Le système immunitaire joue un double rôle dans le cancer: il peut non seulement supprimer la croissance tumorale en détruisant les cellules cancéreuses, mais aussi promouvoir la progression de la tumeur en sélectionnant les cellules tumorales ou en créant un microenvironnement tumoral immunosuppresseur. Au cours de ma thèse, je me suis intéressée à deux populations du système immunitaire : les MDSC (Myeloïd Derived Suppressor Cells) et les lymphocytes Th17. Dans ce travail, nous avons exploré les mécanismes impliqués dans l’activation et dans les fonctions suppressives de ces deux populations cellulaires au cours de la croissance tumorale. Tout d’abord, nous avons montré que les cellules tumorales activaient les MDSC en libérant des exosomes exprimant HSP72 à leur surface. HSP72 à la surface des exosomes permet l’activation de STAT3 via la voie de signalisation TLR2/Myd88 dans les MDSC et favorise leur fonction suppressive. Le facteur de transcription STAT3 joue aussi un rôle fondamental dans la différenciation des lymphocytes Th17. Le rôle des lymphocytes Th17 est controversé dans la croissance tumorale. Nous avons découvert que les lymphocytes Th17 exprimaient les ectonucléotidases CD39 et CD73 soutenant ainsi leur fonction suppressive. L'expression des ectonucléotidases est dépendante de l’activation de STAT3 par l’IL-6 et de l’inhibition de Gfi-1 par le TGFβ lors de leur différenciation. / Immune system plays a dual role in cancer: it can not only suppress tumor growth by destroying cancer cells, but also promote tumor progression by selecting immunoresistant tumor cells or by establishing an immunosuppressive microenvironment.During my thesis, I focused on two populations of the immune system: MDSC (myeloid derived suppressor cells) and Th17 lymphocytes. In this work, we explored the mechanisms involved in the activation of suppressive functions of these two cell populations during tumor growth. First, we showed that tumor cells activated MDSC by releasing exosomes that express HSP72 on their surface. HSP72 on exosomes surface induces STAT3 activation via Myd88/TLR2 signaling pathway in MDSC and promotes their suppressive function. The transcription factor STAT3 also plays a fundamental role in Th17 cells differentiation. The role of Th17 cells is controversial in tumor growth. We reported that Th17 cells express CD39 and CD73 ectonucleotidases and thereby supporting their suppressive function. The expression of ectonucleotidases is dependent on the activation of STAT3 by IL-6 and inhibition of Gfi-1 by TGFβ during their differentiation.

