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Allogeneic Hematopoietic stem cell transplants (HSCT) are used for the treatment of bone marrow aplasias. Allogeneic HSCT is performed by treating the patient with chemotherapy drugs and irradiation and then transplanting hematopoietic stem cells from a healthy donor to restore the immune system and hematopoietic cells. Allogeneic HSCTs has the added benefit of the graft vs leukemia effect (GvL), whereby donor allogeneic T cells are able to mount immune responses against any residual cancer cells. However, alloreactivity towards the mismatched minor and major histocompatibility antigens the patient's healthy tissues leads to graft vs host disease (GvHD). This process is also mediated by Macrophages, Dendritic cells, B cells. Furthermore, a decrease in the number of NK, B, and T regulatory cells exacerbates GvHD. This leads to a state of systemic inflammation, tissue damage and multiorgan fibrosis. Current therapies designed to suppress the immune system have been shown to be efficacious in preventing GvHD but patients become susceptible to infection or experience cancer relapse through the elimination of the GvL response as well. In this thesis, we explore two strategies for targeting T cell activation in two mouse models of GvHD. In the first model, we examined the contribution of donor-derived complement C5 on the induction GvHD. We observed that recipient mice were only protected from GvHD when donor cells were deficient for complement protein C5. Our second strategy involves selective targeting of alloreactive T cells using peptide immunotherapy. For this approach, we first developed a humanized mouse model of GvHD whereby cells from donor mice expressing human class II HLA were reconstituted into recipient mice expressing human class I HLA. We then tested peptide immunotherapy using peptides derived from the human class I HLA. Our initial results were inconclusive and require further optimization. / Thesis / Doctor of Philosophy (PhD) / Graft vs Host Disease is an unwanted side effect of mismatched bone marrow transplant. Donor T cells recognize and attack mismatched tissues of the recipient and this leads to systemic inflammation and tissue scarring. Current treatments primarily target T-cell activation by suppressing the immune system, however, this leaves the patients susceptible to recurrent infections. In this thesis we describe the creation of two mouse models of Graft vs Host Disease and then examine two ways of specifically targeting donor T cell activation that is designed not to affect normal immune responses.

Background The inducible Cre-lox system is a valuable tool to study gene function in a spatial and time restricted fashion in mouse models. This strategy relies on the limited background activity of the modified Cre recombinase (CreER) in the absence of its inducer, the competitive estrogen receptor ligand, tamoxifen. The RIP-CreER mouse (Tg (Ins2-cre/Esr1) 1Dam) is among the few available β-cell specific CreER mouse lines and thus it has been often used to manipulate gene expression in the insulin-producing cells of the endocrine pancreas. Principal Findings Here, we report the detection of tamoxifen-independent Cre activity as early as 2 months of age in RIP-CreER mice crossed with three distinct reporter strains. Significance Evidence of Cre-mediated recombination of floxed alleles even in the absence of tamoxifen administration should warrant cautious use of this mouse for the study of pancreatic β-cells.

Hearing loss (HL) is one of the most common sensorineural disorders and several dozen genes contribute to its pathogenesis. Establishing a genetic diagnosis of HL is of great importance for clinical evaluation of deaf patients and for estimating recurrence risks for their families. Efforts to identify genes responsible for HL have been challenged by high genetic heterogeneity and different ethnic-specific prevalence of inherited deafness. Here we present the utility of whole exome sequencing (WES) for identifying candidate causal variants for previously unexplained nonsyndromic HL of seven patients from four unrelated Altaian families (the Altai Republic, South Siberia). The WES analysis revealed homozygous missense mutations in three genes associated with HL. Mutation c.2168A>G (SLC26A4) was found in one family, a novel mutation c.1111G>C (OTOF) was revealed in another family, and mutation c.5254G>A (RAI1) was found in two families. Sanger sequencing was applied for screening of identified variants in an ethnically diverse cohort of other patients with HL (n = 116) and in Altaian controls (n = 120). Identified variants were found only in patients of Altaian ethnicity (n = 93). Several lines of evidences support the association of homozygosity for discovered variants c.5254G>A (RAI1), c.1111C>G (OTOF), and c.2168A>G (SLC26A4) with HL in Altaian patients. Local prevalence of identified variants implies possible founder effect in significant number of HL cases in indigenous population of the Altai region. Notably, this is the first reported instance of patients with RAI1 missense mutation whose HL is not accompanied by specific traits typical for Smith-Magenis syndrome. Presumed association of RAI1 gene variant c.5254G>A with isolated HL needs to be proved by further experimental studies.

