As células de pluripotência induzida (iPS) foram reportadas pela primeira vez em 2006 por Takahashi e Yamanaka e desde então vem sendo extensivamente estudadas. Por meio da técnica, células somáticas adultas adquirem comportamento muito semelhante às células tronco embrionárias, reduzindo as questões éticas relacionadas ao uso destas em pesquisas. Entretanto, os mecanismos biológicos das células iPS ainda não estão completamente elucidados. Ainda são necessárias pesquisas mais aprofundadas para garantir a segurança e eficácia de sua possível utilização em futuras terapias. Os coelhos, como modelos experimentais, são vantajosos tanto pelo seu tamanho ideal para procedimentos cirúrgicos quanto por sua manutenção fácil e econômica. As células tronco derivadas do tecido adiposo (ADSC) consistem em um tipo de célula tronco mesenquimal multipotente que se destaca pela facilidade, rapidez e segurança de coleta e processamento. As ADSC foram coletadas e caracterizadas por meio de análises de curva de crescimento celular, doubling time, viabilidade após criopreservação, capacidade de formação de colônias fibroblastoides, diferenciação osteogênica, condrogênica e adipogênica, imunocitoquímica e citometria de fluxo. Elas demonstraram possuir as características básicas de células tronco mesenquimais, destacando-se por uma alta e rápida capacidade proliferativa. A indução de pluripotência foi realizada nas ADSC de coelhos pela introdução de quatro fatores de transcrição (OCT4, SOX2, cMYC, e KLF4), pelo vetor lentiviral STEMCCA (Milipore). Cinco protocolos de indução foram testados. Células resultantes da indução foram caracterizadas quanto à atividade da fosfatase alcalina e perfil fenotípico por citometria de fluxo. A proliferação acentuada das ADSC parece ser um fator limitante para a eficácia da reprogramação. A partir destes dados, espera-se contribuir para o desenvolvimento de uma tecnologia brasileira ainda inédita em coelhos e adicionar informações importantes à literatura, acerca das propriedades das células iPS, visando sua utilização como modelo experimental para futuras terapias. / Induced Pluripotent Stem (iPS) cells were first reported in 2006 by Takahashi e Yamanaka and has been intensively studied since then. Through the technique, adult somatic cells acquire behavior very similar to embryonic stem cells, reducing the ethical issues related to the use of such research. However, biological mechanisms of iPS cells are not yet fully elucidated. Thus, further research is needed to ensure the safety and efficacy for their possible use in future therapies. Rabbits, as experimental models, are advantageous by their ideal size for surgical procedures and easy and economic maintenance. Adipose Derived Stem Cells (ADSC) consists of multipotent mesenchymal stem cells that stand for ease, speed and safety of collection and processing. The ADSC were collected and characterized by analysis of cell growth curve, doubling time, viability after cryopreservation, ability of fibroblast colony formation, osteogenic, adipogenic and chondrogenic differentiation, immunocytochemistry and flow cytometry. They showed to have the basic characteristics of mesenchymal stem cells, standing out by a high and fast proliferative capacity. Induction of pluripotency was performed in rabbits ADSC by the introduction of four transcription factors (OCT4, SOX2, cMYC, and KLF4) by lentiviral vector STEMCCA (Millipore). Five protocols were tested and analyzed. The resulting cells after induction were characterized by alkaline phosphatase activity and phenotypic profile by flow cytometry. The great proliferation of ADSC seems to be detrimental for the reprogramming efficiency. From these data, it is expected to contribute to the development of a Brazilian technology still unpublished in rabbits, and to add important information to the literature about the properties of iPS cells, aiming their use as an experimental model for future therapies.

The thesis describes several unknown aspects of the spruce bark beetle, Ips typographus (L.), and the double-spined bark beetle, Ips duplicatus (Sahlberg.), life-cycles and ecophysiology. The first study focuses on I. typographus and its dispersal under epidemic conditions in the National park Šumava and introduces a novel method of fluorescent marking and detection of captured specimens. The second study is focused on ability of I. typographus to establish so-called sister-broods, i.e. re-emergence of females that are capable to continue egg laying without a need to mate again. The importance of sister-broods becomes apparent mainly in recent hot and dry vegetation seasons, which is demonstrated by a comparison of recent and historical data. The third study focuses on temperature-dependent development of I. duplicatus under laboratory conditions by the means of sandwich method. Altogether, the studies underline practical importance of ecophysiological studies on bark beetles as one of the tools for their effective management.

