation de Spak (substrat bien caractérisé de Wnk1). En conclusion, ce travail a permis d’identifier Wnk1 comme un nouveau gène cible de l’angiotensine II au niveau vasculaire et a révélé un nouveau mécanisme mis en jeu au cours de l’hypertension et du remodelage cardiovasculaire qui lui est associé. Cette étude fait l’objet d’un article que je signe en premier auteur et qui est actuellement soumis pour publication. Dans une dernière partie, j’ai étudié le rôle de la culline3 dans la régulationde la voie RhoKinase. Les mutations du gène Cul3 ont très récemment été identifiées comme responsables du syndrome de Gordon. Ce gène code une protéine d’échafaudage d’un complexe d’ubiquitination important et ubiquitaire (CRL3) conduisant à la dégradation protéique. La voie des Rho-kinases joue un rôle majeur dans le tonus vasculaire et sa régulation par les agents relaxants ou constricteurs. Des travauxrécents suggèrent que la dégradation de RhoA implique le complexe culline3-ring-ligase (CRL3). Nous avons voulu établir les liens structuraux et fonctionnels entre ce complexe d’ubiquitination et la voie Rho-kinase dans des modèles cellulaires, pour ainsi expliquer tout ou partie du mécanisme moléculaire conduisant des mutations constitutionnelles du gène Cul3 à produire une hypertension artérielle. Les interactions protéiques entre deux adaptateurs différents et la culline3 nous ont permis de montrer que la culline3 mutée entraine une modification d’affinité spécifique selon ses partenaires. Les interactions entre RhoA et le CRL3 n’ont pas pu être démontré. (...) / Hypertension is a disease due to multiple genetic and environmental factors. Many cardiovascular diseases are associated. During my PhD thesis, I addressed three aspects of the regulation of blood pressure in vivo and in vitro. In the first part, I studied the role of angiotensin II AT1 receptor activation in the development of fibrosis, independently of hypertension. I used animal and cellular models (MEF, HEK293, H295) expressing a constitutively active receptor. The results show that the mutant mice did not develop cardiac or renal fibrosis in a pure C57Bl6 strain. Furthermore, their moderate hypertension has not been normalized with two antihypertensives. The pure C57BL6 genetic background seems to be the cause of this moderate phenotype. Furthermore, MEF cells are not a good model to study fibrosis induced by angiotensin II. Only osteopontin is a marker induced by expression of the mutant receptor. As the mouse models and despite of their originality, these cellular models appear to be inappropriate to study AngII-dependent fibrosis. These limitations together with the weakness of the AT1 mutant phenotype lead to us to stop this project. In the second part of my thesis, a collaborative study allows us to show that Wnk1-haploinsufficiency in mice is responsible for a strong and transitory resistance to angiotensin II (AngII)-induced hypertension associated with a significant reduction of cardiovascular remodeling and a preservation of renal function and aldosterone release. Mechanistically, we unravel a critical role for Wnk1 in the activation of the phosphorylation of Mypt1, a known marker of the Rho-kinase pathway activity. Wnk1-haploinsufficient mice display a significant and transitory decrease of AngII-induced phospho- Mypt1 in the aorta, concomitant to the hypertension-resistance. Importantly, we further evidence that, in the vasculature, AngII chronic infusion induces a significant upregulation in Wnk1 gene expression which causes in vitro a significant increase in Mypt1 phosphorylation independently of spak activation. Our results provide new insight into the downstream vascular signaling pathway of AngII and unravel a previously unsuspected mechanism linking Wnk1 to hypertension and vascular remodeling. In the last part, I studied the role of vascular Cullin3 (Cul3) in the development of hypertension. Mutations in this gene have been recently identified as responsible for a familial hypertension with hyperkalaemia, FHHt. The Cul3 gene encodes an important and ubiquitous ubiquitin scaffold protein participating to a protein degradation complex (CRL3). The Rho-kinase pathway plays a major role in vascular tone and its regulation by relaxing or vasoconstricting agents. Recent studies suggest that the degradation of RhoA involves the CRL3 complex. I started to analyze the structural and functional links between this ubiquitination complex and the Rho kinase pathway in cellular models to explain all or part of the molecular mechanisms leading constitutional mutations of Cul3 gene to produce hypertension. I have shown that the Cul3Δ9 mutant presents an increased neddylation compared to the wild form and mofications of its affinity for some adaptors. However, in this preliminary work, its interactions with and its role in the degradation of RhoA have not been demonstrated yet. This PhD thesis has helped to address several aspects of the pathophysiology of the vessels and the role of angiotensin II in these regulations using modern tools, original mouse and cell line models. This has particularly highlighted a new target of angiotensin II and a new WNK1 vascular role.

