There are an estimated 2 billion individuals infected with Mtb, and 37.7 million people living with HIV (PLWH) worldwide. HIV/Mtb co-infection increases the risk of developing active tuberculosis by over 20-fold, and 210,000 of 1.5 million deaths from TB were among co-infected PLWH in 2020. Therefore, development of effective TB vaccination, particularly within the vulnerable PLWH population, is an urgent global issue. With limited in vivo models to study co-infection, humanized NRG (huNRG) mice and humanized DRAG-A2 mice (a next-generation of huNRG mice expressing HLA class I and II transgenes with improved human immune reconstitution, huDRAG-A2) are promising tools for HIV and TB reserach as they develop robust human immune cell populations and recapitulate many aspects of HIV or TB clinical disease. HIV/Mtb co-infection was investigated using huNRG and hu-DRAG-A2 mice in separate experiments where intravaginal (with DMPA pre-treatment) or intraperitoneal HIV-1 infection was administered, respectively, and intranasal infection of Mtb was administered 3.5 weeks later. Both huNRG and huDRAG-A2 mice recapitulated hallmark features of HIV/Mtb co-infection such as severe granuloma pathology, hCD4+ T cell depletion in lung and spleen tissue, and human like lung pathology such as Mtb-infected foamy macrophages in the granuloma. Co-infected huDRAG-A2 mice also displayed significantly higher bacterial burden in the lungs, increased extrapulmonary dissemination into spleen and liver, and significantly lower hCD4+ T cells in the peripheral blood post-Mtb infection when compared to the Mtb-only infected group. To investigate TB vaccine immunogenicity, huNRG and huDRAG-A2 mice were immunized with a novel trivalent vaccine, AdCh68MV. Upon intranasal immunization, both models showed trends of developing higher Mtb antigen-specific hCD4+ T cell responses in the lung and spleen. Overall, this project sets the initial stages of a pre-clinical HIV/Mtb co-infection model in huNRG and huDRAG-A2 mice appropriate for immune investigations, therapeutic and vaccination development. / Thesis / Master of Science in Medical Sciences (MSMS) / There are over 2 billion individuals infected with TB and 37.7 million people living with HIV (PLWH) worldwide. When someone is co-infected with both diseases, the risk of death is greatly increased. Research in co-infection and developing effective TB vaccination for PLWH are urgent global issues. Animal studies are currently limited because studying HIV requires human immune cells. Our lab has established humanized mice (hu-mice) that develop many different human immune cells and are useful for HIV/Mtb co-infection research. When hu-mice were co-infected, they showed more dying lung tissue, immune cell loss, and bacteria in the lungs. Hu-mice were also used to study human immune responses to a novel TB vaccine delivered to the lungs. Trends of higher immune responses towards TB were observed in the lung and spleen of immunized hu-mice. Overall, this project shows the utility of hu-mice as pre-clinical models of HIV/Mtb co-infection and Mtb vaccine studies.

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The activation of the p53 pathway has been associated with neuronal degeneration in different neurological disorders, including spinal muscular atrophy (SMA) where aberrant expression of p53 drives selective death of motor neurons destined to degenerate. Since direct p53 inhibition is an unsound therapeutic approach due carcinogenic effects, we investigated the expression of the cell death-associated p53 downstream targets c-fos, perp and fas in vulnerable motor neurons of SMA mice. Fluorescence in situ hybridization (FISH) of SMA motor neurons revealed c-fos RNA as a promising candidate. Accordingly, we identified p53-dependent nuclear upregulation of c-Fos protein in degenerating motor neurons from the severe SMNΔ7 and intermediate Smn2B/– SMA mouse models. Although motor neuron-specific c-fos genetic deletion in SMA mice did not improve motor neuron survival or motor behavior, p53-dependent c-Fos upregulation marks vulnerable motor neurons in different mouse models. Thus, nuclear c-Fos accumulation may serve as a readout for therapeutic approaches targeting neuronal death in SMA and possibly other p53-dependent neurodegenerative diseases.

