La maladie de Lesch-Nyhan (MLN) est une maladie rare dont la prévalence est estimée à une naissance sur 380 000. Il s’agit d’une maladie métabolique d’origine génétique liée au chromosome X, impliquant le gène de la HGPRT (hypoxanthine-guanine-phosphoribosyl-transférase). Les mutations dans ce gène entrainent un déficit d’activité de l’enzyme qui joue un rôle central dans le métabolisme des purines. Cette baisse d’activité provoque un défaut de fonctionnement de la voie de recyclage des purines induisant l’accumulation d’acide urique dans les articulations (goutte) et les reins (lithiase) qui peut être contrôlée par la prise d’allopurinol. En revanche, ces symptômes sont généralement accompagnés de troubles neurologiques quant à eux encore totalement inexpliqués et non traités. Ces derniers se traduisent par un handicap moteur important avec des mouvements anormaux (dystonie) et des troubles du tonus (hypotonie axiale). Dans les cas les plus graves des troubles du comportement peuvent également survenir se manifestant par des évènements importants d'automutilation (morsure des lèvres et des doigts). Des études menées en imagerie cérébrale, ont permis d’identifier chez les malades une diminution de la concentration cérébrale en dopamine mais aucune étude n’a encore pu lier de façon évidente le déficit en dopamine et la HGPRT, rendant difficile le développement de thérapies efficaces. L’objectif de ce travail a été de tirer avantage des propriétés d’auto renouvellement et de pluripotence des cellules souches induites à la pluripotence humaines (iPSC) pour produire d'authentiques neurones dopaminergiques (nDA) puis de les utiliser afin de déterminer dans quelle mesure la HGPRT est essentielle au développement et à l’homéostasie des nDA. Pour cela nous avons sélectionnés des fibroblastes obtenus à partir de biopsie de peau d’enfants atteints par la MLN que nous avons reprogrammé en iPSC. Ces iPSC ont été caractérisées et en particulier, l’expression protéique et l’activité enzymatique de la HGPRT a été contrôlée afin de valider notre modèle pathologique. Un protocole de différenciation de neurones dopaminergiques à partir d’iPSC a ensuite été mis au point pour permettre l’étude des différents stades de développement des neurones DA. Celui-ci permet d’obtenir des précurseurs exprimant pour 60% des cellules les marqueurs caractéristiques des précurseurs du mésencéphale ventral (MV), tout en respectant les différentes étapes clés du développement des nDA. Après l’induction de la différenciation des précurseurs en neurones, la population neuronale est composée d’au moins 20% de nDA exprimant les deux enzymes de la voie de synthèse de la dopamine TH et AADC. Ces différentes étapes du développement des nDA ont ensuite été analysées en comparant des cellules porteuses de mutations associées à la MLN et des cellules contrôles. Une anomalie neuro-développementale survenant à un stade précoce de la formation des nDA a été identifiée. Lors de l’étape ultime de différenciation, la proportion de précurseurs du MV capables de sortir du cycle cellulaire et donnant des neurones matures est plus faible dans les cultures de cellules porteuses des mutations MLN. Cette étude a permis dans un premier temps de démontrer qu’il était possible de modéliser une maladie impactant un gène essentiel dans le métabolisme à l’aide des iPSC. De plus, nous avons montré qu’il est possible d’identifier l’étape critique dans la genèse des nDA en utilisant des neurones humains dérivés d’iPSC issus d’enfants atteints de MLN. Cette approche apporte ainsi une meilleure compréhension sur les mécanismes responsables de la pathologie, et de nouveaux axes de recherche pour des approches thérapeutiques. / Lesch-Nyhan disease (LND) is a rare genetic disease with a prevalence estimated to 1:380,000. LND is a metabolic Xchromosome linked disorder that essentially affects boys and involves HGPRT gene (hypoxanthine-guanine phosphoribosyltransferase). Mutations in this gene result in a deficit of enzyme activity that plays a central role in the metabolism of purines. This activity deficiency induces a dysfunction on purine recycling pathway and promotes accumulation of uric acid in the joints (gout) and kidneys (stones) controlled using allopurinol. Next to these metabolic symptoms are neurological disorders which are not understood and efficiently controlled. These involve abnormal movements (dystonia) and low tonus (axial hypotonia). A unique feature of LND is the occurrence of self-injurious behaviors known as SIB (biting of lips and fingers). Brain imaging studies have revealed in LND patient a decrease of cerebral dopamine concentration but no study has yet been able to clearly link dopamine defect and HGPRT loss of