La synthèse protéique est une étape importante de la régulation de l'expression des gènes. Dans beaucoup d'espèces animales, les premières étapes du développement embryonnaire sont majoritairement ou exclusivement basées sur l'utilisation des messagers maternels stockés dans l'ovocyte. L'embryon d'oursin est un modèle avantageux pour l'étude du contrôle traductionnel de l'expression des gènes lors du développement précoce. La fécondation provoque l'activation de la machinerie traductionnelle conduisant à une augmentation de synthèse protéique nécessaire à la reprise des cycles mitotiques et au départ du développement embryonnaire. Les modifications touchant la machinerie traductionnelle qui ont lieu à la fécondation sont à l'origine du recrutement polysomal des messagers stockés. Ainsi, l'ensemble des ARNm maternels est-il globalement traduit, ou existe-t-il une sélection des ARNm qui vont être traduits précocement ? Et s'il y en a, quels sont les modes de sélection ? Au cours de ce travail de thèse, nous avons obtenu le répertoire complet des ARNm traductionnellement régulés à la fécondation, et montré que seule une sous-partie du stock de messager est traduite en réponse à la fécondation, avec un enrichissement de messagers codant pour des protéines régulatrices. Enfin, de manière originale, ce travail a permis la mise en évidence de la diversité et de la complexité des voies de signalisation en amont de la régulation traductionnelle, qui concourent à la sélectivité de la traduction. / Protein synthesis is a crucial step for gene expression regulation. In many animal species, the early steps of development are based on translation of stored maternal mRNAs. Sea urchin embryo is a powerful model to study translational control during early development. Fertilization triggers the activation of translational machinery, leading to the increase of protein synthesis which is necessary to cell cycle entry and early embryonic development. Translational machinery modifications are responsible for the polysomal recruitment of the stored maternal mRNAs. Thus, are all the stored maternal mRNAs translated, or is there any selection of the translated mRNAs? If so, what are the mechanisms driving this selectivity? Over this work, we obtained the entire subset of the translationally regulated mRNAs, and demonstrated that only a part of the stored maternal mRNAs is actively translated at sea urchin fertilization, with an important enrichment of mRNAs coding for regulatory proteins. Finally, this work highlighted the diversity and the complexity of the signaling network upstream the selective polysomal recruitment.

Les résultats de la transplantation hépatique (TH) se sont considérablement améliorés mais la survie à long terme est une préoccupation des transplanteurs. Elle dépend de la survenue d’un rejet, de la récidive de l’hépatite C, du traitement immunosuppresseur et de ses complications. Après une TH suite à une complication de l’hépatite C, l'infection du greffon par le virus de l’hépatite C (VHC) est constante, et l'évolution de l'hépatite chronique est plus rapide et agressive que chez les non transplantés. L’implication des cellules T régulatrices (Treg) a été suggérée dans l’induction de tolérance après transplantation et dans la persistance de l’infection par le VHC. Le nombre et la fonction des Treg seraient influencés par le traitement immunosuppresseur (IS). Dans ce contexte, je me suis intéressé au rôle des Treg dans l’évolution de la TH, et les effets des différents traitements utilisés (IS et anti-VHC) sur ces cellules. Les résultats montrent que les Treg, en particulier les cellules régulatrices de type 1 (Tr1), seraient prédictifs de la réponse au traitement antiviral C, et aussi que les Treg pourraient être impliquées dans l’évolution de la récidive virale C après TH. JE montre aussi que les inhibiteurs de mTor induisent une augmentation de taux des Treg chez les patients transplantés hépatiques après changement du traitement d’un anticalcineurine, et que les anticalcinurines réagissent différemment sur l’activité suppressive des Treg in vitro. En conclusion, je précise le rôle majeur des Treg à la fois dans l’évolution de la récidive virale C sur le greffon hépatique, mais également quels sont les IS qui auraient un effet favorable sur le développement des Treg. / The results of liver transplantation (LT) have improved significantly, but long-term graft survival is still a major concern for doctors. It depends on the rejection rates, the recurrence of hepatitis C, and the immunosuppressive treatment and its complications. After LT, the graft reinfection with HCV is constant, and the evolution of chronic hepatitis is faster and more aggressive when compared to the time course before transplantation. It has been suggested the regulatory T cells (Treg) are involved in the induction of tolerance after organ transplantation, and in the persistence of HCV infection by suppressing the HCV-specific T responses. Furthermore, the number and function of Treg are very likely influenced by the immunosuppressive therapy used after transplantation. In this context, my work focuses on the role of Treg cells in the evolution of liver transplantation, and the effects of different treatments used after LT (immunosuppressive and anti-HCV) on this population. The results of my thesis show that the Treg cells (Type-1 regulatory cells, Tr1, in particular) are predictive of the response to the anti-HCV treatment after LT, and that Treg cells are associated with severe evolution of recurrent hepatitis C. I show that mTOR inhibitors have a positive impact on the number of circulating Treg cells in patients who underwent a conversion of therapy from Tacrolimus to a mTOR inhibitor, and that calcinurine inhibitors have different effects on Treg suppressive activity in vitro. In conclusion, we bring evidences on the involvement of Treg cells in HCV recurrence and treatment failure after liver transplantation and in their interaction with immunosuppressive drugs.

