Abnormal activation of brain microglial cells is widely implicated in the pathogenesis of schizophrenia. The disrupted balance of microglia phenotypes has been hypothesized to influence the clinical course of the disease and affect symptom severity. Previously, the pathophysiology of microglial activation was considered to be intrinsic to the central nervous system. We hypothesised that due to their perivascular localization, microglia can also be activated by factors present in circulating blood. We applied a high-content functional screening platform, to characterize alterations in microglial intracellular signalling cascades induced by schizophrenia patient serum relative to control serum. Using automated sample preparation, fluorescent cellular barcoding and flow cytometry, the applied platform is capable of detecting multiple parallel cell signalling responses in microglia. First, we exposed a human microglia cell line to serum isolated from first-onset drug-naïve schizophrenia patients (n=60) and healthy controls (n=79). We were able to show that peripheral blood serum obtained from schizophrenia patients induced differential microglial cell signalling network responses in vitro. We subsequently assessed whether antipsychotic drug-treatment can normalise the abnormal microglial signalling responses previously identified by exposing microglia cells to serum from antipsychotic treated schizophrenia patients (n=15) and controls (n=17). In addition, in order to assess microglia activation in vivo, we obtained positron emission tomography (PET) imaging data from collaborators, who used a radiotracer to assess potential altered microglia activation in patients suffering from schizophrenia. Finally, as a proof of concept study, we attempted to validate these findings by assessing the effect of serum collected from first-onset drug-naïve schizophrenia patients (n=9), controls (n=12) as well as serum isolated from the same patients subjected to six weeks of clinical treatment with the antipsychotic olanzapine (n=9). This study aimed to identify normalisation of previously detected differences in microglia signalling pathways based on successful in vivo treatment. We demonstrate that peripheral blood serum isolated from schizophrenia patients, independent of their treatment status, is sufficient to trigger microglial cell signalling network responses in vitro, which are indicative of altered STAT3 signalling. We further explored the composition of the serum for differential expression of analytes, previously associated with neuropsychiatric disorders, and the utility of the detected microglial cellular phenotype as a target for novel drug discovery.

This thesis investigates the connection between the Sirtuin1 protein (Sirt1) and the Notch signaling pathway in Drosophila melanogaster. We used RNAi to downregulate the Sirt1 gene expression in a tissue specific manner in the muscles, brain, fat body, and wing disc and tested the sensitivity of Notch pathway to dietary amino acids. We identified tissue specific requirements for Sirt1 protein to mediate the sensitivity of Notch pathway to amino acids.

Submitted by Rita de Cassia Siqueira Bruder (bruder.28@hotmail.com) on 2018-03-22T13:20:34Z No. of bitstreams: 1 TESE DE DOUTORADO 2018.pdf: 1906745 bytes, checksum: 78920ae0ca10dab6eec3900df2039470 (MD5) / Approved for entry into archive by ROSANGELA APARECIDA LOBO null (rosangelalobo@btu.unesp.br) on 2018-03-23T19:14:35Z (GMT) No. of bitstreams: 1 bruder_rcs_dr_bot.pdf: 1906745 bytes, checksum: 78920ae0ca10dab6eec3900df2039470 (MD5) / Made available in DSpace on 2018-03-23T19:14:35Z (GMT). No. of bitstreams: 1 bruder_rcs_dr_bot.pdf: 1906745 bytes, checksum: 78920ae0ca10dab6eec3900df2039470 (MD5) Previous issue date: 2018-03-02 / RESUMO Bruder R. Redução da Incidência de Citomegalovírus no esquema Sirolimo associado à Tacrolimo em paciente idoso transplantado renal. Tese (Doutorado). Faculdade de Medicina de Botucatu. Universidade Estadual Paulista, 2018. Introdução: O transplante renal é considerado uma opção de terapia renal substitutiva segura para pacientes acima de 60 anos, entretanto, não