Thesis (doctoral)--Swedish University of Agricultural Sciences, 2002. / Thesis statement in Swedish and English abstract inserted. Appendix reprints four papers and manuscripts, three co-authored with others. Includes bibliographical references. Also issued electronically via World Wide Web in PDF format; online version lacks appendix.

The ability to reprogram adult somatic cells into induced pluripotent stem (iPS) cells could provide a valuable implement for in vitro disease modeling and drug discovery. More importantly, they may potentially serve as an unlimited source of cells for regenerative medicine. However, most of the iPS cells have been generated by retroviral vectors, and therefore they carry the risk of viral integration into the host genome. This problem prevents their use for clinical applications and regenerative medicine. mRNA-mediated delivery of reprogramming factors is an alternative approach for cellular reprogramming. mRNA-based reprogramming offers the advantage of being completely free of genomic integration and is therefore highly suitable for clinical translation. However, there are some limitations which must be overcome so that mRNA can be widely used for successful cellular reprogramming. In the current thesis, the attempt was to generate stable mRNA-iPS cells through overcoming those limitations. Several human donor cells were transfected with mRNA encoding reprogramming factors and the generation of two stable mRNA-iPS cell lines was shown. The resultant mRNA-iPS colonies were assessed for pluripotency markers. Their pluripotency features were evaluated by the viral-iPS cells produced by conventional retroviral vectors. It was noticed that the generation of mRNA-iPS cells was largely affected by the parental cells from which they were derived. However, characterization and evaluation of the generated mRNA-iPS cells proved their pluripotency states comparable to the viral-iPS cells. On the other hand, the aging hallmarks of the iPS cells were assessed in the second part of this thesis. The potential aging signatures of the iPS cells should be conducted before their use in clinical applications. Currently, there are controversial data regarding the ability of reprogramming to fully rejuvenate an aged somatic cell and reverse agerelated changes such as shortened telomeres, dysfunctional mitochondria and DNA damage. Moreover, mixed findings have been published regarding whether the iPS cells are fully rejuvenated or they might retain some of the aging hallmarks from the cells which they were derived. This thesis studied these controversies through the investigation of three hallmarks of aging including telomere length, mitochondrial alteration and DNA damage. Telomere elongation was indicated in the iPS cells. Furthermore, mitochondrial morphology and function were improved into more immature features in iPS cell lines than their corresponding fibroblasts. Moreover, the iPS cell lines were shown to have less amount of DNA damage compared to their parental fibroblasts. In summary, it can be concluded that generation of mRNA-iPS cells is largely affected by the primary donor cells from which they are derived. Furthermore, it seems that reprogramming enables reversion of aging signatures to a more youthful state.