Zelluläre Seneszenz beschreibt einen terminalen Zellzyklus-Arrest. Nach zellulärem Stress u. a. durch aktivierte Onkogene oder DNA-schädigende Chemotherapie wird Seneszenz induziert und kann so zur Tumorsuppression bzw. zum Behandlungserfolg beitragen. Vor kurzem wurde gezeigt, dass der Transkriptionsfaktor NF-kappaB – welcher bisher vor allem durch seine onkogenen Funktionen mit Krebs in Verbindung gebracht wurde - bei der Seneszenz-assoziierten Zytokinausschüttung mitwirkt und den seneszenzten Phänotyp möglicherweise sogar verstärkt, wodurch NF-kappaB potentiell eine tumorsuppressive Rolle zukäme. Ziel dieser Arbeit ist die Untersuchung des NF-kappaB-Signalweges in Seneszenz und Therapie. In der vorliegenden Arbeit zeige ich die deutliche Aktivierung von NF-kappaB nach Therapie-induzierter Seneszenz (therapy-induced senescence, TIS) und erhöhte Expression NF-kappaB-regulierter Zytokine. TIS ist vor allem in vivo mit starker Aktivität des NF-kappaB-Signalweges assoziiert und von selbiger abhängig. Primäre Eµ-myc-transgene Mauslymphome wurden nach ihrer endogenen NF-kappaB-Aktivität klassifiziert bzw. mit inhibierenden und aktivierenden NF-kappaB-Konstrukten modifiziert, welche auch in diffusen großzelligen B-Zell Lymphomen (diffuse large B-cell lymphoma, DLBCL) als natürlich vorkommende Mutationen gefunden wurden. Über einen neuartigen „Cross-Species“-Vergleich wurden Bcl2-hochexprimierende Keimzentrums-B-Zell-DLBCL (germinal center B-cell type, GCB) als klinisch relevante Gruppe identifiziert, welche nach NF-kappaB-Hyperaktivierung signifikant besser auf Therapie ansprach. Diese Ergebnisse zeigen eine kontextspezifische, d. h. von „onkogenen Netzwerken“ abhängige Rolle des NF-kappaB Signalweges unter Chemotherapie. Diese Information könnte für künftige klinische Studien bedeutsam sein, da sie Bedingungen aufzeigt, unter denen NF-kappaB als Vermittler einer erwünschten Therapie-induzierten Seneszenzantwort eher nicht inhibiert werden sollte. / Cellular senescence is a terminal cell-cycle arrest program that is executed in response to cellular stresses, such as activated oncogenes or DNA-damaging anti-cancer chemotherapy, where it serves as a tumor-suppressive mechanism or contributes to treatment outcome, respectively. Recently, transcription factor NF-kappaB which has long been linked to cancer development primarily through its oncogenic functions, has been postulated to participate in a senescence-associated and possibly senescence-reinforcing cytokine response, thereby suggesting a tumor-restraining role for NF-kappaB. The aim of my PhD project was to understand the role of the NF-kappaB pathway in senescence and cancer treatment outcome. In this thesis, I show markedly elevated NF-kappaB activity upon therapy-induced senescence (TIS), associated with strong upregulation of NF-kappaB-controlled cytokines. TIS is associated with and depends on hyper-activated NF-kappaB signaling. By characterization and genetic engineering of primary mouse lymphomas according to distinct NF-kappaB-related oncogenic networks reminiscent of diffuse large B-cell lymphoma (DLBCL) subtypes, Bcl2-overexpressing germinal center B-cell-like (GCB) DLBCL were identified as a clinically relevant subgroup with significantly superior outcome when NF-kappaB is hyperactive. These results demonstrate the context-dependent role of NF-kappaB signaling in cancer therapy and unveil oncogenic scenarios in which NF-kappaB hyperactivity unexpectedly accounts for superior long-term outcome to therapy. This finding has significant ramifications for future clinical trials that aim at inhibiting NF-kappaB activity based on the assumption of its detrimental impact on treatment outcome.