Células neuronais cocultivadas com células-tronco derivadas do tecido adiposo (ADSC) podem induzir estas últimas à transdiferenciação neuronal. No entanto, os processos de comunicação celular envolvidos nessa indução, e a funcionalidade da ADSC transdiferenciada in vivo, são desconhecidos. Recentemente, um novo tipo de comunicação celular mediada por vesículas extracelulares são indicados na modulação de diferentes eventos celulares como a diferenciação. Portanto, a hipótese, nesta proposta, foi identificar se o processo de diferenciação celular é mediado por vesículas extracelulares e se as células diferenciadas são capazes de atuar na regeneração de tecidos nas lesões do sistema nervoso periférico. Para tal, essa pesquisa foi dividida em duas fases: a primeira consiste no processo in vitro, com o objetivo de observar o processo de transição da ADSC para a linhagem neuronal e analisar a função da comunicação celular no processo de diferenciação; a segunda consistiu na avaliação in vivo da possível funcionalidade das ADSC diferenciadas. O camundongo foi o modelo animal utilizado (C57BL/6 e FVB). As ADSC e as células neuronais foram isoladas, cultivadas em cultivo primário e cocultivadas durante três, sete e 14 dias. Para a comprovação das mudanças fenotípicas das ADSC, realizou-se a imunolocalização com beta tubulina III e SNAP25 e PCR em tempo real (RT-qPCR) dos genes Map2 e Snap25. Seguido de analises de genes relacionados com a neurogênese. Adicionalmente, as vesículas extracelulares foram isoladas e utilizados para análises in vitro da diferenciação e análises gênicas e funcionais. Como resultado, verificou-se que as ADSC em cocultivo com neurônios podem se diferenciar em neuronais-like. Além disso, comprovou-se a comunicação por vesículas extracelulares entre neurônios e ADSC, e as vesículas extracelulares foram correlacionadas neste processo, pelo transporte da proteína SNAP25. Após estes resultados prosseguiu-se para a segunda fase deste trabalho, a etapa in vivo, que incidiu na utilização das ADSC cocultivadas por sete dias e avaliação funcional local e sistêmica no processo de regeneração do nervo ciático após neurotmese. Como resultado desta etapa as células-tronco cocultivadas modularam a lesão, e proporcionaram uma melhoria na funcionalidade após lesão. Conclui-se, através desta pesquisa, que as ADSC diferenciadas em neuronais-like, sob indução dos neurônios e suas vesículas extracelulares podem ser uma fonte celular alternativa no auxílio na regeneração de nervos periféricos. / Neuronal cells co-cultured with stem cells derived from adipose tissue (ADSC) can induce the latter to neuronal transdifferentiation. However, the cellular communication processes involved in this induction, and the functionality of the transdifferentiated ADSC in vivo, are unknown. Recently, a new type of cellular communication measured by extracellular vesicles was indicated in the modulation of different cellular events like differentiation. Therefore, the hypothesis in this proposal was to identify if the process of cellular differentiation is mediated by extracellular vesicles and if the differentiated cells are able to act in the regeneration of tissues in the lesions of the peripheral nervous system. For this, the research was divided in two phases: the first one consisted of the in vitro process, with the objective of observing the transition process of the ADSC to the neuronal lineage and analyzing the cellular communication function in the differentiation process; the second consisted in the in vivo evaluation of the possible functionality of the differentiated ADSCs. Murine was the animal model used (C57BL/6 and FVB). ADSCs and neuronal cells were isolated, cultured in primary culture and co-cultured for three, seven and 14 days. To confirm the phenotypic changes of ADSC, immunolocalization with beta tubulin III and SNAP25 and real-time PCR (RT-qPCR) of the Map2 and Snap25 genes was performed, followed by analysis of genes related to neurogenesis. In addition, extracellular vesicles were isolated and used for in vitro differentiation and gene and functional analysis. As a result, it has been found that ADSCs in co-culture with neurons can differentiate into neuronal-like. The communication by extracellular vesicles between neurons and ADSCs was verified, and the extracellular vesicles were correlated in the differentiation process by the transport of the protein SNAP25. After these results, the second phase of this work was continued, the in vivo step, which focused on the use of the co-cultivated ADSCs for seven days and functional local and systemic evaluation in the process of sciatic nerve regeneration after neurotmese. As a result of this step, the co-cultured stem cells modulated the lesion and provided an improvement in functionality after injury. It is concluded that ADSCs can transdifferentiate neuronal lines in co-culture with neurons, the extracellular vesicles play a certain role in this process and the transdifferentiated ADSC may be an alternative to aid in the regeneration of peripheral nerves.