La maladie d’Alzheimer est une maladie très complexe, aujourd’hui encore mal comprise et cette démence est devenue un réel problème de santé publique. La protéine précurseur de l’amyloïde (APP) et la protéine Tau sont deux acteurs majeurs impliqués dans la maladie. De nombreuses recherches se sont investies dans la compréhension du métabolisme, de l’action et de l’implication de ces deux protéines dans les mécanismes pathologiques de la maladie et d’autres maladies neurodégénératives. Elles sont notamment l’objet de la plupart des approches thérapeutiques passées et actuelles, et étudiées pour le diagnostic biologique de la maladie. Dans ce projet de thèse, notre objectif fut d’explorer le métabolisme des protéines APP et Tau au cours de la différenciation neuronale à l’aide d’outils biochimiques et de systèmes innovants d’immunodétection multiplex très sensibles (MSD®) dans plusieurs modèles de culture cellulaire de la maladie. L’objectif était d’obtenir une vision globale des processus physiopathologiques au travers d’analyses d’échantillons générés au cours de la différenciation neuronale de cellules souches pluripotentes induites (iPSC) de patients Alzheimer comparées aux cellules souches embryonnaires humaines (hESC). Nous avons ainsi généré et caractérisé plusieurs lignées cellulaires d’IPSC d’une personne saine contrôle et de patients atteints des formes sporadiques et familiales de la maladie. Ce projet offre l’opportunité unique de combiner des approches innovantes pour tenter de comprendre comment les fragments et les peptides Ab sont générés, ainsi que les modifications de Tau en conditions normales et pathologiques. / Amyloid precursor protein (APP) and Tau protein are two main molecular actors of the Alzheimer’s disease (AD), which is of prime importance in Human Health. Intensive research is ongoing to understand these proteins’ metabolism, action and implication in the pathological mechanism of these affections. They are the target of most therapeutic approaches and are used for biological diagnosis. In the present PhD project, our objective was to investigate neuronal APP and Tau protein processing and metabolism using biochemical tools and innovative multiplex immunodetection system (MSD®) in diverse cell culture models of AD. The goal was to get a comprehensive view oh physiopathological processes based on the analysis of samples generated in neuronal differentiated human embryonic stem cell and induced pluripotent stem cells derived from AD-patients. We generated several cell lines from an healthy control individual, and AD patients showing sporadic and familial forms of the disease. This project offer the unique opportunity to combine state-to-the-art approaches to understand how the APP fragments and peptides are generated as well as the modifications of the Tau protein in normal and pathological situation.

As células de pluripotência induzida (iPS) foram reportadas pela primeira vez em 2006 por Takahashi e Yamanaka e desde então vem sendo extensivamente estudadas. Por meio da técnica, células somáticas adultas adquirem comportamento muito semelhante às células tronco embrionárias, reduzindo as questões éticas relacionadas ao uso destas em pesquisas. Entretanto, os mecanismos biológicos das células iPS ainda não estão completamente elucidados. Ainda são necessárias pesquisas mais aprofundadas para garantir a segurança e eficácia de sua possível utilização em futuras terapias. Os coelhos, como modelos experimentais, são vantajosos tanto pelo seu tamanho ideal para procedimentos cirúrgicos quanto por sua manutenção fácil e econômica. As células tronco derivadas do tecido adiposo (ADSC) consistem em um tipo de célula tronco mesenquimal multipotente que se destaca pela facilidade, rapidez e segurança de coleta e processamento. As ADSC foram coletadas e caracterizadas por meio de análises de curva de crescimento celular, doubling time, viabilidade após criopreservação, capacidade de formação de colônias fibroblastoides, diferenciação osteogênica, condrogênica e adipogênica, imunocitoquímica e citometria de fluxo. Elas demonstraram possuir as características básicas de células tronco mesenquimais, destacando-se por uma alta e rápida capacidade proliferativa. A indução de pluripotência foi realizada nas ADSC de coelhos pela introdução de quatro fatores de transcrição (OCT4, SOX2, cMYC, e KLF4), pelo vetor lentiviral STEMCCA (Milipore). Cinco protocolos de indução foram testados. Células resultantes da indução foram caracterizadas quanto à atividade da fosfatase alcalina e perfil fenotípico por citometria de fluxo. A proliferação acentuada das ADSC parece ser um fator limitante para a eficácia da reprogramação. A partir destes dados, espera-se contribuir para o desenvolvimento de uma tecnologia brasileira ainda inédita em coelhos e adicionar informações importantes à literatura, acerca das propriedades das células iPS, visando sua utilização como modelo experimental para futuras terapias. / Induced Pluripotent Stem (iPS) cells were first reported in 2006 by Takahashi e Yamanaka and has been intensively studied since then. Through the technique, adult somatic cells acquire behavior very similar to embryonic stem cells, reducing the ethical issues related to the use of such research. However, biological mechanisms of iPS cells are not yet fully elucidated. Thus, further research is needed to ensure the safety and efficacy for their possible use in future therapies. Rabbits, as experimental models, are advantageous by their ideal size for surgical procedures and easy and economic maintenance. Adipose Derived Stem Cells (ADSC) consists of multipotent mesenchymal stem cells that stand for ease, speed and safety of collection and processing. The ADSC were collected and characterized by analysis of cell growth curve, doubling time, viability after cryopreservation, ability of fibroblast colony formation, osteogenic, adipogenic and chondrogenic differentiation, immunocytochemistry and flow cytometry. They showed to have the basic characteristics of mesenchymal stem cells, standing out by a high and fast proliferative capacity. Induction of pluripotency was performed in rabbits ADSC by the introduction of four transcription factors (OCT4, SOX2, cMYC, and KLF4) by lentiviral vector STEMCCA (Millipore). Five protocols were tested and analyzed. The resulting cells after induction were characterized by alkaline phosphatase activity and phenotypic profile by flow cytometry. The great proliferation of ADSC seems to be detrimental for the reprogramming efficiency. From these data, it is expected to contribute to the development of a Brazilian technology still unpublished in rabbits, and to add important information to the literature about the properties of iPS cells, aiming their use as an experimental model for future therapies.