Le rôle des lymphocytes T CD4+ dans la progression des tumeurs et dans l'immunité anti-tumorale est de plus en plus reconnu chez l'homme et chez la souris. Les mécanismes effecteurs de l’immunité T CD4+ contre le cancer ont été étudiés principalement dans des systèmes de tumeurs transplantées. Dans ces modèles, de nombreuses cellules tumorales meurent au moment de l'implantation, ce qui conduit à la libération de l'antigène tumoral (Ag) dans un contexte inflammatoire. Ceci contraste avec la croissance lente et non-destructrice des tumeurs humaines aux stades précoces. Nous avons montré que la présentation de l’Ag retreint par le CMH-II DBY, libéré par des cellules tumorales mortes injectées en sous-cutané, peut persister durant plusieurs semaines dans le ganglion drainant. L’activation précoce d’une réponse immune dirigée contre l’Ag lors de l’injection de lignées tumorales peut gêner l’étude des relations entre les tumeurs et le système immunitaire. Par conséquent, nous avons généré une lignée tumorale dans laquelle l’expression de DBY peut être induite in vivo à distance de l’implantation. Nous avons également utilisé un modèle de tumeurs pulmonaires endogènes se développant dans des souris transgéniques KrasG12D, p53flox et exprimant DBY de manière spécifique. Notre objectif était d'étudier dans ces deux modèles l'histoire naturelle de la réponse des lymphocytes T CD4+ spécifiques de la tumeur. Dans le système de tumeur transplantée «Ag inductible », nous avons montré que DBY est présenté de manière efficace à des cellules naïves T CD4+ spécifiques dans les ganglions drainant la tumeur. Les réponses prolifératives et effectrices sont similaires dans les systèmes où DBY est exprimé de façon inductible in vivo ou constitutive. Le récepteur de co-stimulation ICOS, ainsi que les récepteurs co-inhibiteurs PD-1 et BTLA sont régulés positivement sur les lymphocytes T CD4+ spécifiques en réponse à l’Ag. La production de cytokines en réponse à une restimulation in vitro révèle un profil effecteur mixte TH1 /TH17. Notamment, un petit pourcentage de lymphocytes T co-expriment les marqueurs de cytotoxicité LAMP-1 et granzyme B. Ainsi, lorsqu’un Ag apparaît à distance de l'implantation de la tumeur, il n’est pas ignoré et n’induit pas de tolérance immune. D'autres mécanismes doivent être considérés pour expliquer l'absence de rejet de tumeur efficace malgré l’activation et la migration des cellules effectrices T CD4+ dans les tumeurs. Les travaux réalisés sur le modèle de tumeurs pulmonaires endogènes sont en cours. A ce stade, nous avons observé que, comme dans les tumeurs transplantées, l’Ag est présenté aux LT CD4+ naïfs qui prolifèrent dans le ganglion drainant et ne se différencient pas en cellules T régulatrices, même aux stades très avancés de la maladie. La capacité des tumeurs à induire une réponse T effectrice semble liée à leur stade de développement. Durant la phase tumorale précoce, les LT spécifiques de DBY produisent de l’IFN-γ et du granzyme B. En revanche, des LT spécifiques produisant de l'IL-17 sont retrouvés dans les poumons de souris ayant des tumeurs invasives de stade terminal. Bien que l'IL-17 puisse favoriser la progression des tumeurs, y compris dans les modèles induits par l’oncogène KrasG12D, de nouvelles données suggèrent que les cellules effectrices TH17 possèdent un haut degré de plasticité et peuvent présenter une activité anti-tumorale. Notre modèle pourrait être utile pour tester de nouvelles stratégies de ciblage de l’IL-17 dans l'immunothérapie du cancer. / The role of CD4+ T cells in both tumor progression and immunity is being increasingly acknowledged in humans and mice. CD4+ T cell immunity against cancer has been mostly studied using murine transplanted tumor systems. In these models, many tumor cells die at the time of surgical implantation, leading to the release of tumor antigen (Ag) in an inflammatory context. This contrasts with the slow and non-destructive growth of early stage human tumors. Here, we show that the presentation of a MHC class II-restricted model (male, DBY) Ag released by dying fibroblastic tumor cells may last more than 3 weeks in the tumor draining lymph node (dLN). This artificial, early and long lasting priming precludes the study of the interactions between the immune system and tumors at the steady state. We therefore generated a cell line that could be induced to efficiently express DBY as a neoAg after implantation. We also took advantage of a previously described mouse model of genetically engineered, KrasG12D p53flox lung adenocarcinoma