Sutherlandia frutescens is a South African herbal remedy traditionally used for various ailments and lately to improve the overall health in cancer and HIV/AIDS patients. Relatively little is known about the mechanisms of action of the constituents present in S. frutescens. The aim of this project was to examine the in vitro influence of crude ethanolic S. frutescens extracts in human breast adenocarcinoma (MCF-7) and non-tumorigenic breast epithelial (MCF-12A) cells after 48 h of exposure. Dose-dependent studies were conducted on cell numbers and metabolic activity by means of spectrophotometry. Morphological changes were determined with light-, fluorescent- and transmission electron microscopy (TEM). Cell cycle progression and apoptosis were analyzed using flow cytometry. The differential effects of S. frutescens extracts on gene expression levels in both the MCF-7 and MCF-12A cells were conducted utilizing micro array analysis. mTOR kinase activity was measured with an ELISA assay. S. frutescens reduced cell proliferation in both the non-tumorigenic MCF-12A and the tumorigenic MCF-7 cell line in a dose-dependent manner. The tumorigenic MCF-7 cells were more susceptible to S. frutescens treatment compared to the non-tumorigenic MCF-12A cells. Morphological characteristics of apoptosis and autophagy, including cytoplasmic shrinking, membrane blebbing and an increase in autophagic vacuoles were observed in both cell lines with the MCF-7 cells being more susceptible to autophagy and the MCF-12A cells less susceptible to autophagy and apoptotic cell death. TEM confirmed ultrastructural characteristics of autophagy in both cell lines. Flow cytometry revealed a G2/M arrest with no increase in apoptosis in MCF-7 cells and a G2/M arrest with an increase in apoptosis in MCF-12A cells treated with 1.5mg/ml S. frutescens extract. Microarray analyses revealed 325 statistically significantly differentially expressed genes in MCF-7 cells and 1467 genes in MCF-12A cells. The majority of S. frutescens-treated genes were down-regulated when compared to the vehicle-treated control in both cell lines. Several genes involved in DNA replication and repair were differentially expressed in response to S. frutescens exposure. These include Poly (ADP-ribose) polymerase family, member 2 (PARP-2) (down-regulated in both cell lines), PCNA (down-regulated in MCF-7 cells) and growth arrest and DNA-damage-inducible beta (GADD45B) (up¬regulated in MCF-12A cells). This suggests that abrogated expression of genes involved in DNA replication and repair play a role in inducing a G2/M cell cycle arrest in S. frutescens-treated cells. ELISA analysis of the mTOR kinase revealed a decrease in mTOR kinase activity in both cell lines after S. frutescens exposure. Therefore, attenuated mTOR kinase activity as a result of S. frutescens treatment in both cell lines is regarded as a central mediator in inducing autophagy suppressing gene expression and inhibiting ribosome biogenesis. Understanding of in vitro molecular mechanisms of S. frutescens enables researchers to focus on affected cellular mechanisms and identify active compounds with subsequent evaluation as possible candidates for use in anticancer therapy. The current study contributes to the unraveling of the in vitro molecular mechanisms and signal transduction associated with 70% ethanolic S. frutescens extracts, providing a basis for further research on this multi-purpose medicinal plant in Southern Africa. / Dissertation (MSc)--University of Pretoria, 2007. / Physiology / unrestricted

With a five-year survival rate of less than 5%, pancreatic cancer is one of the deadliest cancers. The most common activating mutations in pancreatic cancer are found in the KRAS gene, causing a constitutively-active KRAS protein in approximately 90% of pancreatic ductal adenocarcinomas (PDAC). PDAC-derived cell lines that harbor oncogenic KRAS mutations can be divided into two classes, KRAS dependent (or addicted) cells and KRAS independent cells. Oncogene dependency (or addiction) is a phenomenon where tu-mors require sustained activity of a single aberrantly activated gene despite the accumulation of multiple oncogenic lesions. In the case of PDAC, the single aberrantly activated gene is KRAS. KRAS independent cells have acquired various other oncogenic lesions that confer alternative cell survival signaling pathways to bypass oncogenic KRAS dependency. The Spleen Tyrosine Kinase (Syk) is highly expressed in KRAS dependent cells, while KRAS independent cells have low Syk expression. This pattern suggests that in KRAS dependent cells, constitutively active KRAS and Syk play a role in stimulating pro-survival pathways. One of these pro-survival pathways is known as mTORC1, which causes increased anabolic processes like protein and lipid synthesis. Accordingly, mTORC1 causes suppression of catabolic processes like autophagy. The net effect is in-creased cellular growth and proliferation. However, mTORC1 inhibitors have limited clinical efficacy, and potential therapeutic targets upstream of mTORC1 have drawn interest. Syk is a non-receptor tyrosine kinase that is an upstream activator of the mTORC1 pathway in hematopoietic malignancies. Through Syk inhibition studies using the small molecule