A inserção de fatores de transcrição conhecidos em células somáticas é capaz de reprograma-las levando a um estágio de pluripotência, gerando células-tronco pluripotentes induzidas (iPS). A utilização destas células na medicina regenerativa tem grande potencial tanto na medicina humana quanto na veterinária, e a influência da origem da célula somática utilizada na geração de iPS é discutida atualmente. Mamíferos domésticos, como por exemplo o equino, são bons modelos para o estudo para medicina humana e veterinária, com destaque para terapia celular utilizando células mesenquimais em lesões ortopédicas e articulares. Tendo como hipótese que células equinas com diferentes origens apresentam potenciais de indução à pluripotência e capacidade de diferenciação in vitro variáveis; esta proposta tem como objetivo gerar células iPS equinas obtidas através da transdução viral dos fatores de transcrição OSKM humanos ou murinos em fibroblastos (eFibro) adultos e células mesenquimais derivadas de tecido adiposo (eAdMSC), medula óssea (eMO) e tecido de cordão umbilical (etCU). Foram isoladas e cultivadas 4 linhagens de células do tecido de cordão umbilical, 3 linhagens de fibroblastos equinos, 3 linhagens de células mesenquimais de tecido adiposo equinas e 2 linhagens de células de medula óssea equina. Essas células tiveram seu tempo de duplicação celular calculado em horas (eMO 61,3±15; etCU 53,8±5; eFIBRO 27±2; eAdMSC 18,2±4,5) e foram caracterizadas por diferenciação condrogênica, osteogênica e audiogênica e por imunofenotipagem. A partir destas células foram produzidas 85 linhagens iPS equinas (eiPS- 30 linhagens derivadas de fibroblasto, 33 linhagens derivadas de células de tecido adiposo, 21 linhagens derivadas de células de tecido de cordão umbilical com os fatores humanos e 1 linhagem de derivada de células de tecido adiposo com os fatores murinos). Testes de detecção de fosfatase alcalina e OCT4 foram realizados para células eiPS obtidas de cada linhagem e as células eiPS derivadas de tecido adiposo e fibroblastos foram positivas para detecção de NANOG, TRA1-60, TRA1-81 e as células eiPS derivadas de eAdMSC foram positivas para detecção de SSEA-1. As células eiPS derivadas de cada tipo celular geraram corpos embrióides, que posteriormente foram dissociados para teste de diferenciação espontânea in vitro. Os resultados mostram que células equinas podem ser mais facilmente reprogramadas com fatores humanos quando comparadas com os fatores murinos (P<0.05). Enquanto os fatores murinos produziram apenas uma linhagem de células iPS derivada de eAdMSC, os fatores de reprogramação humanos foram capazes de produzir variadas linhagens de células iPS, sendo a formação de colônias mais eficiente para células derivadas de eAdMSC (322, P<0.01) do que para células derivadas de eFibro (65) e etCU (58), que não diferiram (P=0.95), e não foi possível produzir células eiPS a partir de eMO. Usando o sistema de indução à pluripotência padronizado em nosso laboratório, a utilização de fatores humanos na reprogramação direta gera uma maior eficiência na produção de células iPS equinas quando comparados com fatores murinos. No nosso conhecimento, este é o primeiro relato de células eiPS produzidas unicamente dependentes de bFGF, sem necessidade de adição de LIF no meio de cultivo. / The insertion of known transcription factors into somatic cells is capable of reprogramming them into a pluripotency stage, generating induced pluripotent stem cells (iPS). The influence of the origin of the somatic cell used in the generation of iPS is currently discussed, and the use of these cells in regenerative medicine has great potential in both human and veterinary medicine. Domestic mammals, such as the equine are great models for the study of human and veterinary medicine, highlighting cell therapy using mesenchymal cells in orthopedic and articular injuries. Hypothesizing that equine cells derived from different tissues show variable pluripotency induction potentials and in vitro differentiation capacity, depending on the source of derivation; this proposal aims to generate equine iPS cells obtained by viral transduction of human or murine OSKM transcription factors in adult fibroblasts (eFibro) and mesenchymal cells derived from adipose tissue (eAdMSC), bone marrow(eMO), umbilical cord tissue (etCU). Four umbilical cord tissue cell lines, three equine fibroblast cells lines, three equine adipose tissue