activity, making it difficult to develop effective therapies. The aim of my study was to take advantage of the self-renewal and pluripotency properties of human induced pluripotent stem cells (iPSC) to produce dopaminergic neurons (nDA), then to use them to determine in which extend HGPRT is essential to the development and homeostasis of nDA. To that purpose, we selected fibroblasts obtained from skin biopsies of LND children that we have reprogrammed into iPSC. These iPSC were characterized and in particular, protein expression and enzymatic activity of HGPRT was assessed to validate our pathological model. We developed a protocol to differentiate dopaminergic neurons from iPSC to allow the study of different stages of nDA development. It provides mature precursors of nDA, expressing the typical marker of ventral midbrain (VM), while respecting the different key stages of nDA development. Upon terminal differentiation, these precursors produce at least 20% of nDA that express the two main enzymes of the dopamine synthesis pathway, namely TH and AADC. These different stages of nDA development were analyzed comparing LND and control IPSC. Neurodevelopmental abnormality occurring at an early stage of nDA formation was identified. At the final stage of differentiation, the proportion of MV precursors able to exit the cell cycle and differentiate as mature neurons is lower in LND culture compared to controls. This study provided evidences that it is possible to model a metabolic disease with iPSC and that they are essential tools to study neurodevelopmental disorders. This approach provides a better understanding of mechanisms responsible for the disease, and new research directions for therapeutic approaches.

Les mutations affectant la fonction d'enzymes impliquées dans le cycle des purines sont responsables d'une multitude de syndromes pédiatriques, caractérisés par des atteintes neurologiques et comportementales. A ce jour, aucune stratégie thérapeutique n'a été réellement efficace pour contrôler ces symptômes. La maladie de Lesch-Nyhan (MLN), associée à la perte de fonction de l'enzyme de recyclage HGPRT, constitue un bon modèle d'étude. Mon travail a consisté à utiliser la technologie des cellules souches induites à la pluripotence, reprogrammées à partir de fibroblastes de patients atteints des formes sévères de la MLN, pour identifier des phénotypes neuronaux associés à la perte de fonction de l'HGPRT. Ces marqueurs phénotypiques ont ensuite été utilisés pour identifier, par une approche de criblage à haut débit, de nouvelles molécules chimiques capables de corriger ces défauts. Plus de 3000 molécules ont été testées et 6 composés, tous dérivés de l'adénosine, ont pu être identifiés comme compensant le métabolisme par un mécanisme d'action indépendant de l'HGPRT. De manière intéressante, un des composés, la S-adenosylmethionine (SAM) a par le passé déjà démontré des effets bénéfiques sur les symptômes comportementaux typiques de la MLN dans plusieurs études de cas. Cela démontre que la stratégie abordée ici a permis l'identification de cibles thérapeutiques permettant d'améliorer les symptômes neurospychiatriques de cette pathologie et constitue un modèle réplicable pour différentes pathologies touchant le métabolisme cérébral. / Mutations in genes coding for enzymes involved in purine synthesis or recycling lead to dramatic neurological conditions with poor pharmacological options. Lesch–Nyhan disease (LND) is caused by deficiency of the salvage pathway enzyme HGPRT that compromises recycling of guanine and hypoxanthine into GMP and IMP. LND is characterized by severe neuropsychiatric symptoms that are out of reach of pharmacological treatments. Here we use human cortical neural stem cells and neurons derived from iPSC of children affected by severe forms of LND to identify neural phenotypes associated with HGPRT-deficiency and of interest to develop a target-agnostic based drug screening system. We screened more than 3000 molecules and identified 6 compounds, all possessing an adenosine moiety, that corrected LND related neuronal phenotypes by promoting metabolism compensations in a HGPRT-independent manner. One of these compound, S-adenosylmethionine (SAM), has already been reported as providing amelioration of behavioral symptoms in some LND cases, demonstrating that our screening allowed the identification of pathways that can be relevant therapeutic targets to ease the devastating neuropsychiatric symptoms associated with this pathology. Interestingly, these pathways can be activated in LND patients via simple food supplementation. This experimental paradigm can also be easily adapted to other purine associated neurological disorders affecting normal brain development.