A síntese ribossomal é uma das maiores atividades em células eucarióticas. Este processo inicia-se no nucléolo e é finalizado após a exportação das subunidades 40S e 60S para o citoplasma. Três dos RNAs ribossomais de eucariotos (18S, 5.8S e 25S) são sintetizados como um transcrito primário de 35S, o qual é processado através de uma complexa e ordenada série de modificações nucleotídicas e clivagens endo e exonucleolíticas. Estas reações dependem de aproximadamente 170 proteínas, 80 small nucleolar RNAs e de seqüências no pré-rRNA. Os fatores trans-atuantes envolvidos no processamento podem ser agrupados como RNA-helicases, endonucleases, snoRNPs (small nucleolar ribonucleoprotein complexes) e exonucleases, que incluem o complexo exossomo. O exossomo de levedura é formado por 10 proteínas essenciais que atuam na maturação de rRNAs, snRNAs, snoRNAs, além da degradação de mRNAs incorretamente processados. A estrutura do exossomo de archaea foi descrita recentemente, mas ainda não existem muitas informações sobre a regulação deste complexo e sobre a participação de cofatores que interagem de forma transiente com o exossomo. Diante disso, este trabalho visou a caracterização funcional das proteínas que formam o anel de RNases PH em Saccharomyces cerevisiae, assim como a caracterização estrutural e funcional de possíveis cofatores do exossomo de Saccharomyces cerevisiae, Nop17p e Ylr022p, e do exossomo de Pyrococcus, Pab418p, Pab1135p e aNip7p. Os dados obtidos evidenciam que a atividade exonucleolítica do exossomo de levedura, assim como o de archaea, é dependente da formação de heterodímeros; Ylr022p, uma proteína de levedura com função não caracterizada, liga inespecificamente RNA in vitro, mas mais eficientemente alguns RNAs in vivo. Dentre as proteínas de archaea, Pab418p e aNip7p também ligam RNA, e como demonstrado aqui, aNip7p influencia significativamente a atividade do exossomo de archaea. / The synthesis of ribosomes is one of the major metabolic pathways in eukaryotic cells. This process starts in the nucleolus and ends with the export and final maturation of the ribosomal subunits 40S and 60S in the cytoplasm. Three eukaryotic ribosomal RNAs (18S, 5.8S and 25S) are synthesized as a 35S primary transcript (35S pre-rRNA), which is then processed by a complex and ordered series of nucleotide modifications and endo- and exonucleolytic cleavage reactions. These processing reactions depend on 170 proteins, 80 small nucleolar RNAs and specific pre-rRNA sequences. The trans-acting factors, that take part in the processing can be grouped as RNA-helicases, endonucleases, snoRNPs (small nucleolar ribonucleoprotein complexes) and exonucleases, including the exosome. The yeast exosome is composed of 10 essential proteins that function in the processing of rRNAs, snRNAs, snoRNAs and in the degradation of aberrant mRNAs. Recently, the archaeal exosome structure was determined, but no information is yet available on the regulation of the exosome function or on the possible role of the cofactors that transiently interact with it. The main goals of this work were the functional characterization of the protein components of the Saccharomyces cerevisiae exosome RNase PH ring, as well as the structural and functional characterization of the possible cofactors of that complex, Nop17p and Ylr022p. Since the recent characterization of the Pyrococcus exosome, the study of the archaeal exosome cofactors, Pab418p, Pab1135p and aNip7p, was also included in this work, in order to correlate the data on the complex of these different organisms. Our results show that the exonucleolytic activity of the yeast exosome is dependent on the heterodimers formation, as described for archaea. Although it is not clear how Nip7p affects the exosome function in yeast, aNip7p binds RNA and inhibits a-exosome activity in vitro. Yeast Ylr022p binds RNA inespecificaly in vitro, but coprecipitates specific RNAs more efficiently from total cell extracts. Its archaeal orthologue, Pab418p, also binds RNA, but does not affect significantly a-exosome function.

Em eucariotos, o processamento de pré-rRNA depende de vários fatores como endonucleases, exonucleases, RNA helicases, enzimas modificadoras de rRNA e componentes de snoRNPs. Com o objetivo de caracterizar novas proteínas envolvidas no processamento de pré-rRNA, foi identificada a proteína Nop53p interagindo com a proteína nucleolar Nop17p a partir de uma varredura da biblioteca de cDNAs de Saccharomyces cerevisiae. A cepa condicional contendo a seqüência da ORF NOP53 sob controle do promotor de galactose não cresce em meio contendo glicose, indicando que Nop53p seja uma proteína essencial para a viabilidade celular. Os resultados deste trabalho demonstram que Nop53p está envolvida nas etapas iniciais de clivagem do pré-rRNA, assim como nas clivagens responsáveis pela formação dos rRNAs maduros 5.8S e 25S. Análise mais detalhada do processamento de pré-RNA por Northern blot e \"pulse-chase labeling\", revelou também que Nop53p afeta principalmente o processamento do rRNA intermediário 27S, que origina os rRNAs maduros 5.8S e 25S. Nop53p participa do processamento desses rRNAs afetando a poliadenilação dos precursores dos rRNAs 5.8S e 25S. Experimentos de co-imunoprecipitação de RNA com a proteína de fusão ProtA-Nop53p confirmaram o envolvimento de Nop53p no processamento do 27S rRNA, indicando que essa proteína possa ligar RNA diretamente. A capacidade de Nop53p de ligar RNA foi confirmada através de testes in vitro, enquanto que ensaios de co-imunoprecipitação de cromatina revelaram que Nop53p liga-se ao rRNA 5.8S durante a transcrição. Nop53p regula a função do exossomo através da sua interação direta com a subunidade exclusivamente nuclear deste complexo, Rrp6p. / In eukaryotes, the rRNA processing depends on several factors, such as, endonucleases, exonucleases, RNA helicases, rRNA modifying enzymes and components of the snoRNPs. With the purpose of characterizing new proteins involved in pre-rRNA processing, Nop53p was identified interacting with the nucleolar protein Nop17p in a two hybrid assay. The conditional yeast strain containing the sequence of the ORF NOP53 under the control of the galactose promoter cannot grow in medium containing glucose, indicating that the protein is essential for cell viability. The results of this work demonstrate that Nop53p is involved in the initial steps of pre-rRNA processing and in the cleavages responsible for the formation of the mature rRNAs 5.8S and 25S. A more detailed analysis of the pre-rRNA processing, by Northern blot and pulse-chase labeling, revealed that Nop53p affects the processing of the 27S precursor, that originates the rRNAs 5.8S and 25S. Nop53p participates in the processing of these RNAs by affecting the polyadenylation of the precursors of the rRNAs 5.8S and 25S. RNA co-imunoprecipitation assays with the fusion protein A-Nop53p confirmed the involvement of Nop53p in the processing of the 27S pre-rRNA, indicating that the protein may interact directly with the RNA. The capacity of Nop53p to bind RNA was confirmed by in vitro assays, while chromatin imunoprecipitation assays demonstrated that Nop53p binds the 5.8S rRNA co- transcriptionally. Nop53p regulates the function of the exosome by interacting directly with the exclusively nuclear subunit of the complex, Rrp6p.