mesenchymal cell lines and 2 equine bone marrow cell lines were isolated and cultured. These cells had their doubling time calculated in hours (eMO 61.3 ± 15, etCU 53.8 ± 5, eFIBRO 27 ± 2, and ADMSC 18.2 ± 4.5) and were characterized by chondrogenic, osteogenic and adipogenic and by immunophenotyping. From these cells, 85 equine iPS (eiPS) cell lines were produced (30 fibroblast-derived cell lines, 33 cell lines derived from adipose tissue cells, 21 cell lines derived from umbilical cord tissue cells, with human factors and 1 cell line derived from eAdMSC using murine factors). Alkaline phosphatase and OCT4 detection tests were performed on eiPS cells obtained from each cell line and eAdMSC and eFibro derived iPS cells were positive for the detection of NANOG,TRA1-60, TRA1-81 and eiPS derived from eAdMSC were positive for SSEA-1 detection. Embryonic bodies, which were later dissociated for spontaneous differentiation test in vitro were derived from each cell type. Results show that equine cells can be more easily reprogrammed with human factors when compared to murine factors (P<0.05). While murine factors produced only one eiPS cell line derived from eAdMSC, human reprogramming factors were able to produce multiple eiPS cell lines, and colony formation was more efficient for cells derived from eAdMSC (322 P <0.01) than for cells derived from eFibro (65) and etCU (58), which did not differ (P = 0.95) and It has not yet been possible to produce iPS cells from the eMO cell lines. Using the induction system standardized in our laboratory, the use of human factors in direct reprogramming results in greater efficiency in the production of equine iPS cells when compared to murine factors. To our knowledge, this is the first report of eiPS cells produced solely dependent on bFGF, without the need for addition of LIF in the culture medium.

A geração de células-tronco pluripotentes induzidas (iPS) a partir de células somáticas demonstrou que células adultas de mamíferos podem ser reprogramadas a um estágio de pluripotência através da inserção de fatores de transcrição embrionários. Esta descoberta tem levantado questões fundamentais sobre os mecanismos, que através destes fatores de transcrição, influenciam epigeneticamente as células e seus potenciais de diferenciação após a reprogramação e um normal desenvolvimento. Componentes lipídicos e lipoprotéicos afetam vários aspectos no comportamento celular durante sua manutenção e diferenciação, podendo afetar diretamente fatores essenciais em processos de reprogramação celular, manutenção da pluripotência e perfil epigenético das células. Nesse sentido, esta tese propôs o estudo da composição lipídica com diferentes abordagens entre células iPS, células-tronco embrionárias (H1) e células fibroblastos (BJ). Foram produzidas três linhagens de células pluripotentes induzidas no modelo humano que foram caracterizadas quanto 1a sua pluripotência e utilizadas, juntamente às linhagens H1 e BJ como modelos para o estudo da composição lipídica proposto. Foram identificadas e estudadas um total de 44 espécies lipídicas das classes PC, PE, PI, SM e PS, e discutidas frente a reprogramação celular e manutenção da pluripotência. Foi identificado um padrão de composição fosfolipídica distinta entre células pluripotentes e não pluripotentes, e especulamos que a presença dessas espécies parecem ter um envolvimento fundamental para a manutenção da pluripotência. Este padrão, mostrou pela análise de componente principal, que durante o processo de reprogramação, alterações na composição lipídica ocorrem de forma com que a pluripotência surge durante a reprogramação, evidenciando alterações lipídicas particulares do estádio da pluripotência, sugerindo uma ligação entre estas alterações na composição lipídica com as alterações metabólicas da própria reprogramação celular. O estudo da quantificação de fosfolipídios entre linhagens celulares pluripotentes e não pluripotentes evidenciaram que existe uma diferença fosfolipídica entre estas linhagens, observamos que as linhagens iPS e H1, do ponto de vista das classes observadas e os fosfolipídios quantificados, são similares entre si e diferentes de células não pluripotentes. É evidente que estas moléculas lipídicas, individualmente, não são capazes de modular processos como a reprogramação celular, entretanto, é de extrema importância o entendimento das mesmas dentro da reprogramação celular e manutenção da