to generate a “spontaneous” tumor model expressing DBY. Our aim was to study in these two models the natural history of the tumor-specific CD4+ T cell response. In the transplanted tumor system, we show that the Ag reaches the dLNs and is efficiently presented to naïve specific CD4+ T cells. The proliferative and effector responses were similar in the inducible and constitutively expressed Ag tumor systems. The ICOS co-stimulatory receptor, and the PD-1 and BTLA co-inhibitory receptors were upregulated on the Ag specific CD4+ T cells in the dLN. We did not observe de novo induction of tumor-specific regulatory T cells. Finally, the pattern of secreted lymphokines in the dLN, spleen and tumor after in vitro Ag restimulation was similar, with a mixed TH1/TH17 response. Notably, a small percentage of DBY-specific effector T cells also displayed a cytolytic phenotype marked by the co-expression of granzyme B and LAMP-1. Thus, when the neo-Ag appears at distance of tumor implantation, the tumor was not ignored and did not induce tolerance of naïve CD4+ T cells. Other mechanisms have to be thought to explain the absence of tumor rejection despite efficient priming and migration of effector CD4+ T cells into tumors. Similarly to the strong proliferative response mentioned above, the DBY tumor Ag was efficiently presented in LNs draining “spontaneous” lung tumors, and induced activation and proliferation of adoptively transferred naive T cells. After priming they did not convert into Tregs, even in end-stage disease. This work is still ongoing, but preliminary results show that activated DBY-specific T cells from the dLN and the lungs produced IFN-γ and granzyme B during early stages of the disease. In contrast, IL-17 secreting cells were found exclusively in the lungs from mice with late-stages invasive tumors. Although IL-17 may enhance tumor progression, including in models driven by the Kras oncogen, emerging data strongly suggest that TH17 effector cells demonstrate a high grade of plasticity and can display anti tumor activity. Little is known about the antigen specificity of IL-17 production in lung cancer patients, and our model could be useful to test new strategies targeting either positively or negatively tumor Ag-specific TH17 cells in cancer immunotherapy.

Ovarian cancer is thought to be derived from the ovarian surface epithelium (OSE), but it is often diagnosed during the late stages and therefore the events that contribute to the initiation and progression of ovarian cancer are poorly defined. Epidemiological studies have indicated an association between the female reproductive hormones and ovarian cancer etiology, but the direct effects of 17β-estradiol (E2), progesterone (P4), luteinizing hormone (LH) and follicle stimulating hormone (FSH) on disease pathophysiology are not well understood. A novel transgenic mouse model of ovarian cancer was generated that utilized the Cre/loxP system to inducibly express the oncogene SV40 large and small T-Antigen in the OSE. The tgCAG-LS-TAg mice developed poorly differentiated ovarian tumours with metastasis and ascites throughout the peritoneal space. Although P4 had no effect; E2 significantly accelerated disease progression in tgCAG-LS-TAg mice. The early onset of ovarian cancer was likely mediated by E2’s ability to increase the areas of putative preneoplastic lesions in the OSE. E2 also significantly decreased survival time in ovarian cancer cell xenografts. Microarray analysis of the tumours revealed that E2 mainly affects genes involved in angiogenesis and cellular differentiation, proliferation, and migration. These results suggest that E2 acts on the tumour microenvironment in addition to its direct effects on OSE and ovarian cancer cells. In order to examine the role of the gonadotropins in ovarian cancer progression, the tgCAG-LS-TAg mice were treated with 4-vinylcyclohexene-diepoxide (VCD) to induce menopause. Menopause slowed the progression of ovarian cancer due to a change in the histological subtype from poorly differentiated tumours to Sertoli tumours. Using a transgenic mouse model, it was shown that E2 accelerated ovarian cancer progression, while P4 had little effect on the disease. Menopause (elevated levels of LH and FSH) altered the histological subtype of the ovarian tumours in the tgCAG-LS-TAg mouse model. These results emphasize the importance of generating animal models to accurately recapitulate human disease and utilizing these models to develop novel prevention and treatment strategies for women with ovarian cancer.