PRT062607 (SYKi), we demonstrated that Syk is also involved in activating the mTORC1 pathway in KRAS dependent PDAC cells. However, the mechanism by which Syk-mediated activation of mTORC1 occurs is currently unknown. Moreover, it is unclear whether SYK kinase activity is required for the activation of the mTORC1 pathway. To address this issue, we introduced a single nucleotide mutation in the kinase do-main of Syk to render it kinase-inactive and found that Syk requires its kinase function to activate mTORC1. Studies using Syki also revealed that mTORC1 activity was also inhibited in KRAS independent PDAC cells that lack significant Syk expression. Interestingly, substrate specificity studies indicate that Syki also binds to and inhibits structurally similar protein tyrosine kinases such as the SRC Family Kinases (SFKs). Therefore, we designed an experiment to look for Syk and SFK cooperativity in regards to mTORC1 activation in PDAC cells. Our results indicate that the SFKs, Yes1 and Src display the most significant cooperative effect with Syk in activating the mTORC1 pathway. Src and Yes1 may even be involved in the upstream activation of Syk. To establish the physiological significance of Syk signaling in pancreatic cancer, it is important to establish model organisms that could be used for future studies. Thus, we test-ed Syk expression and function in PDAC cell lines derived from genetically-engineered mouse models (GEMM), which develop pancreatic cancer via oncogenic mutations in KRAS and TP53. We found that Syk is indeed expressed in murine PDAC cell lines and that the use of Syki in the murine PDAC cell lines results in decreased mTORC1 activity. These results recapitulate those obtained in human KRAS dependent PDAC cell lines. In summary, our studies show that Syk is a key regulator of mTORC1 signaling in human and mouse-derived pancreatic cancer cells. Syk kinase activity is required for mTORC1 activation. Finally, SFKs cooperate with Syk to promote robust mTORC1 activation. The mechanisms of SFK and Syk cooperativity in mTORC1 pathway activation will require further investigation. Additionally, our findings provide a strong rationale to study the effects of Syk kinase inhibition in physiologically-relevant murine models of pancreatic cancer. / 2021-06-08T00:00:00Z

Orientador: Luís Gustavo Modelli de Andrade / Resumo: 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 mi... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: 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. T... (Complete abstract click electronic access below) / Doutor

BK virus (BKV) is a ubiquitous polyomavirus known to asymptomatically reside in the renal tissues of up to 90% of the human population. BK virions reactivate during periods of intense immunosuppression and can cause disease in renal transplant recipients, such as BKV-associated nephropathy (BKVAN). BKVAN can lead to loss of the transplanted renal grafts. For this reason, the study of BKV biology is of importance to the transplant community. Previous studies have shown that BKV upregulates the pro-growth mTOR pathway in host cells, thereby increasing BKV replicative efficiency. Downstream effectors of the mTOR pathway, particularly p70S6 kinase, control the basal rate of protein translation, in part through regulation of ribosomal biogenesis. It was hypothesized that viral upregulation of the mTOR pathway is beneficial for viral replication due to an increase in the number of ribosomes available to translate viral proteins. Therefore, inhibition of the mTOR pathway could reduce viral replication. This study investigated whether host cell mTOR inhibition could reduce BK viral replication in an in vitro model. We utilized the dual PI3K/mTOR inhibitor NVP BEZ-235 (Novartis Pharmaceuticals), which potently downregulates expression of both upstream (PI3K) and central (mTOR) effectors of the mTOR pathway. Immortalized renal epithelial cells were exposed to varying concentrations of BEZ-235 for a period of 48 hours, infected with BK virus for three hours, and allowed to grow for a further 48 hours. Cell populations were then assayed via quantitative PCR (qPCR), Western blotting and fluorescent immunohistochemical staining to determine the effect of BEZ-235 on BK viral replication. Western blot experiments confirmed the effectiveness of BEZ-235's inhibition of the mTOR pathway in a renal epithelial cell culture model, as well as downregulation of the mTOR pathway during BK viral infection. Western blotting for the key BK replicative protein Large T antigen showed a dose-dependent decrease in expression, with increasing concentrations of BEZ-235. Fluorescent immunohistochemical staining showed a dose-dependent decrease in expression of Large T antigen staining in host cell nuclei. qPCR results were inconclusive, in that no clear pattern in the number of BKV genomes per cell population could be observed across the range of BEZ-235 concentrations tested. While results from our study indicate that BEZ-235 can reduce BKV replication in vitro, further in vitro experimentation, including repetition of approaches already carried out as well as novel approaches, will be needed to definitively confirm inhibition of the mTOR pathway as a viable antiviral strategy.