Aristolochic acids (AA) are carcinogenic and nephrotoxic alkaloids from Aristolochia species. Aristolochic acid I (AAI), the major component of AA, causes the development of Aristolochic acid nephropathy (AAN) and Balkan endemic nephropathy (BEN). These two diseases cause total renal failure and urothelial malignancies. The fact that these diseases have not been developed in all persons, who have been exposed to their action, might be causd by different activities and protein levels of the enzymes metabolizing AAI. Thus, the identification of enzymes involved in the metabolism, and detailed knowledge of their expression and catalytic specifities is a major importance. Aristolochic acid I (AAI) can be metabolized by several types of reactions. Like most nitroaromatics, the main activation pathway of AAI is reduction of its nitro group to form a cyclic acylnitrenium ion, which can bind to the purine bases, thereby forming AAI-DNA adducts. The detoxication pathway of AAI is its oxidative demethylation by cytochromes P450 forming detoxication metabolite 8-hydroxyaristolochic acid Ia (AAIa). In the present thesis, using rat and human enzymes and as well as several mice models, the metabolism of AAI in vitro and in vivo was investigated. The first model has deleted gene for NADPH:cytochrome P450...

Beyond its essential roles in embryonic development, the Wnt-mediated signal transduction cascade is critically implicated in homeostasis of adult tissues. In the gastrointestinal epithelium, the threshold of active Wnt signaling is kept in a physiological range by a spectrum of regulatory networks and loops, thereby balancing the opposing processes of cell fate determination, proliferation and stem cell self-renewal. Furthermore, compelling evidence undoubtedly link an aberrant Wnt activity to the onset of bowel cancer. Understanding the principle causes and effects secondary to excessive Wnt signaling can provide valuable insights into the pathology of the malignant transformation of the colorectum. The proposed thesis attempts to focus on novel modes of the Wnt pathway modulation; both general and context-specific nuances of the Wnt level adjustment are thereby delineated. The results are presented in three distinct research publications and one review article. The first study examines the contribution of the distinct post-translational modifications, which the Wnt proteins undergo, to their proper processing, secretion and signaling activity. First, we investigated the sequential order and mutual interdependence of cysteine and serine-linked fatty acylation and N-linked glycosylation of murine...

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.