há consenso sobre o melhor esquema imunossupressor para o paciente transplantado renal idoso. Objetivo: Avaliar a incidência de infecção por citomegalovírus na combinação de tacrolimo e sirolimo em doses reduzidas comparada com a associação tacrolimo e micofenolato. Métodos: Estudo prospectivo randomizado de centro único comparando a combinação de tacrolimo e sirolimo em dose reduzida (grupo sirolimo) contra tacrolimo e micofenolato (grupo micofenolato). Foram incluídos todos os pacientes transplantados renais maiores de 60 anos. Foram avaliadas a incidência de infecção por citomegalovírus (CMV), a sobrevida do paciente e enxerto, taxas de rejeição e função renal em 12 meses de seguimento. Resultados: Foram randomizados 46 pacientes e analisados 44 casos (dois casos excluídos por não terem sido transplantados). As características basais dos grupos foram semelhantes sendo todos os casos transplantados com doador falecido e a maioria induzida com basiliximab. O grupo micofenolato (n=23) e o grupo sirolimo (n=21) apresentaram sobrevida do paciente e enxerto censurado óbito respectivamente de 95,7% e 100% para o micofenolato e 87,3% e 90,5% para o sirolimo sem diferenças estatísticas. A infecção por CMV ocorreu em 60,9% no grupo micofenolato e 16,7% no grupo sirolimo apresentando um risco relativo 3,54 [1,2 – 10,3] vezes maior no grupo micofenolato, p=0,004. A incidência de rejeição e função renal ao longo de 12 meses foi semelhante entre os grupos. A infecção de ferida operatória, retardo de função do enxerto e proteinúria foram semelhantes entre os grupos. O colesterol total e HDL colesterol foram maiores no grupo sirolimo. Conclusão: No grupo de transplantados renais idosos a combinação de tacrolimo e sirolimo em doses reduzidas resultou na redução de 40% da incidência de infecção por CMV. A função renal em 12 meses, taxa de rejeição e sobrevida do enxerto e paciente foram semelhantes. / ABSTRACT Bruder R. Reduced incidence of cytomegalovirus in the Sirolimus associated with tacrolimus in elderly kidney transplant patient. Tese (Doutorado). Faculdade de Medicina de Botucatu. Universidade Estadual Paulista, 2018. Introduction: Renal transplantation is considered safe for patients over 60 years, however, there is no consensus on the best immunosuppressive regimen in elderly. Objective: To evaluate the incidence of cytomegalovirus infection in the combination of tacrolimus and sirolimus in reduced doses compared to the combination tacrolimus and mycophenolate. Methods: A single-center prospective randomized study comparing the combination of tacrolimo and sirolimo in reduced dose (sirolimo group) against tacrolimo and mycophenolate (mycophenolate group). We included all kidney transplant patients over 60 years of age. The incidence of cytomegalovirus (CMV) infection, patient survival and graft, rejection rates and renal function were evaluated at 12 months of follow-up. Results: 46 patients were randomized and we analyzed 44 cases (two cases excluded because they were not transplanted). Baseline characteristics were similar between groups, all patients were transplanted with deceased donor, and the majority were induced with basiliximab. Mycophenolate group (n = 23) and sirolimo group (n = 21) had patient survival, and death censored graft survival respectively 95.7% and 100% for mycophenolate and 87,3% e 90,5% for the sirolimo without statistical differences. The incidence of CMV infection occurred in 60.9% and 16.7% in mycophenolate and sirolimo group respectively. The relative risk of CMV were 3.54 [1, 2 - 10.3] times greater in mycophenolate group, p=0.004. The incidence of acute rejection and kidney function over 12 months was similar between the groups. Infection of the surgical wound, delayed graft function and proteinuria were similar between groups. The total cholesterol and HDL cholesterol were higher in the sirolimo group. Conclusion: In the elderly renal transplant group, the combination of tacrolimus and sirolimus at reduced doses resulted in a 40% reduction in the incidence of CMV infection. Renal function at 12 months, rejection rate and graft and patient survival were similar.