SLMAP is a tail anchored membrane protein that alternatively splices to generate three isoforms, SLMAP1, SLMAP2 and SLMAP3. Previous studies in our lab have shown that the postnatal cardiac-specific overexpression of SLMAP1 results in intracellular vesicle expansion and enhanced endosomal recycling. I generated a postnatal cardiac-specific knockout model using the Cre-Lox system to nullify all three SLMAP isoforms and further evaluate its role in the mouse myocardium. SLMAP knockdown and knockout mouse hearts were analyzed with western blotting and qPCR. I found that only SLMAP3 was nullified and phenotypic evaluation through echocardiography indicated that young and old SLMAP3 knockout animals showed no remarkable changes in cardiac function. Furthermore, challenge with stressor isoproterenol had a similar response to wildtype and knockout mice in cardiac structure and function. Surprisingly the level of expression of SLMAP1 and SLMAP2 was maintained in the myocardium from SLMAP3 deficient mice. Interestingly the machinery involved in endosomal recycling was not impacted by the loss of SLMAP3. These data indicate that loss of SLMAP3 does not alter cardiac structure and function in the postnatal myocardium in the presence of SLMAP1 and SLMAP2.

No description available.

Fragestellung: Um die Auswirkungen von genetischen Varianten für CASP1 in vivo analysieren zu können, sollte in dieser Arbeit ein Tiermodell generiert werden. Dadurch könnten die zugrundeliegenden Pathomechanismen der an „ICE-Fieber“ leidenden Patienten in einem gesamten Organismus aufgeklärt werden, da die Untersuchung von Patientenmaterial nur sehr eingeschränkt möglich ist. Ein Mausmodell stellt eine der besten Alternativen zur Analyse von Primärmaterial dar, da die Immunsysteme von Maus und Mensch sehr ähnlich sind. Ergebnisse: Um die natürliche Expression und Regulation der Procaspase-1 im Mausmodell zu gewährleisten, wurde für die Generierung ein BAC-Transgen verwendet. Mittels homologer Rekombination wurde die künstliche Variante C284A von Casp1 inseriert. Diese führt zu einer Zerstörung des enzymatischen Zentrums der Caspase-1 und zum vollständigen Verlust der enzymatischen Aktivität. Nach zwei Pronukleusinjektionen konnten lediglich drei Gründertiere mit mosaikartiger Expression des zufällig im Genom integrierten Transgens Casp1C284A und in der F1-Generation nur ein Tier mit stabil integriertem Transgen identifiziert werden. Die Nachkommen dieses transgenen Tieres zeigten keine basale Expression von Casp1C284A, jedoch konnte nach Stimulation von BMDCs mit LPS in vitro die Expression sowohl auf RNA- wie auch auf Proteinebene nachgewiesen werden. Ebenfalls eine erhöhte Sekretion der proinflammatorischen Zytokine TNF-α und IL-6 wurde in den Zellen der transgenen Tiere detektiert. Gleichfalls konnte nach Stimulation mit LPS in vivo eine gesteigerte Entzündungsreaktion in den Tieren mit Casp1C284A gezeigt werden, da ein stärkerer und länger anhaltender Abfall der peripheren Körpertemperatur und außerdem eine gesteigerte Sekretion von TNF-α und IL-6 zu verzeichnen war. Eine gesteigerte Inflammation des fetalen Gewebes, ausgelöst durch die integrierte künstliche Variante der Procaspase-1, könnte die Frage nach der geringen Anzahl der generierten Gründertiere beantworten. Hierfür wurde weiterführend eine Maus mit einem konditionalen Casp1C284A-Konstrukts generiert, welches zusätzlich eine zeit- als auch zelltyp-spezifische Expression der Procaspase-1 mit zentraler Mutation ermöglicht. Nach erfolgreichem Screening der ES-Zellen konnten diese in Blastozysten mikroinjiziert und Chimäre identifiziert werden. Eine