Submitted by Luciana Sebin (lusebin@ufscar.br) on 2016-10-11T12:04:16Z No. of bitstreams: 1 DissACMF.pdf: 2760486 bytes, checksum: 27824647d350872bddb22f28a9206da8 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-10-20T13:38:00Z (GMT) No. of bitstreams: 1 DissACMF.pdf: 2760486 bytes, checksum: 27824647d350872bddb22f28a9206da8 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-10-20T13:38:07Z (GMT) No. of bitstreams: 1 DissACMF.pdf: 2760486 bytes, checksum: 27824647d350872bddb22f28a9206da8 (MD5) / Made available in DSpace on 2016-10-20T13:38:15Z (GMT). No. of bitstreams: 1 DissACMF.pdf: 2760486 bytes, checksum: 27824647d350872bddb22f28a9206da8 (MD5) Previous issue date: 2016-02-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / The mansoni schistosomiasis is the most important of human helminthiasis. Despite advances in its control this disease continues to spread to new geographical areas. It currently affects more than 250 million people. However, limited options are available for and Praziquantel is the drug of choice. Various authors have been searching new drugs and vaccines to control schistosomiasis. This study aimed to evaluate the effects of a prior immunization with recombinant enzymes of Schistosoma mansoni: Adenosine Kinase (AK) and Hypoxanthine-guanine Phosphoribosyltransferase (HGPRT), which are important for parasite purine metabolism, as well as a MIX of these enzymes, and subsequent challenge with cercariae of the parasite in the control of murine infection. Female Balb/c mice were divided into 5 groups. The groups were enzyme-immunized in three doses and 15 days after the last immunization, animals were infected with S. mansoni. After infection in the 47º day egg count were carried in mice faeces and in the 48º day mice were sacrificed for evaluation of leukocyte numbers (blood and peritoneal cavity), worm burden, antibodies production, cytokines quantification and histopathological analysis of the liver of these animals. Our results strongly suggest that, immunization with a MIX originated in these animals reduction in the number of eggs in faeces by 46% when compared with the animals of the infected group. Animals of the groups immunized with AK, HGPRT and/or MIX seem to induce a reduction in the number of eosinophils in the peritoneal cavity when compared to the animals of the infected group. Concerning worm burden, the animals of the MIX group presented greater reduction (31.27%) when compared to the animals of the infected group. The animals of the immunized groups, AK, HGPRT and/or MIX were capable of producing IgG1 antibodies and IgE anti the enzymes and anti the parasite proteins. The animals of the immunized group MIX showed a slight increase in IL-4 production and observed reduction of IL-10, and in the HGPRT group induced a slight increase on IFN-γ production when in compared with the infected group. In addition, the animals of the AK group showed a decrease in the number of hepatic granulomas in tissue (44,55%) and the eggs present in liver (42,31%). Therefore, it suggests that immunization with these enzymes can contributes to schistosomiasis control, as well as it might helps to modulate experimental infection inducing reduction of physiopathology of this parasitosis. / A esquistossomose mansônica é a mais importante das helmintíases humanas. Apesar dos avanços no seu controle continua se espalhando para novas áreas geográficas. Atualmente afeta mais de 250 milhões de pessoas. Entretanto, opções limitadas estão disponíveis para o tratamento da doença e o único fármaco de escolha é o Praziquantel. Assim, vários estudos têm sido propostos para encontrar novos fármacos e vacinas para combater a esquistossomose. Dessa forma, o presente estudo teve como proposta avaliar os efeitos da imunização prévia com as enzimas recombinantes de Schistosoma mansoni Adenosina Quinase (AK) e Hipoxantina-Guanina Fosforibosiltransferase (HGPRT), que participam do metabolismo de purinas do parasito, bem como com o MIX das duas enzimas, e posterior desafio com cercárias do parasito, para o controle da infecção murina. Camundongos fêmea Balb/c foram divididos em 5 grupos. Os grupos imunizados receberam três doses das enzimas e após 15 dias da última imunização, os animais foram infectados com S. mansoni. Após a