京都大学 / 新制・課程博士 / 博士(理学) / 甲第23049号 / 理博第4726号 / 新制||理||1677(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 大野 睦人, 教授 青山 卓史, 教授 川口 真也 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

Les gliomes, tumeurs cérébrales primaires du système nerveux central, sont souvent de pronostic défavorable, d'autant plus que l'absence de critères indiscutables pour les identifier rend leur diagnostic et leur prise en charge particulièrement difficiles. L’analyse conjointe, d’une cohorte française de 64 patients porteurs de gliomes et d’une cohorte internationale de 671 patients issus du TCGA, a permis de mettre en évidence deux groupes pronostiques constitués par un panel d’expression différentielle de 26 gènes (p = 0,007). Cette stratification en deux groupes pronostiques a été confirmée quels que soient le grade et le groupe moléculaire de la tumeur (p < 0,0001). Nous avons établi une nouvelle stratégie diagnostique à partir de la classification moléculaire des gliomes en intégrant deux biomarqueurs pronostiques CHI3L1 et NTRK2. L’analyse multivariée confirme que ces biomarqueurs sont indépendants du statut IDH et du grade tumoral. Si nous avons mis en évidence par l’analyse protéique de CHI3L1 une concordance avec les transcrits, les résultats divergent pour TrkB. Ainsi, une expression élevée de TrkB et son corécepteur p75NTR serait liée à l’agressivité tumorale indépendamment du statut IDH. Enfin, TrkB et p75NTR sont présents aussi bien dans les exosomes issus du plasma de témoins sains et de patients atteints de gliomes mais leur expression augmente en fonction de l’agressivité de la tumeur / Gliomas, primary brain tumours of the central nervous system, are often of poor prognosis.The absence of clear criteria to identify them makes their diagnosis and management particularly difficult. The combined analysis of a cohort of 64 glioma patients and an international cohort of 671 patients from the TCGA revealed two prognostic groups of a differential expression panel of 26 genes (p = 0.007). This stratification into two prognostic groups was confirmed independently of the grade and molecular group of the tumor (p <0.0001). We have established a new diagnostic strategy based on the molecular classification of gliomas by integrating two prognostic biomarkers CHI3L1 and NTRK2. Multivariate analysis confirms that these biomarkers are independent of IDH status and tumor grade.While we have demonstrated by the protein analysis of CHI3L1 concordance with the transcripts, the results are different for TrkB. Therefore, a high expression of TrkB and its p75NTR co-receptor would be associated with tumor aggressiveness regardless of IDH status. Lastly, TrkB and p75NTR are present in exosomes from plasma of healthy controls and glioma patients, but their expression increases with the aggressiveness of tumor.

Exosomes are nanosized vesicles that contain proteins and nucleic acids. They are released and taken up by many different cell types as a way of cell-to-cell communication. It has previously been recognized that exosomes released by cancer cells promote tumor progression. Moreover, recent studies have increasingly found evidence that microRNA contained within cancer derived exosomes plays an important role in tumor progression. In this manuscript, the current knowledge on exosomes, the sorting of microRNA into exosomes as cargo, the gene regulatory mechanisms of microRNA, and recent findings on the tumor promoting actions of exosomal microRNAs on gastric cancer, hepatocellular carcinoma and breast cancer, is reviewed. The information gathered emphasizes the importance of exosomal microRNAs that increase tumor growth in the development of cancer treatment and research.