By 2040, the World Health Organization expects neurodegenerative diseases, such as Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), and Parkinson’s disease, to surpass cancer as the second most common cause of death worldwide. Currently, only treatments for symptoms of these diseases are available. Thus, research is critical to alleviate this public health burden by elucidating the pathogenic processes and developing novel therapies. While exact mechanisms by which these heterogeneous neuropathological conditions become manifest in patients remain unclear, growing evidence suggests that oxidative stress (OS) makes a significant contribution to neuronal dysfunction and apoptosis in all major neurodegenerative diseases. Recently, the gene oxidation resistance 1 (Oxr1) has emerged as a critical regulator of neuronal survival in response to OS. Oxr1 is expressed throughout the central nervous system, and its highly conserved TLDc domain protects neurons from oxidative damage through an unknown mechanism. This thesis aimed to define mechanisms by which Oxr1 confers neuronal sensitivity to OS, and to determine its role in neurodegenerative diseases. I found that Oxr1 mediates cytoplasmic localization of ALS-associated proteins Fused in Sarcoma (FUS) and transactive response DNA binding protein 43 kDa (TDP-43) through a TLDc domain- and arginine methylation-dependent pathway. Next, I investigated in vivo neuroprotective functions of Oxr1, and demonstrated that neuronal Oxr1 over-expression extends survival and ameliorates behavioural dysfunction and pathology of an ALS mouse model. In particular, neuronal Oxr1 over-expression strikingly delays neuroinflammation during ALS pathogenesis. Finally, I characterised a mouse model that specifically deletes Oxr1 from motor neurons. While loss of Oxr1 in ChAT-positive motor neurons does not cause overt neurodegeneration in the spinal cord, constitutive loss of Oxr1 leads to neuroinflammation in the cerebellum and spinal cord. Taken together, these studies illuminate functions of Oxr1 in the complex antioxidant defence network and present implications for future therapeutic strategies.

La polykystose rénale autosomique dominante (ADPKD) est une des maladies génétiques les plus communes. ADPKD se manifeste le plus souvent au stade adulte par la présence de kystes rénaux, et bien souvent de kystes hépatiques, avec une progression très variable. ADPKD mène à une insuffisance rénale: les seuls recours sont la dialyse puis la transplantation rénale. Les mutations dispersées sur les gènes PKD1 (majoritairement; la protéine polycystine-1, PC1) et PKD2 (la protéine polycystine-2, PC2) sont responsables de l’ADPKD. Le mécanisme pathogénétique de perte de fonction (LOF) et donc d’un effet récessif cellulaire est évoqué comme causatif de l’ADPKD. LOF est en effet supporté par les modèles murins d’inactivation de gènes PKD1/PKD2, qui développent de kystes, quoique in utéro et avec une rapidité impressionnante dans les reins mais pas dans le foie. Malgré de nombreuses études in vitro, le rôle de PC1/PC2 membranaire/ciliaire reste plutôt hypothétique et contexte-dépendant. Ces études ont associé PC1/PC2 à une panoplie de voies de signalisation et ont souligné une complexité structurelle et fonctionnelle exceptionnelle, dont l’implication a été testée notamment chez les modèles de LOF. Toutefois, les observations patho-cellulaires chez l’humain dont une expression soutenue, voire augmentée, de PKD1/PC1 et l’absence de phénotypes extrarénaux particuliers remet en question l’exclusivité du mécanisme de LOF. Il était donc primordial 1) d’éclaircir le mécanisme pathogénétique, 2) de générer des outils in vivo authentiques d’ADPKD en terme d’initiation et de progression de la maladie et 3) de mieux connaitre les fonctions des PC1/PC2 indispensables pour une translation clinique adéquate. Cette thèse aborde tous ces points. Tout d’abord, nous avons démontré qu’une augmentation de PKD1 endogène sauvage, tout comme chez l’humain, est pathogénétique en générant et caractérisant en détail un modèle murin transgénique de Pkd1 (Pkd1TAG). Ce modèle reproduit non seulement les caractéristiques humaines rénales, associées aux défauts du cil primaire, mais aussi extrarénales comme les kystes hépatiques. La sévérité du