embryonale Letalität des transgenen Konstrukts konnte durch die Verpaarung der ki-Tiere R26_Casp1C284A mit PGK-Cre-Tieren, die Cre-Rekombinase ubiquitär exprimieren, nahezu ausgeschlossen werden, da die Nachkommen alle lebensfähig waren und eine basale Expression des „knock-ins“ in mehreren Organen und auch in BMDCs nachgewiesen wurde. Ferner konnte nach Induktion einer Inflammation in vivo mit einer subletalen Dosis von LPS ein gesteigerter und länger anhaltender peripherer Temperaturabfall in den ki-Tieren ähnlich zu den transgenen Tieren detektiert werden. Desgleichen wurde eine Tendenz zu einer gesteigerten Sekretion der Zytokine TNF-α und IL-6 verzeichnet. Schlussfolgerungen: Mit dem transgenen Casp1C284A-Mausmodell als auch mit dem konditionalen R26_Casp1C284A-Modell konnte gezeigt weren, dass eine inaktive Variante der Procaspase-1 zur Entstehung einer gesteigerten Inflammation in einem gesamten Organismus führen kann. Somit können die im Rahmen dieser Arbeit generierten Tiermodelle zur Analyse der Pathomechanismen der an „ICE-Fieber“ leidenden Patienten herangezogen werden. In künftigen Studien kann ferner geklärt werden, ob die Entzündungsreaktionen durch eine verstärkte Interaktion von Casp1C284A mit der Kinase RIP2 verursacht werden und dadurch ähnlich wie im Patienten eine Aktivierung des proinflammatorischen Moleküls NFκB ausgelöst wird. Im Anschluss könnte eine spezifische Inhibierung des RIP2-Signalweges in diesen Mausmodellen getestet werden und schließlich im Patienten Anwendung finden. Die in dieser Arbeit generierten Mausmodelle könnten somit zur Erprobung zukünftiger therapeutischer Konzepte dienen.:Inhaltsverzeichnis Abkürzungsverzeichnis 1 Einleitung 1.1 Das angeborene und adaptive Immunsystem 1.2 Sensoren des angeborenen Immunsystems 1.2.1 Membran-gebundene Rezeptoren 1.2.2 Intrazelluläre PRRS 1.2.3 Inflammasome als Multiproteinkomplexe 1.2.4 Das NLRP3-Inflammasom 1.2.5 Metabolische Krankheiten, die mit Inflammasom-Aktivität assoziiert werden 1.2.6 Inflammasom-assoziierte autoinflammatorische Erkrankungen 1.2.6.1 Das Cryoporin-assoziierte periodische Syndrom (CAPS) 1.2.6.2 Familian Mediterranean Fever (FMF) 1.2.6.3 Pyogenic arthritis, pyoderma gangrenosum and acne syndrome (PAPA) 1.3 Caspase-1 1.3.1 Caspasen im Allgemeinen 1.3.2 Der Caspase-1 Gen-Lokus und das Caspase-1 Gen 1.3.3 Das Caspase-1 Protein 1.3.4 Funktionen von Caspase-1 1.3.4.1 Prozessierung der Zytokine pro-IL-1ß und pro-IL-18 1.3.4.2 Induktion von Pyroptose 1.3.4.3 Aktivierung der Caspase-1 durch ER-Stress 1.3.4.4 Aktivierung des Transkriptionsfaktors NFκB 1.3.4.5 weitere Funktionen 1.3.5 Caspase-1 Genvarianten und Entzündung 1.4 Mausmodelle 1.4.1 NLRP3 1.4.2 Pyrin 1.4.3 Caspase-1 2 Zielsetzung 3 Material und Methoden 3.1 Material 3.1.1 Antibiotika 3.1.2 Antikörper 3.1.3 Bakterienstämme 3.1.4 BAC-Klon 3.1.5 Plasmide 3.1.6 Zelllinie 3.1.7 Chemikalien und Substanzen 3.1.8 Enzyme 3.1.9 Größenstandards 3.1.10 Oligonukleotide 3.1.11 Kommerzielle Kits 3.1.12 Puffer und Lösungen 3.1.13 Mauslinien 3.1.14 Medien 3.1.15 Geräte 3.1.16 Software 3.2 Molekularbiologische Methoden 3.2.1 Agarosegelelektrophorese 3.2.2 Amplifikation von Nukleinsäuren mittels Polymerasekettenreaktion 3.2.3 Aufreinigung von DNA-Fragmenten aus Agarosegelen 3.2.4 Aufreinigung von BAC-DNA mittels Elektroelution 3.2.5 Aufreinigung von Subklon-Plasmid-DNA für die Elektroporation von ES-Zellen 3.2.6 Bestimmung der Konzentration und der Reinheit von DNA und RNA 3.2.7 c-DNA-Synthese 3.2.8 Dephosphorylierung von 5´-Phosphatresten 3.2.9 DNA-Isolierung 3.2.9.1 DNA-Isolierung aus Bakterienzellen 3.2.9.2 DNA-Isolierung aus eukaryotischen