Submitted by PAULA DALSOGLIO GARCIA null (pauladgarcia@gmail.com) on 2016-09-22T14:54:06Z No. of bitstreams: 1 Doutorado Final Pós Defesa Setembro 2016.pdf: 934309 bytes, checksum: 2f5f46040168ed2b3e80c62144f20b52 (MD5) / Approved for entry into archive by Felipe Augusto Arakaki (arakaki@reitoria.unesp.br) on 2016-09-26T17:54:28Z (GMT) No. of bitstreams: 1 garcia_pd_dr_bot.pdf: 934309 bytes, checksum: 2f5f46040168ed2b3e80c62144f20b52 (MD5) / Made available in DSpace on 2016-09-26T17:54:28Z (GMT). No. of bitstreams: 1 garcia_pd_dr_bot.pdf: 934309 bytes, checksum: 2f5f46040168ed2b3e80c62144f20b52 (MD5) Previous issue date: 2016-08-19 / Introdução: O transplante renal é a melhor opção de tratamento para doença renal crônica estadio V. Melhora substancial da sobrevida do enxerto no primeiro ano ocorreu principalmente pela drástica redução dos índices de rejeição aguda com o uso de imunossupressores potentes, destacando-se os inibidores de calcineurina. Porém essa não se acompanhou de melhora da sobrevida do paciente e do enxerto em longo prazo. A mortalidade cardiovascular continua sendo a principal causa de morte no transplante renal. A hipertrofia do ventrículo esquerdo (HVE) e a aterosclerose são muito prevalentes nessa população e constituem fatores de risco para eventos cardiovasculares. Os inibidores da mTOR (mammalian target of rapamycin inhibitor) parecem ser drogas promissoras na redução da HVE e na redução e prevenção de aterosclerose no campo experimental, porém seu efeito nos pacientes transplantados renais ainda é controverso. Objetivo: Comparar a ação do everolimo com o tacrolimo na redução do índice de massa do ventrículo esquerdo (iMVE) e da espessura médio intimal das carótidas (EMIC) em pacientes transplantados renais. Material e Métodos: Ensaio clínico prospectivo, randomizado, unicêntrico, cego para o cardiologista que realizou os ecocardiogramas. Os pacientes receberam imunossupressão inicial com Tacrolimo (Tac), Micofenolato sódico (MFS) e Presnisona (PDN). Após 12±4 semanas, pacientes que preencheram critérios de inclusão e exclusão foram randomizados 1:1 nos grupos: TACRO (controle) que manteve a imunossupressão inicial e no grupo EVERO que foi convertido de tacrolimo para everolimo com manutenção de MFS e PDN. Dados clínicos e laboratoriais foram obtidos na randomização e 3, 6, 9 e 12 meses após. Os ecocardiogramas para cálculo do iMVE e EMIC foram realizados no momento da randomização, 6 e 12 meses após. Resultados: A despeito de maior proteinúria e maior colesterol total, o grupo EVERO apresentou redução significativa da EMIC ao final de 12 meses (p=0,012). A porcentagem de redução da EMIC no grupo EVERO foi quase o dobro da encontrada no grupo TACRO, com p=0,0528. Não houve diferença entre o grupo EVERO e o grupo TACRO em relação à redução da iMVE ao final de um ano. A função renal dos dois grupos foi semelhante e considerada excelente ao final do estudo. Em longo prazo talvez o everolimo possa ajudar na redução da mortalidade cardiovascular destes pacientes. Conclusão: O everolimo, mas não o tacrolimo, reduziu a EMIC ao final de um ano de seguimento. Não houve diferença quanto a redução de iMVE e a função renal foi semelhante entre grupos. / Introduction: Kidney transplantation is the gold standard treatment for end-stage renal disease. Allograft survival after one year of transplantation has had a significant improvement. However, there is a lack of improvement in patient and allograft long term survival. The mTOR inhibitors (mammalian target of rapamycin inhibitor) are the newest drugs available to prevent allograft rejection and they seem to have potential benefits in reducing myocardial hypertrophy and atherosclerosis in experimental studies. This benefits for kidney transplanted patients is still controversial. Objective: To compare the effect of everolimus to the tacrolimus in reducing left ventricular mass index (LVMi) and carotid intima-media thickness (IMT) after one year in kidney transplanted patients. Material and Methods: This randomized, open-label, controlled trial compared the effect of everolimus to tacrolimus in reducing the LVMi and the IMT in kidney transplanted patients after one year of these immunosuppressive therapies. After initial immunosuppression with tacrolimus, mycophenolate sodium (MFS) and prednisone (PDN), patients were randomly assigned at 12±4 weeks in a 1:1 ratio to undergo conversion from tacrolimus to everolimus (EVERO group) and maintenance of MFS and PDN or continue on standard tacrolimus-based treatment (TACRO group). Each patient was randomized only after a kidney biopsy with no evidence of rejection or inflammation. Clinical and laboratorial data were collected on randomization day and 3, 6, 9, and 12 months after randomization. Echocardiograms were done at the randomization and 6 and 12 months after. Results: Everolimus significantly reduced IMT after one year (p=0,012), despite higher levels of proteinuria and cholesterol. The percentage of reduction of the IMT on the EVERO group was almost twice the reduction of the TACRO group (p=0,0528). No difference was found on the reduction of LVMi between groups. The renal function was excellent in both groups at one year, with no superiority of any drug. Everolimo may contribute to the reduction of cardiovascular mortality of this population in long term. Conclusion: Everolimus reduced IMT at one year in kidney transplanted patients. No difference between groups was found in the reduction of iMVE or increase on renal function in the end of the study.