infecção, no 47° dia foi realizada a contagem de ovos nas fezes e no 48° dia foi realizada a eutanásia dos animais para avaliação de resposta leucocitária (sangue e lavado da cavidade peritoneal), carga parasitária, produção de anticorpos, quantificação de citocinas e análise histopatológica do fígado desses animais. Os resultados demonstraram que, a imunização com o MIX promoveu nesses animais redução do número de ovos nas fezes de 46% quando comparado com os animais do grupo somente infectado. Os animais dos grupos imunizados com AK, HGPRT e/ou MIX apresentaram diminuição na quantidade de eosinófilos na cavidade peritoneal quando comparados com os animais do grupo somente infectado. Em relação à carga parasitária, os animais do grupo imunizado com o MIX apresentaram maior redução (31,27%) quando comparados aos animais do grupo somente infectado. Os animais dos grupos imunizados com AK, HGPRT e/ou MIX foram capazes de produzir anticorpos IgG1 e IgE anti as enzimas e anti as proteínas do parasito. Os animais do grupo imunizado com o MIX apresentaram aumento discreto de IL-4 e foi observada redução de IL-10, e no grupo imunizado com HGPRT houve aumento discreto de IFN-γ, quando comparados com os animais do grupo somente infectado. Além disso, os animais do grupo imunizado com AK apresentaram redução do número de granulomas hepáticos (44,55%) e de ovos no fígado (42,31%), quando comparados com o grupo somente infectado. Assim, sugere-se que a imunização com essas enzimas pode contribuir para o controle da esquistossomose, bem como auxiliar na modulação da infecção experimental, induzindo redução da fisiopatologia desta parasitose.

An estimated 1,735,350 new cancer diagnosis and 609,640 cancer related deaths are predicted to occur in the United States in 2018. To improve patient prognosis, biomarkers are needed to identify cancer in early stages. When diagnosed at an early stage, cancer is more likely to respond to treatments and patients have a higher survival rate. Consequently, there is an ever-present need to identify biomarkers that can aid in the detection of cancer. Additionally, there is a paradigm shift in the field of cancer treatment towards immunotherapy. Traditional cancer treatments include chemotherapy, radiation, and hormone therapy and are not cancer-specific, which leads to bystander effects on the patient<&trade>s normal organs that often harm the patient and create unnecessary hardship. To alleviate this, immunotherapy utilizes a patient<&trade>s own immune cells to attack and destroy cancer cells via cancer-specific biomarkers. These biomarkers are ideally on the surface of cancer cells and absent from the patient<&trade>s normal cells to avoid healthy tissue destruction. With this new therapy, there is a recent push to find surface antigens for immunotherapy techniques.This dissertation describes the characterization of HPRT as a diagnostic and therapeutic biomarker for the detection and possible treatment of hematological and solid malignancies. We describe the general upregulation of HPRT upon malignancy and show that this elevation in protein expression is independent of stage, which indicates that it would be useful as an early stage diagnostic companion tool. We have preliminarily linked the elevation in HPRT to a mutation in one of its prime transcription factors, p53. Specific mutation in p53 called Gain of Function mutations have shown to influence salvage pathway enzyme expression, and we have shown that mutations in p53 are relevant to the elevated levels of HPRT within several cancer types. In addition, we also found that HPRT associates significantly with the membrane of several cancer cell lines as well as patient samples. We found that HPRT has insignificant expression on normal cells, which suggests it may be useful as a targetable biomarker for immunotherapy. Throughout our analysis, we also determined that HPRT might have a role in immune regulation as an elevation of the protein correlates to the decrease of several pro-inflammatory genes involved in immune activation. The knowledge gained from the data presented in this dissertation have opened up new functions for HPRT outside of simple nucleotide production and have confirmed that HPRT has a unique role in cancer that has not been previously reported.