Zellen 3.2.10 DNA-Sequenzierung 3.2.11 Enzymatische Restriktionsspaltung von DNA-Fragmenten 3.2.12 Homologe Rekombination von DNA-Sequenzen 3.2.13 Ligation von DNA-Fragmenten mit linearisierten Vektoren 3.2.14 Phenol-Chloroform-Extraktion 3.2.15 Pulsfeldgelelektrophorese 3.2.16 Quantitative Real-Time-PCR (qRT-PCR) 3.2.17 RNA-Isolation aus eukaryotischen Zellen und Geweben 3.2.18 Southern Blot 3.3 Mikrobiologische Methoden 3.3.1 Kultivierung von Bakterien 3.3.2 Hitzschock-Transformation von E.coli 3.3.3 Kryokonservierung 3.4 Proteinbiochemische Methoden 3.4.1 CBA (cytokine bead assay) 3.4.2 Proteinbestimmung 3.4.3 Immunpräzipitation mit Caspase-1 p10 (M-20) Antikörper von Santa Cruz 3.4.4 Immunpräzipitation mit AntiFlag M2 Affinity Gel von Sigma 3.4.5 Diskontinuierliche SDS-Polyacrylamid-Gelelektrophorese (SDS-PAGE) 3.4.6 Western Blot 3.4.7 Immunchemische Detektion von Proteinen 3.5 Zellbiologische Methoden 3.5.1 Bestimmung der Zellzahl 3.5.2 Isolierung und Generierung von BMDCs 3.5.3 Kryokonservierung von Zellen 3.5.4 Kultivierung von Zelllinien 3.6 Tierexperimentelle Methoden 3.6.1 Genotypisierung 3.6.2 Haltung 3.6.3 Hautbiopsie 3.6.4 Retroorbitale Blutentnahme und Herzpunktion 3.6.5 Injektion von LPS 3.6.6 Organentnahme 4 Ergebnisse 4.1 Generierung eines Casp1C284A-BAC-Transgens 4.1.1 Identifikation und Verifizierung des BAC-Klons 4.1.2 Klonierung des Casp1C284A-BAC-Transgens 4.1.2.1 Entfernung der wt-loxP-Stelle vom Vektorrückrat 4.1.2.2 Insertion eines Flag-Tags 4.1.2.3 Einführung der Punktmutation C284A 4.1.3 „Shaving“ des generierten Casp1C284A-BAC-Konstrukts 4.1.4 Aufreinigung des Casp1C284A-BAC-Konstrukts und Pronukleusinjektion 4.1.5 Genotypisierung der transgenen Casp1C284A Gründertiere 4.2 Charakterisierung der transgenen Casp1C284A-Tiere (tg) 4.2.1 In vitro-Stimulation von BMDCs mit LPS 4.2.2 In vivo-Stimulation der transgenen Casp1C284A-Tiere 4.3 Generierung eines konditionalen Casp1C284A-Konstrukts 4.3.1 Klonierung des Targetingvektors pSerc_Casp1C284A_Flag und des Kontroll-Targetingvektors pSerc_Casp1_Flag 4.3.2 Screening von ES-Zellen 4.3.3 Genotypisierung der Chimäre und Verpaarung mit PGK-Cre Tieren 4.3.4 Expressionsanalyse der R26_Casp1C284A del-Tiere 4.3.5 In vivo-Stimulation der R26_Casp1C284A del-Tiere 5 Diskussion 5.1 Entwurf eines Mausmodells für ICE-Fieber 5.2 Konstruktion des Casp1C284A-BAC-Transgens 5.3 Identifizierung von Casp1C284A-transgenen Tieren 5.4 Funktionelle Analyse des Casp1C284A-BAC-Transgens 5.5 Konstruktion des konditionalen Mausmodells R26_Casp1C284A 5.6 Aktivierung des ki-Mausmodells R26_Casp1C284A durch Cre-mediierte Rekombination in vivo 5.7 Funktionelle Analyse des ki-Mausmodells R26_Casp1C284A 5.8 Relevanz eines Mausmodells für den Patienten 6 Zusammenfassung 7 Literaturverzeichnis 8 Anhang A. Abbildungsverzeichnis B. Tabellenverzeichnis / Problem: In order to recapitulate the effects of the CASP1 variants found in the patients a mouse model should be generated. The analysis of material from the patients unfortunately is very restricted and therefore the generation of a mouse model represents the best alternative to see if the in vitro hypothesis of the IFG group really applies to an in vivo situation. Results: To generate a transgenic mouse model the artificial variant Casp1C284A was inserted into a BAC to enable a natural expression and regulation of Casp1C284A. This mutation results in a disruption of the active centre and to a complete loss of the enzymatical activity of caspase-1. After two pronuclei injections we received 180 pubs of TG mice. Only three of them harboured transgenic sequences and only one animal in the F1 