Novas informações assimiladas pelo sistema nervoso primeiramente ficam em um estado de labilidade para depois se estabilizarem através de um processo conhecido como consolidação, que envolve síntese de proteínas. Depois da reativação, uma memória previamente consolidada retorna ao seu estado de labilidade, e para que volte a ser estável, é necessário que haja novamente síntese de proteínas. Este segundo processo é chamado de reconsolidação. Recentemente os mecanismos moleculares e celulares envolvidos na regulação da síntese protéica relacionados à formação de memória de longa duração vêm sendo esclarecidos. A proteína alvo da rapamicina em mamíferos (mTOR) modula a plasticidade sináptica pela regulação da fosforilação de dois alvos: a proteína ribossomal S6K e a proteína de ligação 4E. A amígdala basolateral e o hipocampo dorsal são parte integrante do sistema neural envolvido na formação e expressão de diversos tipos de memórias. Estudos indicam que a via de sinalização da mTOR no hipocampo tem um papel importante na consolidação da memória de ratos submetidos a tarefa de esquiva inibitória e reconhecimento de objetos e na reconsolidação da memória de medo contextual condicionado. Contudo, estudos anteriores não avaliaram o efeito da inibição de mTOR amigdalar sobre a memória de esquiva inibitória e reconhecimento de objetos. O objetivo do presente trabalho é investigar o efeito da inibição de mTOR na amígdala basolateral por rapamicina na consolidação e reconsolidação da memória de esquiva inibitória e reconhecimento de objetos e comparar estes resultados com a inibição de mTOR no hipocampo. Ratos Wistar machos foram submetidos à cirurgia estereotáxica para implantação de cânulas na amígdala basolateral e hipocampo dorsal. Os animais foram submetidos à tarefa de esquiva inibitória, um modelo animal de memória de caráter aversivo, e a tarefa de reconhecimento de objetos, um modelo animal de memória de caráter pouco aversivo. Para investigar o efeito da inibição de mTOR na consolidação e reconsolidação da memória, os animais receberam microinfusões de rapamicina intra-amigdalar e intra-hipocampal em diferentes tempos em torno do treino e do teste. Nós demonstramos que a via de sinalização de mTOR na amígdala basolateral é necessária para consolidação da memória de esquiva inibitória e de reconhecimento de objetos. Nós também mostramos que a reativação torna a memória novamente suscetível e sensível à inibição de mTOR por rapamicina. / Memory formation requires protein synthesis, but only recently the cellular and molecular mechanisms involved in the regulation of protein synthesis related to the formation of long term memory has been elucidated. During memory formation, new information is acquired by the central nervous system as an initially fragile trace that over time becomes stable through a process known as consolidation. After reactivation, previously consolidated memories might return to a labile state, requiring a new round of protein synthesis to be restabilized. This second process is called reconsolidation. The basolateral amygdala and dorsal hippocampus are part of the neural systems involved in the formation and expression of several types of memory. One key regulator of protein synthesis is mTOR, a protein critical for different forms of synaptic plasticity by regulation of two targets: S6K and 4EBP. Evidence indicates that the mTOR signaling pathway in hippocampus has an important role in consolidation in rats of inhibitory avoidance and object recognition in rats, as well as in reconsolidation of contextual fear conditioning. However, previous studies have not examinated the effect of amygdalar mTOR inhibition on reconsolidation of inhibitory avoidance and object recognition. The aim of the present study was to evaluate the effect of amygdalar mTOR inhibition by rapamycin on consolidation and reconsolidation of inhibitory avoidance and object recognition, and compare the results with those obtained with hippocampal mTOR inhibition. Male rats Wistar underwent stereotaxic surgeries for cannulae implantation above the basolateral amygdala or dorsal hippocampus. After recovery, the animals were trained in inhibitory avoidance, an aversive memory task, or object recognition, a less aversive task. To investigate the effect of mTOR inhibition on memory consolidation and reconsolidation, we administered rapamycin, a specific mTOR inhibitor, into the basolateral amygdala or the dorsal hippocampus before or after training or reactivation. Our results provide evidence that mTOR in the basolateral amygdala and hippocampus might play a role in inhibitory avoidance and object recognition memory formation and reconsolidation.