phénotype corrèle avec le niveau d’expression de Pkd1 ce qui supporte fortement un modèle de dosage. Dans un deuxième temps, nous avons démontré par les études de complémentations génétiques que ces deux organes reposent sur une balance du clivage GPS de Pc1, une modification post-traductionelle typique des aGPCR, et dont l’activité et l’abondance semblent strictement contrôlées. De plus, nous avons caractérisé extensivement la biogénèse de Pc1 et de ses dérivés in vivo générés suite au clivage GPS. Nous avons identifié une toute nouvelle forme et prédominante à la membrane, la forme Pc1deN, en plus de confirmer deux fragments N- et C-terminal de Pc1 (NTF et CTF, respectivement) qui eux s’associent de manière non-covalente. Nous avons démontré de façon importante que le trafic de Pc1deN i.e., une forme NTF détachée du CTF, est toutefois dépendant de l’intégrité du fragment CTF in vivo. Par la suite, nous avons généré un premier modèle humanisant une mutation PKD1 non-sens tronquée au niveau du domaine NTF(E3043X) en la reproduisant chez une souris transgénique (Pkd1extra). Structurellement, cette mutation, qui mimique la forme Pc1deN, s’est également avérée causative de PKD. Le modèle Pkd1extra a permis entre autre de postuler l’existence d’une cross-interaction entre différentes formes de Pc1. De plus, nos deux modèles murins sont tous les deux associés à des niveaux altérés de c-Myc et Pc2, et soutiennent une implication réelle de ces derniers dans l’ADPKD tou comme une interaction fonctionnelle entre les polycystines. Finalement, nous avons démontré un chevauchement significatif entre l’ADPKD et le dommage rénal aigüe (ischémie/AKI) dont une expression augmentée de Pc1 et Pc2 mais aussi une stimulation de plusieurs facteurs cystogéniques tel que la tubérine, la β-caténine et l’oncogène c-Myc. Nos études ont donc apporté des évidences cruciales sur la contribution du gène dosage dans l’ADPKD. Nous avons développé deux modèles murins qui serviront d’outil pour l’analyse de la pathologie humaine ainsi que pour la validation préclinique ADPKD. L’identification d’une nouvelle forme de Pc1 ajoute un niveau de complexité supplémentaire expliquant en partie une capacité de régulation de plusieurs voies de signalisation par Pc1. Nos résultats nous amènent à proposer de nouvelles approches thérapeutiques: d’une part, le ciblage de CTF i.e., de style chaperonne, et d’autre part le ciblage de modulateurs intracellulaires (c-Myc, Pc2, Hif1α). Ensemble, nos travaux sont d’une importance primordiale du point de vue informatif et pratique pour un avancement vers une thérapie contre l’ADPKD. Le partage de voies communes entre AKI et ADPKD ouvre la voie aux approches thérapeutiques parallèles pour un traitement assurément beaucoup plus rapide. / Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common genetic diseases. ADPKD is manifested by the presence of renal cysts detected most often in the adult stage, and frequently liver cysts, with highly variable progression. ADPKD leads to kidney failure with the only recourse of dialysis and eventual kidney transplantation. Mutations dispersed throughout the PKD1 gene (major player, the polycystin-1 protein, PC1) and the PKD2 gene (polycystin-2 protein, PC2) are responsible for ADPKD. The loss of function (LOF) pathogenetic mechanism, and therefore a cellular recessive effect, has been suggested as causative of ADPKD. LOF is indeed supported by the PKD1/PKD2 gene inactivation mouse models, which develop cysts, although in utero with impressive speed in the kidney but not in the liver. Despite many in vitro studies, the membrane/ciliary role of PC1/PC2 remains rather hypothetical and context-dependent. These studies have associated PC1/PC2 to a variety of signaling pathways and underlined exceptional structural and functional complexity, whose involvement has been tested especially in LOF models. However, pathocellular observations in humans with sustained and even increased expression of PKD1/PC1, and the absence of particular human extrarenal phenotypes questions the exclusivity of the LOF mechanism. It was therefore essential 1) to clarify the pathogenetic mechanism, 2) to generate in vivo tools authentic of ADPKD in terms of initiation and progression of the disease and 3) to better understand the