L'ischémie cérébrale est une pathologie complexe impliquant une cascade de mécanismes cellulaires qui conduisent, entre autres, à une augmentation de l’autophagie dans les neurones. Bien que l’activation de l’autophagie dans l’AVC ischémique soit aujourd’hui un fait avéré, le rôle de l'activateur tissulaire du plasminogène (tPA ; médicament utilisé dans la phase aigüe de l’AVC ischémique et neuromodulateur du système nerveux central) n’a jamais été décrit. Le tPA est une sérine protéase initialement découverte dans le compartiment vasculaire jouant un rôle important dans la fibrinolyse. Mais le tPA est aussi exprimé dans le parenchyme cérébral où il intervient dans le système glutamatergique, la plasticité synaptique et la survie neuronale. Afin de mieux comprendre les effets moléculaires du tPA dans l’autophagie, nous avons utilisé un modèle in vitro d'ischémie cérébrale consistant à sevrer en oxygène et en glucose (OGD) puis à réoxygéner des neurones corticaux primaires murins avec ou sans tPA. Nous avons confirmé, dans un premier temps, que l’OGD induit une autophagie délétère via une diminution de l’axe PI3K/Akt/mTORC1. Nous avons ensuite étudié l’effet du tPA sur l’autophagie induite par l’OGD. Nos résultats démontrent que le tPA protège les neurones de la mort induite par l’OGD en réduisant l’autophagie via l’activation du récepteur du facteur de croissance à l'insuline (IGF-1R, un récepteur tyrosine kinase) et de la voie PI3K/Akt/mTOR. Ce travail de thèse a donc permis de décrire le rôle neuroprotecteur et anti-autophagique du tPA, et d’identifier un nouveau récepteur cible du tPA : IGF-1R. / Cerebral ischemia is a complex pathology involving a cascade of cellular mechanisms leading, among other things, to an increase of neuronal autophagy. The activation of autophagy in ischemic stroke conditions is now well accepted, but the role of tissue-type plasminogen activator (tPA, a drug used in the acute phase of ischemic stroke, and a neuromodulator) on this pathway has never been studied. tPA is a serine protease originally discovered in the vascular compartment, that plays an important role in fibrinolysis. Interestingly, tPA is also expressed in the cerebral parenchyma where it is involved in the glutamatergic neurotransmission, synaptic plasticity and neuronal survival. To better understand molecular effects of tPA on autophagy, we used an in vitro model of cerebral ischemia consisting in an oxygen and glucose deprivation (OGD) followed by reoxygenation, on murine primary cortical neurons with or without tPA. First we reported that OGD enhances deleterious autophagy through the decrease of PI3K/Akt/mTOR pathways. Then, we investigated the effect of tPA on OGD-induced autophagy. Our results demonstrate that tPA protects neurons from OGD-induced death by reducing autophagy through Insulin Growth Factor Receptor (IGF-1R, a tyrosine kinase receptor) and an increase of PI3K/Akt/mTOR pathways. This thesis has made it possible to describe the neuroprotective and anti-autophagic effect of tPA, and to identify a new target receptor for tPA: IGF-1R.