pluripotência. Nossos dados sugerem que a composição lipídica de células pluripotentes tem importante papel para o desenvolvimento e evolução do processo de reprogramação celular e o entendimento da manutenção da pluripotência / The generation of induced pluripotent stem cells (iPS) from adult somatic cells has shown that mammalian cells can be reprogrammed to a pluripotent state by the insertion of embryonic transcription factors. This finding has raised questions about the fundamental mechanisms through which these transcription factors epigenetically influence cells, their potential of differentiation after reprogramming and normal development. Lipid and lipoprotein components affect numerous aspects of cell behavior during its maintenance and differentiation, which can directly affect main factors in cell reprogramming processes, maintenance of pluripotency and epigenetic profile of the cells. Thus, this thesis proposed to study, with different approaches, the lipid composition of iPS cells, embryonic stem cells (H1) and fibroblast cells (BJ). Three induced pluripotent cell lines were produced in the human model. They were characterized regarding their pluripotency and used along with H1 and BJ cell lines, as models for the proposed lipid composition study. A total of 44 species of lipid from the classes PC, PE, PI, PS and SM have been identified, studied and discussed regarding cellular reprogramming and maintenance of pluripotency. A different phospholipid composition pattern was observed between pluripotent and non-pluripotent cells, and it is speculated that the presence of these species appears to have a major involvement on the maintenance of pluripotency. This array showed, by the principal component analysis, that during the reprogramming process changes in the lipid composition occur, so that pluripotency takes place during reprogramming, highlighting lipid changes particular of the pluripotency state, suggesting a connection between these changes in lipid composition and the metabolic changes of cell reprogramming. The study of the quantitation of phospholipids from pluripotent and non-pluripotent cell lines indicated a phospholipid difference between these cell lines when considering the observed classes and quantified phospholipid. It was eminent that iPS lines and H1 are similar and differ from non-pluripotent cells. It is clear that these lipid molecules are not individually capable of modulating processes such as cell reprogramming, however, it is extremely important to understand them within cellular reprogramming and maintenance of pluripotency. Our data suggests that the lipid composition of pluripotent cells has important role in the development and evolution of cellular reprogramming process and the understanding the maintenance of pluripotency

Les syndromes progéroïdes regroupent un ensemble de pathologies caractérisées par un vieillissement précoce et accéléré. Le syndrome le plus connu et étudié est la progéria de Hutchinson-Gilford dont l'incidence est de 1 cas sur 8 millions ce qui en fait une maladie très rare. Nous avons étudié trois symptômes progéroïdes dont le syndrome HGPS, un syndrome HGPS-like ainsi qu'un syndrome APS. Ces pathologies ont de nombreux symptômes en commun dont une ostéolyse, une lipodystrophie, ainsi qu'une atteinte cardiovasculaire. Ces trois syndromes sont provoqués par différentes mutations du gène LMNA qui code pour les Lamines A et C. Nous avons utilisé le modèle des iPSCs afin d'étudier in vitro la physiopathologie de ces trois syndromes en les comparant à des cellules contrôles. Les cellules dérivées de la voie mésenchymateuse étant majoritairement altérées dans ces pathologies, nous avons créé des modèles in vitro d'étude de la différentiation en MSCs. De plus, ces patients présentant des altérations arterio-veineuses, nous avons analysé la différenciation en VSMCs. Le phénotype des ces cellules a été analysé et les profils transcriptomiques comparés pour les différentes lignées. Des gènes communs, impliqués dans le stress oxydatif et dans des systèmes de réparation géniques ont été retrouvés comme étant altérés. De plus, nous avons mis en évidence des altérations de voies de signalisation indispensables à la survie et à la prolifération cellulaire en comparant les cellules progéroïdes aux contrôles. Certaines de ces voies biologiques ouvrent de nouvelles perspectives dans la compréhension des symptômes observés chez ces