Novas informações assimiladas pelo sistema nervoso primeiramente ficam em um estado de labilidade para depois se estabilizarem através de um processo conhecido como consolidação, que envolve síntese de proteínas. Depois da reativação, uma memória previamente consolidada retorna ao seu estado de labilidade, e para que volte a ser estável, é necessário que haja novamente síntese de proteínas. Este segundo processo é chamado de reconsolidação. Recentemente os mecanismos moleculares e celulares envolvidos na regulação da síntese protéica relacionados à formação de memória de longa duração vêm sendo esclarecidos. A proteína alvo da rapamicina em mamíferos (mTOR) modula a plasticidade sináptica pela regulação da fosforilação de dois alvos: a proteína ribossomal S6K e a proteína de ligação 4E. A amígdala basolateral e o hipocampo dorsal são parte integrante do sistema neural envolvido na formação e expressão de diversos tipos de memórias. Estudos indicam que a via de sinalização da mTOR no hipocampo tem um papel importante na consolidação da memória de ratos submetidos a tarefa de esquiva inibitória e reconhecimento de objetos e na reconsolidação da memória de medo contextual condicionado. Contudo, estudos anteriores não avaliaram o efeito da inibição de mTOR amigdalar sobre a memória de esquiva inibitória e reconhecimento de objetos. O objetivo do presente trabalho é investigar o efeito da inibição de mTOR na amígdala basolateral por rapamicina na consolidação e reconsolidação da memória de esquiva inibitória e reconhecimento de objetos e comparar estes resultados com a inibição de mTOR no hipocampo. Ratos Wistar machos foram submetidos à cirurgia estereotáxica para implantação de cânulas na amígdala basolateral e hipocampo dorsal. Os animais foram submetidos à tarefa de esquiva inibitória, um modelo animal de memória de caráter aversivo, e a tarefa de reconhecimento de objetos, um modelo animal de memória de caráter pouco aversivo. Para investigar o efeito da inibição de mTOR na consolidação e reconsolidação da memória, os animais receberam microinfusões de rapamicina intra-amigdalar e intra-hipocampal em diferentes tempos em torno do treino e do teste. Nós demonstramos que a via de sinalização de mTOR na amígdala basolateral é necessária para consolidação da memória de esquiva inibitória e de reconhecimento de objetos. Nós também mostramos que a reativação torna a memória novamente suscetível e sensível à inibição de mTOR por rapamicina. / Memory formation requires protein synthesis, but only recently the cellular and molecular mechanisms involved in the regulation of protein synthesis related to the formation of long term memory has been elucidated. During memory formation, new information is acquired by the central nervous system as an initially fragile trace that over time becomes stable through a process known as consolidation. After reactivation, previously consolidated memories might return to a labile state, requiring a new round of protein synthesis to be restabilized. This second process is called reconsolidation. The basolateral amygdala and dorsal hippocampus are part of the neural systems involved in the formation and expression of several types of memory. One key regulator of protein synthesis is mTOR, a protein critical for different forms of synaptic plasticity by regulation of two targets: S6K and 4EBP. Evidence indicates that the mTOR signaling pathway in hippocampus has an important role in consolidation in rats of inhibitory avoidance and object recognition in rats, as well as in reconsolidation of contextual fear conditioning. However, previous studies have not examinated the effect of amygdalar mTOR inhibition on reconsolidation of inhibitory avoidance and object recognition. The aim of the present study was to evaluate the effect of amygdalar mTOR inhibition by rapamycin on consolidation and reconsolidation of inhibitory avoidance and object recognition, and compare the results with those obtained with hippocampal mTOR inhibition. Male rats Wistar underwent stereotaxic surgeries for cannulae implantation above the basolateral amygdala or dorsal hippocampus. After recovery, the animals were trained in inhibitory avoidance, an aversive memory task, or object recognition, a less aversive task. To investigate the effect of mTOR inhibition on memory consolidation and reconsolidation, we administered rapamycin, a specific mTOR inhibitor, into the basolateral amygdala or the dorsal hippocampus before or after training or reactivation. Our results provide evidence that mTOR in the basolateral amygdala and hippocampus might play a role in inhibitory avoidance and object recognition memory formation and reconsolidation.