L’étude d’échantillons humains montre que les voies de signalisation RAS/MAPK et PI3K/AKT/mTOR sont fréquemment activées de manière aberrante dans les tumeurs de la prostate, d’autant plus dans les phases de résistance aux traitements. Ces deux voies de signalisation sont sensibles aux facteurs de croissances et impliquées dans la régulation de processus cellulaires fondamentaux tels que la prolifération, la croissance ou encore la différenciation cellulaire. Ces données suggèrent qu’elles ont un rôle essentiel dans la tumorigenèse prostatique. Cependant, le rôle respectif de chacune de ces voies dans la carcinogenèse prostatique, particulièrement dans les phases précoces, n’est pas clairement établit. L’objectif de ma thèse est donc de définir le rôle possible de ces deux voies dans l’initiation et la progression du cancer de la prostate, ainsi que les mécanismes impliqués dans leur co-dérégulation. Cette étude est réalisée dans un modèle in vivo alternatif, la drosophile, qui possèdent un équivalent fonctionnel de la prostate : les glandes accessoires. La première partie des travaux réalisés montre que seule la suractivation de la voie RAS/MAPK dans la glande accessoire conduit à un processus de tumorigenèse, avec la production de masses cellulaires récapitulant de nombreuses caractéristiques cancéreuses : croissance cellulaire et prolifération incontrôlée, expression de métalloprotéases, perte de l’expression de marqueurs épithéliaux et formation de nouvelles trachées. Cependant, les deux voies de signalisation sont nécessaires à la tumorigenèse, mais avec des rôles différents : la voie RAS/MAPK est activée précocement et est capable de recruter la voie PI3K/AKT/TOR grâce à la mise en place de deux boucles autocrines de régulation. La première dépend de spitz (dEGF) et du récepteur EGFR pour amplifier l’activation de la voie RAS/MAPK. La seconde dépend de l’activation d’ILP6 (dIGF1), produit suite à l’activation de la voie RAS/MAPK, et permet le recrutement de la voie PI3K/AKT/TOR par l’intermédiaire du récepteur à l’insuline InR. La deuxième partie des travaux réalisés montre que l’activation de la voie RAS/MAPK conduit à la production de MMP1 dans les cellules qui seront à l’origine des tumeurs avant leur extravasation hors de l’épithélium. Cette expression temporelle contrôlée correspond à une étape où une réorganisation du cytosquelette a lieu et où le microenvironnement est altéré. Ces données placent donc la dérégulation de la voie RAS/MAPK comme un évènement précoce de la tumorigenèse prostatique, capable de recruter la voie PI3K/AKT/TOR et d’entrainer la production de MMP1, pour in fine conduire à l’extravasation des cellules et à la formation de tumeurs. / Clinical studies have demonstrated that, in prostate cancer, RAS/MAPK and PI3K/AKT/TOR signaling pathways are often aberrantly co-activated in tumors, their activation levels increasing again in resistance phases. These pathways, that are regulated by growth factors, are implicated in fundamental cellular processes regulation such as proliferation, growth and cellular differentiation. These data suggest that they are likely implicated in prostate tumorigenesis. However, the relative implication of each of these two pathways during prostate tumorigenesis, especially during early phases, is not fully understood. Thus, the aim of my thesis is to define the possible implication of these pathways in prostate cancer initiation and progression and which molecular mechanisms are implicated in their co-deregulation. Therefore, we have developed an alternative in vivo model of prostate tumorigenesis in drosophila, where accessory glands are a functional equivalent of the human prostate. The first part of my work shows that only the hyperactivation of the RAS/MAPK pathway in accessory glands can promote tumorigenesis, with the formation of cell masses that recapitulate many cancer hallmarks including uncontrolled cell growth and proliferation, enhanced matrix metalloproteinases expression, loss of epithelial markers expression, neovascularization-like tracheogenesis. However, both pathways are necessary to tumorigenesis, even though they display different roles: the RAS/MAPK pathway is activated earlier and is able to recruit the PI3K/AKT/TOR pathway thanks to the formation of two feedback loops. The first depend on Spitz (dEGF) and EGFR receptor to amplify RAS/MAPK pathway activation. The second depends on ILP6 (dIGF1) activation, produced following RAS/MAPK pathway activation and allow PI3K/AKT/TOR pathway recruitment via insulin receptor InR. The second part of the work shows that RAS/MAPK pathway activation allows MMP1 production restricted to the cells that will be the origin of the tumors, before their actual extravasation. This temporally controlled step of MMP1 expression corresponds to a time window where the cells show strong cytoskeletal reorganization and where microenvironment is disturbed. These data place the RAS/MAPK pathway deregulation as an early event of prostate tumorigenesis, able to recruit the PI3K/AKT/TOR pathway and to induce MMP1 production to allow cell extravasation and tumor formation.