Skeletal muscle functions as an endocrine organ. Exosomes, small vesicles containing mRNAs, miRNAs, and proteins, are secreted from muscle cells and facilitate cell-to-cell communication. Our recent work found greater exosome release from oxidative compared to glycolytic muscle. Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) is a key driver of mitochondrial biogenesis, a characteristic of oxidative muscle. It was hypothesized that PGC1α regulates exosome biogenesis and secretion in skeletal muscle. The purpose of this study is to determine if PGC-1α regulates skeletal muscle exosome biogenesis and secretion. On day 4 of differentiation, human primary myotubes from vastus lateralis biopsies from lean donors (BMI < 25.0 kg/m2) were exposed to adenovirus encoding human PGC-1α or GFP control. On day 6 of differentiation, culture media was replaced with exosome-free media. On day 8, cells were collected for mRNA and protein analysis, and culture media was collected for exosome isolation. Overexpression of PGC-1α increases regulators of exosome biogenesis in the endosomal sorting complexes required for transport (ESCRT) pathway: Alix (CON: 1.0 ± 0.2 vs. PGC-1α: 7.6 ± 3.8), TSG-101 (CON: 1.0 ± 0.1 vs. PGC-1α: 7.3 ± 2.1), CD63 (CON: 1.0 ± 0.17 vs. PGC-1α: 3.7 ± 0.4), Clathrin (CON: 1.0 ± 0.2 vs. PGC-1α: 11.6 ± 2.5), and the secretion pathway: Rab27b (CON: 1.0 ± 0.3 vs. PGC-1α: 3.2 ± 0.3), STAM (CON: 1.0 ± 0.3 vs. PGC-1α: 7.3 ± 0.6), and VTA1 (CON: 1.0 ± 0.1 vs. PGC-1α: 7.3 ± 2.4). Exosome count and total extracellular vesicle count were not significantly different from control. Overexpression of PGC-1α increases gene 9 expression of regulators of exosome biogenesis and secretion in human primary myotubes. In the future, in vitro studies assessing exosomal content from PGC-1 OE cells as well as in vivo effects of PGC-1 OE on exosome production and release should be investigated to further understand the role PGC-1 plays in exosome secretion.

Multiple regulatory mechanisms work to ensure that eukaryotic transcription maintains mRNA pools and subsequent protein synthesis. When errors in transcription occur, deleterious effects on cellular fitness can develop. RNA degradation as well as histone modifications, specifically at promoter proximal nucleosomes, play a critical role in maintaining transcription, but, exact mechanisms are not fully understood. In this dissertation, I investigate the role of RNA degradation and chromatin dynamics in transcription regulation as well as further understand, through biochemical analysis, a critical histone deacetylase. Using various genome-wide methodologies in Saccharomyces cerevisiae, we find a functional interaction between the nuclear RNA exosome and histone variant H2A.Z that maintains mRNA levels. There is a reduction in RNA polymerase II nascent transcription following RNA exosome subunit Rrp6 depletion that is further globally accentuated with H2A.Z deposition loss. To understand the mechanism leading to this global reduction, we identify the mRNA of Sirtuin histone deacetylase Hst3 as a target of the RNA exosome, revealing a means to link degradation to the transcription machinery. These findings show that even slight changes in deacetylase or acetylase activity can have significant effects on transcription. Additionally, we reveal a global impact of H2A.Z on transcription. We further investigate the functional and structural significance of human surtuin histone deacetylase SIRT6 (yeast homolog Hst3). Using histone deacetylase assays, we confirm the significance of specific residues of SIRT6 in nucleosome binding and deacetylase activity. Additionally, we show SIRT6 has reduced deacetylase activity in vitro on acetylated lysine 56 as compared to acetylated lysine 9 on histone H3. Finally, we confirm structural findings that the histone tail of H2A impacts SIRT6 H3K9Ac deacetylation activity. Together, these findings indicate a critical importance of histone deacetylase activity in maintaining transcription, a novel role of H2A.Z in global transcription regulation that furthers our understanding of SIRT6 structure and function.

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