La maladie rénale chronique (MRC) par ses conséquences organiques et psychologiques représente unenjeu majeur de santé publique. Sa physiopathologie reste mal connue, mais il est établi que toute atteinte rénale,quelle qu’en soit la cause, aboutit à une réduction du nombre de néphrons fonctionnels. Cette réductionnéphronique est responsable de processus adaptatifs complexes des néphrons sains restants pour maintenir unefonction rénale satisfaisante. Si la perte néphronique est suffisamment importante, le parenchyme rénal vas’altérer progressivement aboutissant au remplacement des néphrons sains par un tissu fibreux puis au déclin dela fonction rénale. Les mécanismes moléculaires impliqués, tant dans l’adaptation à la réduction néphronique,que dans la dégradation progressive du parenchyme rénal sont mal connus et les possibilités d’interventionsthérapeutiques limitées.La voie AKT/mTOR est une voie de signalisation intracellulaire ubiquitaire, très conservée, jouant unrôle central dans l’homéostasie cellulaire par sa fonction de régulation de la croissance, de l’apoptose et du cyclecellulaire. L’extraordinaire complexité de son mode de recrutement témoigne du rôle de carrefour de cette voie.Elle intègre des signaux multiples et très variés (facteurs de croissance, acides-aminés, niveau énergétiquecellulaire, disponibilité de l’oxygène) modulant l’anabolisme cellulaire. Notre compréhension du rôle de cettevoie dans la progression de la MRC est encore très limitée et les données sont peu nombreuses et controversées.Mon travail de thèse a consisté à évaluer, en utilisant un modèle expérimental de réduction néphroniquechez la souris, le rôle de la voie AKT/mTORC au cours de la progression des MRC.Nous résultats démontrent que AKT, et plus précisément AKT2, est une molécule essentielle àl’adaptation podocytaire aux contraintes imposées par la réduction néphronique. En appliquant plusieursmodèles de réduction néphronique à des souris génétiquement modifiées, nous avons pu établir la fonctioncruciale de cette isoforme. En effet, l’inactivation d’Akt2, soit systémique soit conditionnelle dans le podocyte,est responsable d’une hyporéactivité de la voie AKT podocytaire, d’un remodelage du cytosquelette, d’uneaugmentation de l’apoptose glomérulaire avec raréfaction podocytaire et de lésions de glomérulosclérose.Transposant nos données à la pathologie humaine, nous avons mis en évidence une activation podocytaired’AKT2 après transplantation rénale chez les patients présentant une altération importante de la fonction rénale.De façon marquante, la survenue d’une protéinurie en réponse à un traitement par sirolimus s’associait à uneperte de cette activation et à une augmentation de l’apoptose glomérulaire.Parallèlement, nous avons évalué le rôle de cette voie chez l’homme au cours d’une MRC trèsparticulière secondaire à la présence d’anticorps antiphospholipides. Cette néphropathie est caractérisée par laprésence de lésions vasculaires sévères prolifératives, hypertrophiques et progressivement obstructivesaboutissant à la destruction du parenchyme rénal. Jusqu'à maintenant, aucun lien formel n’avait été établi entre laprésence de ces anticorps et le développement des lésions vasculaires, et aucune thérapeutique n’était disponible.Nos résultats indiquent que ces anticorps sont directement pathogènes pour l’endothélium induisant l’activationde la voie AKT/mTORC. L’activation de cette voie stimule la prolifération des cellules endothéliales mais aussi .... / Pas de résumé en anglais

La néphropathie diabétique (ND), principale cause d’insuffisance rénale chronique, est caractérisée par une dysfonction des podocytes rénaux. Cette dysfonction podocytaire peut être causée par une résistance à l’insuline induite suite à l’exposition des podocytes aux acides gras libres (AGL). L’un des mécanismes par lequel les AGL réduisent les actions de l’insuline serait l’activation de la voie de Mammalian target of rapamycin (mTOR). Les objectifs sont de caractériser les mécanismes de résistance à l’insuline par les AGL dans les podocytes et d’étudier l’implication de la voie du complexe mTORC1 menant à la ND dans un modèle de diabète de type 2. In vivo, la fonction et la pathologie rénale des souris diabétiques de type 2 (db/db) ont été évaluées. Des podocytes murins ont été cultivés pendant 96 h en conditions normales (5,6 mM, NG) ou élevées (25 mM, HG) de glucose avec ou sans palmitate (25 μM) pour les dernières 24 h. In vitro, les podocytes exposés en HG ont montré une diminution de l’activation d’Akt induite par l’insuline. Le palmitate seul a diminué de 50% l’activation d’Akt alors que la combinaison HG + palmitate a accentué cette diminution en la réduisant de 72%. Cette inhibition se ferait via la phosphorylation en sérine d’IRS1. En effet, en présence de palmitate, la