patients. / Progeroid syndromes are a group of pathologies characterized by accelerated and early aging. One of the most studied of these diseases is HGPS, with an estimated incidence of 1 in 8 millions birth making it an extremely rare disease. We focused our attention on three different progeroid syndromes including classic HGPS, a HGPS-like and an atypical progeroid syndrome. These pathologies share many symptoms, including osteolysis, lipodystrophy, and cardiovascular alterations. These 3 syndromes are caused by 3 different mutations in the LMNA gene that encodes A- and C-type lamins, inducing production of a truncated Lamin A in HGPS and HGPS-like and production of a mutated Lamin with a p.T528M substitution in APS. We produced hiPSCs to create a model of these different diseases and investigate in vitro the physiopathology of these syndromes by comparing them to control cells. Cells derived from mesenchymal stem cells being the most impaired type of tissue, we established in vitro models in order to study the differentiation of hiPSCs into MSCs. In addition given the massive cardiovascular defects in these patients, we also investigated differentiation toward the VSMCs. Cell phenotypes were carefully characterized and we compared the transcripttomic profile of the different cell types. We identified dysregulation in genes involved in oxidative stress response and in DNA repair in progeroid cells. In addition, pathways essential for cell survival and proliferation are also modified when comparing progeroid and controls cells. Altogether, these results might explain some of the symptoms observed in progeroid patients but also reveal pathways involved in ageing.

Avec l'évolution exponentielle de l'Internet des Objets (IoT), assurer la sécurité des réseaux est devenue un grand défi pour les administrateurs réseaux. La sécurité des réseaux est basée sur de multiples équipements indépendants tels que Firewall, IDS/IPS, NAC dont le rôle principal est de contrôler les informations échangées entre le réseau de l'entreprise et l'extérieur. Or, l'administration de ces équipements peut s'avérer très complexe et fastidieuse si elle est réalisée manuellement, équipement après équipement. L'introduction du concept de Software Defined Networking (SDN) depuis ces dernières années, et du protocole OpenFlow, offre beaucoup d'opportunités pour l'amélioration de la sécurité des réseaux en proposant une administration centralisée et programmable.Dans le cadre de cette thèse, nous avons proposé une nouvelle approche de sécurisation des échanges dans un réseau en fonction des événements détectés et de manière automatisée. Cette solution basée sur l'approche SDN couplé avec un système de détection d'intrusion permet d’analyser, de détecter et de supprimer des menaces de sécurité dans un réseau et de manière automatisée. En implémentant cette solution, nous contribuons à faire évoluer la manière de sécuriser les échanges dans un réseau avec du SDN couplé avec un IDS à travers la mise en place d'une architecture réelle de cas d'usage. Ainsi, la gestion de la sécurité du réseau devient simplifiée, dynamique et évolutive. / With the exponential evolution of the Internet of Things (IoT), ensure the network security has become a big challenge for networkadministrators. Traditionally, the network security is based on multiple independent devices such as firewall, IDS/IPS, NAC where the main role is to monitor the information exchanged between the inside and the outside perimeters of the enterprises networks. However, the administration of these network devices can be complex and tedious with an independent manual configuration. Recently, with the introduction of the Software Defined Networking concept (SDN) and the OpenFlow protocol offers many opportunities by providing a centralized and programmable network administration.As part of this research work, we proposed a new approach to secure the network traffic flows exchanges based on a method of events detection, in an automated manner. This solution is based on the SDN approach coupled to an intrusion detection system which allows analyze, detect and remove security threats. With the implementation, we contribute to change the paradigm of secure the network traffic flows exchanges using the SDN principle, coupled with an IDS in a real use case architecture. In this way, the management of network security becomes simplified, dynamic and scalable.