generation harboured the complete Casp1C284A sequence. Expression analyses of the offspring of this mouse revealed no basal transcriptional expression of the transgene. Hence, protein expression could not be detected in unstimulated cells. However, stimulation with LPS upregulated transcription and low-level translation of Casp1C284A in BMDCs and an elevated secretion of the proinflammatory cytokines TNF-α and IL-6 was detected as well. Likewise, after in vivo stimulation of transgenic mice with LPS i.p. the drop of body temperature was significantly enhanced in comparison to the control mice. And also the level of the proinflammatory cytokines was increased. Furthermore, a conditional R26_Casp1C284A construct allowing a temporal or a celltype specific expression of the caspase-1 with the central mutation was generated. Positive screened ES cell clones were injected into blastocysts and thereafter chimera could be identified. An embryonic lethality due to the integration of the enzymatically inactive caspase-1 could be excluded by the crossing with ubiquitious expressing PGK-Cre mice. All the corresponding mice were alive and a basal transcriptional as well translational expression was demonstrated. Concordantly with the results of the transgenic mice the conditional R26_Casp1C284A mice showed an enhanced drop of the body temperature in comparison to control mice after stimulation with sublethal dose of LPS in vivo. Likewise, a trend to an elevated secretion of TNF-α and IL-6 was observed. Conclusion: With the generated transgenic Casp1C284A as well as the conditional R26_Casp1C284A animal models we showed that an inactive variant of the procaspase-1 could result in a proinflammatory cytokine response and a development of an increased inflammation of a whole organism. Thus, these data support the previous postulated model of the IFG group for proinflammatory effects induced by variants of procaspase-1 with reduced enzymatic activity. Hence, the mouse models established in this work are suited for further analysis of the pathomechanism in the patients with ICE fever. For instance the cellular mechanism could be examined if the inflammation is provoked by an increased interaction of the mutated procaspase-1 with the kinase RIP2 and therefore the NFκB activation is increased, respectively. Also a further medicinal inhibition of the RIP2 signaling or other therapeutic testings in this mouse models are conceivable.:Inhaltsverzeichnis Abkürzungsverzeichnis 1 Einleitung 1.1 Das angeborene und adaptive Immunsystem 1.2 Sensoren des angeborenen Immunsystems 1.2.1 Membran-gebundene Rezeptoren 1.2.2 Intrazelluläre PRRS 1.2.3 Inflammasome als Multiproteinkomplexe 1.2.4 Das NLRP3-Inflammasom 1.2.5 Metabolische Krankheiten, die mit Inflammasom-Aktivität assoziiert werden 1.2.6 Inflammasom-assoziierte autoinflammatorische Erkrankungen 1.2.6.1 Das Cryoporin-assoziierte periodische Syndrom (CAPS) 1.2.6.2 Familian Mediterranean Fever (FMF) 1.2.6.3 Pyogenic arthritis, pyoderma gangrenosum and acne syndrome (PAPA) 1.3 Caspase-1 1.3.1 Caspasen im Allgemeinen 1.3.2 Der Caspase-1 Gen-Lokus und das Caspase-1 Gen 1.3.3 Das Caspase-1 Protein 1.3.4 Funktionen von Caspase-1 1.3.4.1 Prozessierung der Zytokine pro-IL-1ß und pro-IL-18 1.3.4.2 Induktion von Pyroptose 1.3.4.3 Aktivierung der Caspase-1 durch ER-Stress 1.3.4.4 Aktivierung des Transkriptionsfaktors NFκB 1.3.4.5 weitere Funktionen 1.3.5 Caspase-1 Genvarianten und Entzündung 1.4 Mausmodelle 1.4.1 NLRP3 1.4.2 Pyrin 1.4.3 Caspase-1 2 Zielsetzung 3 Material und Methoden 3.1 Material 3.1.1 Antibiotika 3.1.2 Antikörper 3.1.3 Bakterienstämme 3.1.4 BAC-Klon 