BACKGROUND: Lymphangioleiomyomatosis (LAM) is a rare disease characterized by cystic destruction of the lung. It occurs in 80% of people with Tuberous Sclerosis Complex disorder (TSC), a multisystem, autosomal dominant disorder caused by mutations in tumor suppressor genes TSC1 and TSC2. Spontaneous biallelic mutations in these genes can give rise to sporadic LAM. Mammalian target of rapamycin complex I (mTORC1), a master regulator of cellular anabolic metabolism is hyperactivated in LAM cells. Upregulation of protein synthesis and downregulation of autophagy creates a state of starvation stress that upregulates pathways of extracellular nutrient acquisition. Macropinocytosis, a form of clathrin-independent endocytosis, is upregulated in TSC2-deficient cells. We performed a high-throughput compound screen utilizing a repurposing drug library. We identified that ritanserin, a diacylglycerol kinase alpha (DGKA) inhibitor, synergizes with Chloroquine (CQ) to selectively inhibit proliferation of TSC2-deficient mouse embryonic fibroblasts (MEFs) compared to TSC2+/+ MEFs. OBJECTIVE: We hypothesized that TSC2-deficient cells rely on macropinocytosis to support their growth during the periods of stress and starvation and that ritanserin synergizes with CQ to inhibit proliferation in TSC2-deficient cells by inhibiting macropinocytosis. METHODS: Crystal violet-based proliferation assays were used to monitor the effect of pharmacological and genetic inhibition of DGKA on cell proliferation. Immunoblotting was used to measure the expression levels of TSC2, tS6R, pS6R, Cleaved PARP, Cleaved Caspase 3 and Actin. siRNA induced Htr2a knockdown and shRNA induced DGKA knockdown cell culture models were used to define the dual functions of ritanserin and observe their effects on macropinocytosis and cell proliferation. LC/MS was used to measure cell lipid content and how it changes in response to ritanserin. Fluorophore-labeled BSA and 70-kDa Dextran were used to measure macropinocytosis. Lysotracker was used to measure the number of lysosomes, while DQ-BSA was used to measure lysosomal functionality. RESULTS: TSC2-deficient cells express higher levels and show upregulated activity of DGKA. Genetic and pharmacologic inhibition of DGKA prevents TSC2-deficient cells from acquiring nutrients via macropinocytosis. Phospholipid metabolism is altered in TSC2-deficient cells, marked by the accumulation of phosphatidic acid and ceramides. Treatment with ritanserin leads to the accumulation of diacylglycerol and phospholipids, as well as a reduction in phosphatidic acid. CONCLUSIONS: TSC2-deficient cells rely on macropinocytosis to meet their metabolic needs. Diacylglycerol kinase alpha (DGKA) is required for macropinocytic nutrient uptake. Pharmacologic or genetic inhibition of DGKA creates metabolic stress in TSC2-deficient cells, which ultimately leads to increased apoptotic response to treatment with CQ. This project identifies a novel connection between mTOR signaling, lysosome metabolism and macropinocytosis, and a vulnerability that allows the selective targeting of LAM cells. / 2021-06-07T00:00:00Z

The eIF4E is an important eukaryotic translation initiation factor, because of its ability to bind cap at 5'end of mRNA. There are three members of this protein family found in humans: eIF4E1, eIF4E2 and eIF4E3. eIF4E1 also plays role in in export of some mRNA from nucleus to cytoplasm. This protein is mostly regulated by mTOR signaling pathway and malfunctions in regulation leads to increased cell proliferation and thus tumorogenesis. eIF4E2 plays a role in regulating of translation during embryogenesis and it is known to mediate translation in terms of hypoxia. Role of eIF4E3 is so far shrouded in mystery. Some studies suggest it might be able to suppress tumor growth, but no studies have been done on human eIF4E3. Big potential of our work is, that all proteins we work with, are human. Based on our results, the endogenous amount of eIF4E3 protein is higher than it was thought. This is one of the reasons, why this protein should not escape our attention. In my diploma thesis, I have studied physiological characteristics of cell cultures overexpressing eIF4E proteins after mTOR inhibition treatment. I have realized that the most efficient inhibitor in all tested cell cultures is PP-242, which binds directly into active site of mTOR kinase. I have cloned 3xC FLAG tagged eIF4Es constructs and used...