phosphorylation d’IRS1 (ser307) est augmentée d’environ 2 fois. De plus, la phosphorylation d’IRS1 par le palmitate est corrélée à une augmentation de la phosphorylation de mTOR (ser2448) et de son substrat S6 (ser240/244). L’inhibition de la voie de signalisation de l’insuline par la voie mTOR serait due à l’activation de la PKC-α suite à une stimulation au palmitate. Pour ce qui est de mTORC2, la phosphorylation inhibitrice de Rictor (thr1135) augmente de 47% en présence de palmitate. In vivo, dans les souris db/db, l’augmentation des marqueurs de la ND (albuminurie, expansion du mésangium, hypertrophie du glomérule et expression de TGF-beta) est associée à une élévation de la p-mTOR, p-Rictor et de p-S6 dans les glomérules rénaux. En conclusion, le phénomène de résistance à l’insuline par les AGL dans les podocytes serait causé par l’activation de PKC-α/mTORC1 menant à la phosphorylation d’IRS1 en sérine 307, un mécanisme complémentaire aux actions de l’hyperglycémie, et contribuant de façon indépendante à la progression de la ND. De plus, l’inhibition du complexe mTORC2 contribue à la diminution de la signalisation de la voie de l’insuline. / Abstract : Diabetic nephropathy (DN) is the leading cause of chronic renal failure in diabetic patients and is characterized by the dysfunction of podocytes. Our laboratory has shown that hyperglycemia caused podocyte insulin unresponsiveness and cell death via the upregulation of PKC- and SHP-1, a tyrosine phosphatase. In contrast, free fatty acids (FFA)-induced insulin resistance in podocytes is not associated with SHP-1 expression. Thus, other signaling pathways could be implicated including the activation of the Mammalian target of rapamycin (mTOR) complexes pathway. The aim of this study was to investigate the insulin resistance mechanisms caused by FFA in podocytes leading to DN in type 2 diabetes. In vitro, cultured podocytes were exposed to normal (5.6 mmol/L; NG) or high glucose (25 mmol/L; HG) levels for 96 h and to palmitate (25 µmol/L) the last 24h with or without insulin stimulation (10 nmol/L). As previously showed, podocytes exposed to HG decreased Akt activation upon insulin stimulation. Palmitate treatment alone reduced insulin-induced Akt phosphorylation by 50% while a combination of palmitate and HG blunted Akt activation by 72%. The inhibition of Akt by palmitate was associated with the increase of PKC- activation leading to mTOR phosphorylation and its substrate S6. Moreover, the mTORC1 complex activation enhanced the serine 307 phosphorylation of IRS1 known to de-activate IRS1. Furthermore, palmitate also mediated the mTORC2 complex inhibition via the Thr1135 phosphorylation of Rictor. In vivo, the implication of mTORC1 complex in DN development was evaluated using 25 weeks old type 2 diabetes mice (db/db). Mice developed increased albuminuria, mesangial cell expansion and glomerular hypertrophy compared to non-diabetic mice, which correlated with the phosphorylation of mTOR, Rictor and S6. In conclusion, elevated FFA levels caused activation of PKC-/mTORC1 pathway and inhibition of mTORC2 leading to insulin resistance in podocytes and DN progression.

7Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by accumulation and deposition of extracellular beta-amyloid peptides (Aβ) and intra-neuronal hyperphosphorylated tau in the brain. The insulin signalling pathway begins upstream at the insulin receptor (IR), where the intracellular insulin receptor substrate 1 (IRS1) is phosphorylated, thus propagating the signal downstream to the PI3K/Akt signalling pathway, which affects both the glycogen synthase kinase 3 beta (GSK3β), which is a tau kinase, and mTOR, which is a critical part of the mTORC1 and mTORC2 complexes that not only mediate a wide range of cell functions, but also feed back upstream to regulate Akt. Increasing evidence builds a strong case for the role of soluble Aβ oligomers (AβOs) in the impairment of insulin signalling in AD. Our in vitro studies with neuroblastoma 2a (N2a) cells stably transfected with human APP695 gene (N2a-APP), which secrete excess Aβ, show that the phosphorylation and expression of several but not all critical signalling proteins along the insulin signalling pathway are dysregulated in the cells in comparison to the parental N2a cells. N2a-APP cells were also found to be phenotypically insulin resistant. Subsequently, N2a-APP cells were treated with the Aβ binding peptide (ABP), which binds Aβ oligomers. The ABP treatment was observed to enhance insulin signalling response compared to untreated controls. The results suggest that Aβ may be responsible for inducing the insulin resistant phenotype in N2a-APP cells, and that the removal of Aβ oligomers is a potential treatment consideration for dysfunctional insulin signalling involved in Alzheimer’s disease.