essential functions of PC1/PC2 for an adequate clinical translation. This thesis addresses all of these issues. First, we demonstrated that an increase in endogenous PKD1, just like in humans, is pathogenetic by generating and characterizing in detail a transgenic mouse model of Pkd1 (Pkd1TAG). This model not only reproduces the renal human characteristics associated with defects of the primary cilium, but also the extrarenal, namely, liver cysts. The severity of the phenotype correlates with the expression level of Pkd1, which strongly supports a dosage model. Secondly, we have demonstrated with genetic complementation studies that these two organs rely on a balance of Pc1 GPS cleavage, a typical post-translational modification of aGPCR, whose activity and abundance seem strictly controlled. Furthermore, we have extensively characterized Pc1 biogenesis and its derivatives in vivo generated upon GPS cleavage. We have identified a new form, predominantly on the membrane, the Pc1deN form, in addition to confirming the two N- and C-terminal Pc1 fragments (NTF and CTF, respectively), which associate non-covalently. Importantly, we have demonstrated that traffic of Pc1deN i.e., the NTF form detached from the CTF, is still dependant on the integrity of the CTF fragment. Next, we generated a first model humanizing a PKD1 nonsense truncated mutation at the level of the NTF(E3043X) domain by reproducing it in a transgenic mouse (Pkd1extra). Structurally, this mutation, which mimics Pc1deN, has also been shown to be causative of PKD. The Pkd1extra model allowed the proposition of the existence of a cross-interaction between different forms of Pc1. In addition, our two mouse models are both associated with altered levels of c-Myc and Pc2, which is supportive of their involvement in ADPKD and a functional interaction between the polycystins. Finally, we have shown a significant overlap between ADPKD and acute renal injury (ischemia/AKI) namely increased expression of Pc1 and Pc2 but also stimulation of several cystogenic factors such as tuberin, β-catenin and the oncogene c-Myc. Our studies have therefore given crucial evidence to the contribution of PKD1 gene dosage mechanism in ADPKD. We have developed two mouse models, which can serve as a tool for the analysis of human pathology as well as for preclinical validation of ADPKD. The identification of a new form of Pc1 adds an additional level of complexity in part explaining the regulation capacity of Pc1 on several signaling pathways. Our findings lead us to propose new therapeutic approaches: firstly, targeting the CTF i.e., chaperone style, and also targeting intracellular modulators (c-Myc, Pc2, Hif1α). Together, our work is of paramount importance in an informative point of view and practical perspective for progress towards a therapy for treating ADPKD. The sharing of common pathways between AKI and ADPKD paves the way for parallel therapeutic approaches for assured much faster treatment.

Aristolochic acids (AA) are human carcinogens which have also very strong nephrotoxic properties. A mixture of AA is present in Aristolochiacae plant species. These plants were and still are used in traditional medicine in some countries, particularly in Asia. Aristolochic acids participate in development of two types of nephropathies. The first disease is designated as Aristolochic Acid Nephropathy (AAN), the second one is Balkan Endemic Nephropathy (BEN). Both nephropathies are associated with urothelial malignancies, which are caused by AA. One of the common features of ANN and BEN is that not all individuals exposed to AA suffer from nephropathy and tumour development. One cause for these different responses may be individual differences in the activities and expression levels of the enzymes catalyzing the biotransformation of AAI, the major toxic component of AA contained in Aristolochia species. Detailed knowledge of enzymes which participate in metabolism of AAI may contribute to elucidation of inter-individual susceptibility to AAN, BEN and later urothelial malignancies. Aristolochic acid I is either oxidative detoxicated or reductive activated by biotransformation enzymes. Reductive bioactiovation of AAI leads to formation of covalent AA-DNA adducts in organism which result in producing of...