3.1.5 Plasmide 3.1.6 Zelllinie 3.1.7 Chemikalien und Substanzen 3.1.8 Enzyme 3.1.9 Größenstandards 3.1.10 Oligonukleotide 3.1.11 Kommerzielle Kits 3.1.12 Puffer und Lösungen 3.1.13 Mauslinien 3.1.14 Medien 3.1.15 Geräte 3.1.16 Software 3.2 Molekularbiologische Methoden 3.2.1 Agarosegelelektrophorese 3.2.2 Amplifikation von Nukleinsäuren mittels Polymerasekettenreaktion 3.2.3 Aufreinigung von DNA-Fragmenten aus Agarosegelen 3.2.4 Aufreinigung von BAC-DNA mittels Elektroelution 3.2.5 Aufreinigung von Subklon-Plasmid-DNA für die Elektroporation von ES-Zellen 3.2.6 Bestimmung der Konzentration und der Reinheit von DNA und RNA 3.2.7 c-DNA-Synthese 3.2.8 Dephosphorylierung von 5´-Phosphatresten 3.2.9 DNA-Isolierung 3.2.9.1 DNA-Isolierung aus Bakterienzellen 3.2.9.2 DNA-Isolierung aus eukaryotischen Zellen 3.2.10 DNA-Sequenzierung 3.2.11 Enzymatische Restriktionsspaltung von DNA-Fragmenten 3.2.12 Homologe Rekombination von DNA-Sequenzen 3.2.13 Ligation von DNA-Fragmenten mit linearisierten Vektoren 3.2.14 Phenol-Chloroform-Extraktion 3.2.15 Pulsfeldgelelektrophorese 3.2.16 Quantitative Real-Time-PCR (qRT-PCR) 3.2.17 RNA-Isolation aus eukaryotischen Zellen und Geweben 3.2.18 Southern Blot 3.3 Mikrobiologische Methoden 3.3.1 Kultivierung von Bakterien 3.3.2 Hitzschock-Transformation von E.coli 3.3.3 Kryokonservierung 3.4 Proteinbiochemische Methoden 3.4.1 CBA (cytokine bead assay) 3.4.2 Proteinbestimmung 3.4.3 Immunpräzipitation mit Caspase-1 p10 (M-20) Antikörper von Santa Cruz 3.4.4 Immunpräzipitation mit AntiFlag M2 Affinity Gel von Sigma 3.4.5 Diskontinuierliche SDS-Polyacrylamid-Gelelektrophorese (SDS-PAGE) 3.4.6 Western Blot 3.4.7 Immunchemische Detektion von Proteinen 3.5 Zellbiologische Methoden 3.5.1 Bestimmung der Zellzahl 3.5.2 Isolierung und Generierung von BMDCs 3.5.3 Kryokonservierung von Zellen 3.5.4 Kultivierung von Zelllinien 3.6 Tierexperimentelle Methoden 3.6.1 Genotypisierung 3.6.2 Haltung 3.6.3 Hautbiopsie 3.6.4 Retroorbitale Blutentnahme und Herzpunktion 3.6.5 Injektion von LPS 3.6.6 Organentnahme 4 Ergebnisse 4.1 Generierung eines Casp1C284A-BAC-Transgens 4.1.1 Identifikation und Verifizierung des BAC-Klons 4.1.2 Klonierung des Casp1C284A-BAC-Transgens 4.1.2.1 Entfernung der wt-loxP-Stelle vom Vektorrückrat 4.1.2.2 Insertion eines Flag-Tags 4.1.2.3 Einführung der Punktmutation C284A 4.1.3 „Shaving“ des generierten Casp1C284A-BAC-Konstrukts 4.1.4 Aufreinigung des Casp1C284A-BAC-Konstrukts und Pronukleusinjektion 4.1.5 Genotypisierung der transgenen Casp1C284A Gründertiere 4.2 Charakterisierung der transgenen Casp1C284A-Tiere (tg) 4.2.1 In vitro-Stimulation von BMDCs mit LPS 4.2.2 In vivo-Stimulation der transgenen Casp1C284A-Tiere 4.3 Generierung eines konditionalen Casp1C284A-Konstrukts 4.3.1 Klonierung des Targetingvektors pSerc_Casp1C284A_Flag und des Kontroll-Targetingvektors pSerc_Casp1_Flag 4.3.2 Screening von ES-Zellen 4.3.3 Genotypisierung der Chimäre und Verpaarung mit PGK-Cre Tieren 4.3.4 Expressionsanalyse der R26_Casp1C284A del-Tiere 4.3.5 In vivo-Stimulation der R26_Casp1C284A del-Tiere 5 Diskussion 5.1 Entwurf eines Mausmodells für ICE-Fieber 5.2 Konstruktion des Casp1C284A-BAC-Transgens 5.3 Identifizierung von Casp1C284A-transgenen Tieren 5.4 Funktionelle Analyse des Casp1C284A-BAC-Transgens 5.5 Konstruktion des konditionalen Mausmodells R26_Casp1C284A 5.6 Aktivierung des ki-Mausmodells R26_Casp1C284A durch Cre-mediierte Rekombination in vivo 5.7 Funktionelle Analyse des ki-Mausmodells R26_Casp1C284A 5.8 Relevanz eines Mausmodells für den Patienten 6 Zusammenfassung 7 Literaturverzeichnis 8 Anhang A. Abbildungsverzeichnis B. Tabellenverzeichnis