La méthylation de l'ADN est un processus vital pour le développement des mammifères. Sa distribution anormale,notamment au niveau des régions répétées du génome, est une signature pathologique. La découverte de maladies héréditaires touchant la stabilité du génome a permis des avancées considérables dans l'identification des acteurs et des mécanismes. Nous avons choisi d'étudier le syndrome ICF (Immunodéficience, instabilité Centromérique et anomalies Faciales), première maladie génétique identifiée avec des défauts de la méthylation de l’ADN, liés à une instabilité chromosomique. Lorsque j'ai commencé ma thèse, des mutations dans les gènes DNMT3B et ZBTB24 avaient été décrites comme causes génétiques du syndrome. Cependant, d'autres causes génétiques restaient inconnues. Nos travaux ont permis d'identifier deux nouveaux gènes, CDCA7 et HELLS, dont les mutations sont responsables du syndrome. J'ai montré que leur perte de fonction dans les cellules somatiques entraîne un défaut de méthylation des répétitions centromériques, suggérant leur rôle dans le maintien de la méthylation de l'ADN. Par conséquent, l'étude de l'étiologie d'une maladie génétique rare a permis d'identifier de nouveaux « gardiens » de la stabilité du génome, avec des fonctions jusqu'alors insoupçonnées dans les processus de méthylation de l'ADN et dans le développement. Au cours de mon doctorat, j'ai établi des cartes de méthylation des cellules de patients ICF afin d'identifier les cibles communes et distinctes de ces facteurs, ainsi que leurs caractéristiques génomiques et épigénomiques. Contrairement aux mutations de DNMT3B,celles de ZBTB24, CDCA7 et HELLS affectent la méthylation dans des régions pauvres en CpG, dans des régions intergéniques et dans des répétitions d'ADN intercalées. Plus généralement, ce sont les régions d'hétérochromatine qui sont les plus touchées et en particulier des clusters des gènes codants et non codants, dont certains sont exprimés de manière monoallélique. Pour mieux caractériser le rôle de ZBTB24 dans le développement et la méthylation de l'ADN,nous avons généré un modèle murin mutant qui nous a permis de monter que ZBTB24 était essentielle pour le développement embryonnaire précoce. De plus, ZBTB24 jouerait un rôle dans l'établissement de la méthylation des séquences répétées de l'ADN, à la fois en tandem ou intercalé. Fait intéressant, ZBTB24 semble être également impliqué dans l'établissement de la marque répressive H3K9me3, suggérant un rôle de la protéine dans le "dialogue" entre la méthylation de l'ADN et celle des histones. Dans l'ensemble, mon travail met l'accent sur la façon dont la méthylation de l'ADN et les marques d'hétérochromatine sont établies et maintenues à des gènes uniques et des répétitions de l'ADN, et fournit de nouveaux acteurs et mécanismes à considérer dans les études sur le maintien de la stabilité du génome. / DNA methylation is an essential process for the development of mammals. Its abnormal distribution, particularly at the level of the repeated regions of the genome, is a pathological signature. The discovery of hereditary diseases affecting DNA methylation and the stability of the genome allowed a considerable progress in the identification of their actors and mechanisms. We chose to study the ICF (Immunodeficiency, Centromeric Instability and Facial Abnormalities) syndrome, the first genetic disorder identified with defects in the distribution of DNA methylation, linked to chromosomal instability. When I started my PhD, mutations in two genes had been described to cause the ICF syndrome: DNMT3B and ZBTB24. However, the genetic origin of a subset of ICF patients remained unknown. We identified mutations in CDCA7 and HELLS as causative of the ICF syndrome. I showed that their loss of function in somatic cells results in the loss of DNA methylation at centromeric repeats, strongly suggestive of a role DNA methylation maintenance. Hence, the study of the aetiology of a genetic disease provided new candidate “guardians” of DNA repeats and genome stability, with virtually unknown functions but with exciting potential roles in the DNA methylation machinery and in development. During my PhD, I established methylation maps in ICF patients cells to identify common and distinct targets of these factors, as well as their genomic and epigenomic characteristics. In contrast to DNMT3B mutations, those in ZBTB24, CDCA7 and HELLS affect methylation at CpG-poor regions in intergenic genomic locations and at interspersed DNA repeats, and more generally, at genomic locations with heterochromatic features. Their integrity is required for the methylated status of coding and non-coding clusters of genes, some of which are expressed in a monoallelic manner. To better characterize the role of ZBTB24 in development and DNA methylation pathways, we generated a mouse model carrying mutations in ZBTB24. We showed that ZBTB24 is essential for early development, while it seemed to be dispensable for in vitro differentiation of murine ES cells. We implicated ZBTB24 in the establishment of DNA methylation at DNA repeats, both in tandem or interspersed, in differentiating ES cells. Interestingly, ZBTB24 seems to be also implicated in the establishment of the repressive mark H3K9me3 suggesting that ZBTB24 may indirectly control DNA methylation through an interplay with histone marks. As a whole, our work sheds light on how DNA methylation and heterochromatin marks are established and maintained at unique genes and DNA repeats, and provides new actors and mechanisms to consider in studies of the maintenance of genome stability.