Abnormal thyroid cell proliferation causes human diseases, such as goiter, thyroid adenoma or carcinoma and primary hypothyroidism resulting from hypoplasia. Thyrotropin (TSH), mainly acting through cAMP and cAMP-dependent protein kinases (PKA), is considered the main regulator of thyrocyte proliferation and differentiation. The general aim of this thesis was to get new mechanistic insights in the regulatory action of the cAMP pathway on various functions of thyrocytes, including proliferation.<p><p>During the first part of this thesis work, we have collaborated to a study of the effects of the TSH/cAMP pathway on small G proteins of the Rho family and their impact on the actin cytoskeleton and thyroid cell function. This study, performed in canine thyrocytes, showed for the first time, that the TSH/cAMP/PKA pathway inactivates the three small G proteins RhoA, Rac1 and Cdc42 and the RhoA/ROCK/LIMK/cofilin pathway. Inactivation of the latter appeared both necessary and sufficient to mediate the action of TSH and PKA on the reorganization of actin microfilaments and its morphological impact. Moreover, this inactivation by PKA of Rho-mediated actin polymerization also played an important role in the cAMP-dependent expression of thyroid differentiation genes. On the other hand, a residual RhoA activity appeared to be required for mitogenesis. This dependence of DNA synthesis on RhoA activity was not mediated by ROCK-dependent events nor by the integrity of the actin cytoskeleton. Indeed, DNA synthesis induction was unexpectedly resistant to actin depolymerisation in canine thyrocytes, which explains how it could be compatible with the cAMP-dependent microfilament disruption. <p><p>This first study did not provide new insights on how the cAMP/PKA-dependent mitogenic stimulus can trigger cell cycle progression, which, in thyrocytes, depends on the phosphorylation of pRb by the cyclin D3-CDK4 complex. In the various in vitro thyroid models, the only convergent early signaling event found in response to TSH/cAMP, insulin and growth factors was the phosphorylation and activation of p70 S6K1 which largely depends on mTOR (mammalian Target Of Rapamycin). The main part of the work in this thesis has been devoted to investigation of the action of TSH/cAMP on the mTOR pathway. mTOR is a therapeutic target for a wide variety proliferative disorders and rapamycin derivates are now considered in anti-cancer treatments.<p><p>We have shown for the first time in PC Cl3 rat thyroid cells that TSH, through cAMP, activates mTORC1, leading to phosphorylation of S6K1 and 4E-BP1. mTORC1-dependent S6K1 phosphorylation in response to both insulin and cAMP required amino acids, whereas inhibition of AMPK and GSK3 enhanced insulin but not cAMP effects. Unlike insulin, TSH/cAMP did not activate PKB, nor induce TSC2 phosphorylation at Thr1462 and Tyr1571. However, like insulin, TSH/cAMP produced a stable increase in mTORC1 kinase activity associated with augmented 4E-BP1 binding to raptor. This could be caused in part by Thr246-phosphorylation of PRAS40, which was found as an in vitro substrate of PKA, but other regulatory events likely remain to be uncovered. Both in PC Cl3 cells and primary dog thyrocytes, rapamycin inhibited DNA synthesis and pRb phosphorylation induced by TSH and insulin. Rapamycin reduced cyclin D3 accumulation but not the abundance of cyclin D3-CDK4 complexes. However, rapamycin inhibited the activity of these complexes and the activating Thr172-phosphorylation of CDK4 stimulated by both TSH and insulin. We propose that mTORC1 activation by TSH, at least in part through PKA-dependent phosphorylation of PRAS40, crucially contributes to mediate cAMP-dependent mitogenesis by regulating CDK4 Thr172-phosphorylation. / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished