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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
41

mRNA degradation factors as regulators of the gene expression in Saccharomyces cerevisiae / mRNA nedbrytningsfaktorer som regulatorer av genexpression i Saccharomyces cerevisiae.

Muppavarapu, Mridula January 2016 (has links)
Messenger RNA degradation is crucial for the regulation of eukaryotic gene expression. It not only modulates the basal mRNA levels but also functions as a quality control system, thereby controlling the availability of mRNA for protein synthesis. In Saccharomyces cerevisiae, the first and the rate-limiting step in the process of mRNA degradation is the shortening of the poly(A) tail by deadenylation complex. After the poly(A) tail shortens, mRNA can be degraded either through the major 5' to 3' decapping dependent or the 3' to 5' exosome-mediated degradation pathway. In this thesis, we show some of the means by which mRNA decay factors can modulate gene expression. First, Pat1 is a major cytoplasmic mRNA decay factor that can enter the nucleus and nucleo-cytoplasmically shuttle.  Recent evidence suggested several possible nuclear roles for Pat1. We analyzed them and showed that Pat1 might not function in pre-mRNA decay or pre-mRNA splicing, but it is required for normal rRNA processing and transcriptional elongation. We show that the mRNA levels of the genes related to ribosome biogenesis are dysregulated in the strain lacking Pat1, a possible cause of the defective pre-rRNA processing. In conclusion, we theorize that Pat1 might regulate gene expression both at the level of transcription and mRNA decay. Second, Edc3 and Lsm4 are mRNA decapping activators and mRNA decay factors that function in the assembly of RNA granules termed P bodies. Mutations in mRNA degradation factors stabilize mRNA genome-wide or stabilize individual mRNAs. We demonstrated that paradoxically, deletion of Edc3 together with the glutamine/asparagine-rich domain of Lsm4 led to a decrease in mRNA stability. We believe that the decapping activator Edc3 and the glutamine/asparagine-rich domain of Lsm4 functions together, to modify mRNA decay pathway by altering cellular mRNA decay protein abundance or changing the mRNP composition or by regulating P bodies, to enhance mRNA stability. Finally, mRNA decay was recently suggested to occur on translating ribosomes or within P bodies. We showed that mRNA degradation factors associate with large structures in sucrose density gradients and this association is resistant to salt and sensitive to detergent. In flotation assay, mRNA decay factors had buoyancy consistent with membrane association, and this association is independent of stress, translation, P body formation or RNA. We believe that such localization of mRNA degradation to membranes may have important implications in gene expression. In conclusion, this thesis adds to the increasing evidence of the importance of the mRNA degradation factors in the gene expression.
42

Isolierung und Charakterisierung funktioneller Exosomen durch sequentielle Filtration

Heinemann, Mitja Leonard 04 October 2016 (has links) (PDF)
Exosomen aus Zellen nehmen eine immer größere Rolle in aktuellen Erkenntnissen zu Tumorwachstum und Metastasierung ein. Die Funktionen dieser circa 40-100 nm großen, aktiv sezernierten Vesikel sind bisher noch weitesgehend ungeklärt. Für die Untersuchung von Exosomen sind optimierte und schonende Isolierungsmethoden notwendig. Zur Zeit gibt es jedoch weder eine standardisierte Methode zur routinemäßigen Isolation von Exosomen noch zur Isolation von funktionell intakten Exosomen für die anschließende Funktionsanalyse. Ziel dieser Arbeit war es, eine vereinfachte, größenbasierte und standardisierbare Methode zur Isolation von funktionell intakten Exosomen zu entwickeln und die isolierten Exosomen qualitativ und quantitativ zu charakterisieren. Die Ergebnisse dieser Arbeit konnten in einer internationalen Fachzeitschrift publiziert werden. Die Publikation liegt dieser Arbeit bei (Heinemann et al. 2014).
43

Análise estrutural e funcional de cofatores do exossomo em Saccharomyces cerevisiae e Pyrococcus / Structural and functional analysis of exosome cofactors in Saccharomyces cerevisiae and Pyrococcus

Luz, Juliana Silva da 25 August 2006 (has links)
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.
44

Caracterização da função da proteína Nop53p de Saccharomyces cerevisiae / Study of the function of the protein Nop53p in Saccharomyces cerevisiae

Granato, Daniela Campos 07 December 2007 (has links)
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.
45

Comunicação exossomal na transdiferenciação de células-tronco em cocultivo com células neuronais / Exosome communication in the transdifferentiation of stem cells in co-culture with neuronal cells

Roballo, Kelly Cristine Santos 22 September 2017 (has links)
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.
46

Perfil de exossomos periféricos nas fases aguda e crônica do acidente vascular encefálico

Magalhães, Amanda de Souza January 2016 (has links)
O acidente vascular encefálico (AVE) é uma doença comum e de grande impacto para a saúde da população, uma vez que é considerado a principal causa de incapacidades neurológicas, principalmente motora e cognitiva. Considerando que os exossomos e o seu conteúdo podem ser marcadores prognóstico de demência em pacientes com Doença de Alzheimer, avaliamos o perfil exossomal circulante em pacientes pós AVE isquêmico com e sem comprometimento cognitivo. Ainda, é de conhecimento que os pacientes tratados com trombolíticos apresentam redução na dependência funcional a longo prazo, assim é de interesse avaliar os exossomos periféricos destes pacientes. Este trabalho visou investigar o perfil de exossomos circulantes em pacientes após diagnóstico de AVE isquêmico (AVEI) nas fases aguda e crônica. Foram determinados o conteúdo de proteína total e a atividade da acetilcolinesterase (AChE), marcadores de exossomos, além de correlacionar escores de comprometimento neurológico com os parâmetros bioquímicos. Ainda, considerando que o estresse oxidativo tem um papel central na fisiopatologia da isquemia cerebral, determinamos o conteúdo de espécies reativas e a atividade da enzima superóxido dismutase (SOD) em exossomos circulantes. Os fatores comprometimento cognitivo e o uso do trombolítico na fase aguda da doença não mostraram ter influência sobre os parâmetros estudados. Os resultados sugerem que uma diminuição de exossomos circulantes, já que houve uma redução na quantificação de proteínas totais na fase crônica, pode sugerir um prejuízo no sistema de remoção de materiais tóxicos levando ao acúmulo de materiais indesejados disso, o estado oxidativo exossomal, representado pelos níveis de espécies reativas e a atividade da SOD, foi alterado na fase crônica quando comparado com a fase aguda. Estes resultados sugerem que há uma alteração do perfil dos exossomos ao longo do tempo nos pacientes com AVE isquêmico. Nossos dados indicam que os níveis de espécies reativas na fase aguda podem predizer alterações no perfil dos exossomos na fase crônica de pacientes com comprometimento cognitivo, especificamente nas atividades das enzimas SOD e AChE e na concentração de proteínas totais. Estas associações não foram observadas em pacientes sem comprometimento cognitivo. Correlacionamos os parâmetros bioquímicos com as escalas de déficit neurológico, os escores do Mini-Exame do Estado Mental (MEEM) foram inversamente proporcionais aos níveis de AChE naqueles pacientes que não receberam o tratamento trombolítico na fase aguda. Assim, nossos dados demonstram que o prejuízo cognitivo (menores escores de MEEM) está associado a maiores níveis de atividade da AChE em exossomos circulantes. O papel dosexossomos na fisiopatologia da isquemia cerebral, assim como na predição de diagnóstico de comprometimento cognitivo precisa ser melhor investigado. / Stroke is a common disease and has a major impact on the public health, since it is considered the main cause of neurological disabilities, mainly motor and cognitive impairment. Considering that exosomes’ content may be prognostic markers of dementia in patients with Alzheimer's disease we evaluated the circulating exosomal profile in patients with ischemic stroke with and without cognitive impairment. Furthermore, it is known that thrombolytic therapy reduces long-term disability, so it is interesting to evaluate the peripheral exosomes of these patients. This work aimed to investigate the profile of circulating exosomes in patients after diagnosis of ischemic stroke in the acute and chronic phases. Total protein content and acetylcholinesterase activity (AChE), markers of exosomes were determined, also it was correlated the scores of cognitive function tests with biochemical parameters. Considering that oxidative stress plays a central role in the pathophysiology of cerebral ischemia, we determined the content of reactive species and the activity of the superoxide dismutase enzyme (SOD) in circulating exosomes. The factors cognitive impairment and the use of thrombolytic in the acute phase of the disease did not show influence on the studied parameters. The results suggest that a decrease in circulating exosomes, since there was a reduction in the quantification of total proteins in the chronic phase, may suggest a damage in the system of removal of toxic materials leading to the accumulation of unwanted materials. The exosomal oxidative state, represented by the reactive species levels and the SOD activity, was altered in the chronic phase when compared to the acute phase These results suggest that the profile of the exosomes is altered over time in patients with ischemic stroke. Our data indicate that reactive species levels in the acute phase may predict changes in the exosome profile in the chronic phase of patients with cognitive impairment, specifically in the activities of SOD and AChE enzymes and in the concentration of total proteins. These associations were not observed in patients without cognitive impairment. We correlated the biochemical parameters with the the scores of the assessment of cognitive function tests, the Mini-Mental State Examination (MMSE) scores were inversely proportional to the AChE levels in those patients who did not receive the thrombolytic treatment in the acute phase. Thus, our data demonstrate that cognitive impairment (lower MMSE scores) is associated with higher levels of AChE activity in circulating exosomes. The role of exosomes in the pathophysiology of cerebral ischemia, as well as in the prediction of diagnosis of cognitive impairment, needs to be better investigated.
47

Encapsulation of Explant-Derived Cardiac Stem Cells in Agarose Nanoporous Gel Cocoons to Enhance Cardiac Repair

Kanda, Pushpinder 27 March 2019 (has links)
Micro-encapsulation of heart explant-derived stem cells (EDCs) within protective nanoporous gel (NPG) cocoons improves cardiac function and long-term retention of transplanted cells after ischemic injury by limiting detachment induced cell death and vascular clearance of intramyocardial injected cells. Although cocooned EDCs boost cardiac function, the fundamental mechanism is unclear. Here, we investigate the effects of altering cocoon stiffness and size on human EDC mediated repair of damaged myocardium using an immunodeficient mouse model of ischemic cardiomyopathy. First, we found that increasing cocoon stiffness by altering NPG content boosted cell viability and migration; effectively forcing cocooned cells to adopt a migratory, invasive phenotype. Although cocooning improved retention of transplanted cells, increasing cocoon stiffness had no additional effects on long-term engraftment despite markedly improving cardiac function and fibrosis after myocardial infarction. Given increased cocoon stiffness boosted the production and microRNA cargo within EDC nanovesicles, the observed benefits in post-ischemic function are likely dependent more on paracrine production of transplanted cells rather than simply increasing the number of cells retained. The effect of cocoon diameter on EDC phenotype and cell mediated repair of ischemic myocardium was evaluated using microfluidic-based cocooning enabling deterministic encapsulation within defined cocoon size and intracapsular cell number while maintaining a fixed cocoon stiffness. Increased cocoon size enhanced post-ischemic cardiac function by reducing clearance of transplanted cells and increased paracrine stimulation of endogenous repair. The latter being attributable to microfluidic cocooning closely following the expected Poisson distribution with smaller cocoons having a greater proportion of single cells while larger cocoons contained greater proportions of multicellular aggregates which enhanced cell-cell interactions to increase the amount and breadth of cytokines/nanoparticles delivered to injured myocardium. In conclusion, altering the biophysical properties of NPG surrounding cocooned cells provides a straightforward means of boosting the regenerative potential of heart EDCs for repair of injured myocardium.
48

Embryonic Stem Cell-Derived Exosomes Increase the Antiproliferative Activity of Doxorubicin in Breast Cancer

Hirsch, Alexander M 01 January 2019 (has links)
The field of cancer research has grown immensely in recent decades and has led to a better understanding of the causes of the disease, as well as greatly improved treatment for various types of cancers, especially breast cancer. One of the most effective treatments involves the chemotherapeutic drug doxorubicin (DOX). DOX is an effective tool against all types of breast cancer, especially against triple negative breast cancer. However, DOX causes adverse side effects that include damage to the heart and skeletal muscle, particularly above specific cumulative doses. Recent evidence suggests that embryonic stem cell-derived (ES) exosomes, nanoscale extracellular vesicles that carry proteins, messenger RNA, and microRNAs, may be able to mitigate some of the cardio- and cytotoxic effects of DOX without reducing its efficacy. The present study examined the effects of combined treatment with DOX (1 μM) and ES exosomes (10 μg/mL) on three cancer cell lines, MCF7, MDA-MB-231, and MDA-MB-468. The DOX/ES exosomes treatment increased cell death and increased apoptosis specifically compared to control, as measured via dye exclusion assay and flow cytometry. The treatment also decreased cell growth compared to control, as measured via MTS cell proliferation assay. In addition, DOX/ES exosomes treatment also increased expression of pro-apoptotic Bax while decreasing the expression of anti-apoptotic Bcl-2, as measured via Western blot. Finally, the DOX/ES exosomes treatment decreased expression of miR-200c, a microRNA associated with preventing epithelial-mesenchymal transition, a process that is integral to metastasis. Although increased cell death and apoptosis and decreased cell proliferation implies that the DOX/exosomes treatment is effective against cancer, the decrease in miR-200c expression may suggest the opposite and will be investigated further in future studies. Even so, the results of this study suggest that exosomes may be an important component to reduce the harmful effects of cancer treatment in the future.
49

Transfert du CFTR par vecteurs de gènes dérivés des adénovirus ou par trogocytose de microparticules membranaires : mécanismes moléculaires et applications à la mucoviscidose

Gonzalez, Gaëlle 14 December 2011 (has links) (PDF)
La mucoviscidose est une maladie génétique due à des mutations du gène CFTR, conduisant à une altération de la fonction de canal à ions chlorure de la glycoprotéine transmembranaire CFTR associée à une atteinte pulmonaire sévère. Plusieurs études récentes ont amené à reconsidérer l'utilisation des vecteurs adénoviraux (Ad) de sérotype 5 (Ad5) dans la mucoviscidose, lesquels induisent non seulement des réactions immunes anti-adénovirales mais aussi des effets cytopathiques indésirables. (1) Dans une première partie de notre étude, nous avons étudié l'entrée et le transit intracellulaire de l'Ad5/F35, vecteur chimérique portant les fibres de l'Ad sérotype 35 sur une capside de sérotype 5. Nous avons montré que la protéine fibre est déterminante dans l'internalisation et le trafic intracellulaire de ce vecteur. Le vecteur Ad5/F35 exprimant la fusion GFP-CFTR s'est révélé (i) être dépourvu de cytotoxicité, (ii) transduire efficacement les cellules épithéliales pulmonaires par voie apicale, et (iii) restaurer l'activité de canal à chlorure dans les cellules CFTR(-). Il constitue donc un vecteur de transfert du gène CFTR potentiellement utilisable en thérapie génique de la mucoviscidose. (2) Dans une seconde partie, nous avons exploré une stratégie alternative de transfert de la protéine CFTR par trogocytose. Nous avons fait l'hypothèse que le canal CFTR pouvait être véhiculé par des microvésicules ou microparticules membranaires (MP) émanant de la membrane cellulaire et libérées dans le milieu de culture. En utilisant un système d'expression stable de la protéine CFTR étiquetée par la protéine fluorescente GFP (GFP-CFTR) dans des cellules donneuses, nous avons pu démontrer que les MP sont capables de prendre en charge et délivrer la protéine GFP-CFTR à des cellules réceptrices, mais ce transfert n'est assuré que par une population réduite de MP (≤ 8 %), et la durée de vie du GFP-CFTR n'est que transitoire (≤ 24h). En fait, la majorité des MP transfèrent des molécules d'ARN messager ou polysomal GFP-CFTR. La protéine GFP-CFTR néosynthétisée à partir de ces ARNm est exprimée plus tardivement (> 48h) mais de façon prolongée (≥ 10 jours). La fonctionnalité du canal CFTR ainsi néosynthétisé est en cours d'évaluation. Les MP constituent donc un nouveau type de vecteurs de transfert non génique du CFTR qui pourraient être employés en thérapie de la mucoviscidose.
50

Rôle des exosomes sécrétés par le muscle strié squelettique au cours de la myogenèse et en situation d'insulino-résistance

Forterre, Alexis 19 December 2012 (has links) (PDF)
Les exosomes sont des nanovésicules de 30 à 100nm sécrétées dans le milieuextracellulaire par une grande majorité de types cellulaires. Entourés d'une bicouchelipidique similaire aux radeaux lipidiques, ils contiennent des protéines, de l'ARNm et desmicroARNs. Récemment, il a été montré que les exosomes pourraient participer auxdialogues moléculaires inter-organes, au même titre que les protéines solubles (hormones etcytokines). Au cours de cette thèse, nous avons émis l'hypothèse que le muscle squelettiquepourrait utiliser les exosomes comme mode de communication intercellulaire, en plus desmyokines qu'il sécrète. Nous avons montré en couplant des techniques de microscopieélectronique, de génomique, de biologie cellulaire et moléculaire, et d'analysesprotéomiques, que le muscle était capable de sécréter des exosomes, dont la compositionvariait au cours de la myogenèse. De plus, nous avons montré que les exosomes sécrétéspar les cellules prolifératrices et différenciées avaient des rôles distincts au cours de ladifférenciation myogénique, via le transfert des microARNs notamment.En parallèle, nous nous sommes intéressés aux exosomes sécrétés par le musclesquelettique en situation d'insulino-résistance induite par du palmitate. En utilisant unedouble approche in vitro et in vivo, nous avons montrés que les exosomes sécrétés par lacellule musculaire insulino-résistante ont une morphologie et une composition luminalemodifiée. Enfin, ces exosomes sécrétés sembleraient transmettre un signal délétère àd'autres cellules musculaires différenciées, et aux autres tissus insulino-sensibles, comme lepancréas.

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