La Granulomatose Septique Chronique (CGD) est une maladie génétique rare de l’immunodéficience innée affectant les cellules phagocytaires (neutrophiles, macrophages). Elle est causée par des mutations dans les sous-unités du complexe NADPH oxydase formé du cytochrome b558 membranaire (NOX2 associé à p22phox) et de facteurs cytosoliques (p47phox, p67phox et p40phox). La déficience de ce complexe enzymatique va conduire à l’absence de formation de formes réactives de l’oxygène (FRO) microbicides et donc à l’apparition d’infections graves et récurrentes très tôt dans l’enfance. La chimioprophylaxie à vie permet de protéger ces patients mais peut être responsable d’effets indésirables. La seule thérapie curative est la transplantation de moelle osseuse mais tous les patients ne peuvent en bénéficier, et la thérapie génique n’est pas encore envisageable. Il y a donc un manque réel de nouvelles thérapies pour cette maladie. Cependant pour développer de nouveaux traitements, il faut disposer de modèles physiopathologiques pertinents. Or, les modèles existants sont imparfaits ou manquants. Le but de notre travail est donc de produire des modèles cellulaires et animaux de la CGD pour développer dans un second temps, une nouvelle approche thérapeutique basée sur l’utilisation de protéoliposomes.Grâce à leurs propriétés de pluripotence et d’auto-renouvellement à l’infini, les cellules souches pluripotentes induites (iPS) sont un outil puissant pour la modélisation physiopathologique. Ainsi, à partir de fibroblastes de patients atteints de CGD reprogrammés en cellules iPS, nous avons mis au point un protocole efficace de différenciation hématopoïétique in vitro en neutrophiles et macrophages. Nous avons montré que ces cellules phagocytaires sont matures et reproduisent parfaitement le phénotype déficient en FRO des patients CGD. Nous avons donc obtenu des modèles cellulaires pertinent modélisant trois formes génétiques de CGD, la CGD liée à l’X et deux formes autosomiques récessives, CGDAR22 et CGDAR47.Nous avons ensuite réalisé la preuve du concept de l’efficacité de protéoliposomes thérapeutiques sur les macrophages modélisés de la forme CGDX, la forme génétique la plus fréquente (70 % des cas) due à l’absence du cytochrome b558 membranaire (NOX2/p22phox). Grâce à une collaboration avec la start-up Synthelis SAS, des liposomes contenant le cytochrome b558 au niveau de la membrane lipidique ont été produits dans un système d’expression acellulaire basé sur l’utilisation d’extraits d’Escherichia coli. Ces liposomes NOX2/p22phox sont capables de reconstituer une enzyme NADPH oxydase fonctionnelle in vitro et de délivrer le cytochrome b558 à la membrane plasmique des macrophages CGDX qui présentent alors une restauration de l’activité NADPH oxydase avec la production de FRO.Enfin, nous nous sommes proposés de générer des souris dites « humanisées » par transplantation de cellules souches hématopoïétiques CD34+ capables de prise de greffe et de reconstitution hématopoïétique dans des souris immunodéficientes. A partir de cellules iPS saines, nous avons réussi à produire des cellules hématopoïétiques CD34+ possédant un potentiel hématopoïétique in vitro. Cependant, malgré des résultats encourageants, aucune prise de greffe in vivo n’a pu être réellement confirmée à ce jour.Pour conclure, nous avons donc montré au cours de ce projet, la production de modèles cellulaires de trois formes génétiques de CGD à partir de cellules iPS. Puis le modèle de macrophages CGDX nous a permis de faire la preuve de l’efficacité d’une nouvelle thérapie in vitro, une « enzymothérapie substitutive liposomale », qui pourrait à terme, offrir une alternative thérapeutique pour le traitement des infections aigües pulmonaires des patients CGD réfractaires aux traitements antibiotiques et antifongiques conventionnels. / Chronic Granulomatous Disease (CGD) is a rare inherited pathology of the innate immune system that affects the phagocytic cells (neutrophils, macrophages). This disease is caused by mutations in the subunits of the NADPH oxidase complex composed of the membrane cytochrome b558 (NOX2 associated with p22phox) and the cytosolic components (p47phox, p67phox et p40phox). Dysfunction in this enzymatic complex leads to the absence of microbicidal reactive oxygen species (ROS) and therefore to the development of recurrent and life-threatening infections in early childhood. Life-long prophylaxis is used to protect these patients but it may be responsible for side effects. Bone marrow transplantation is the only curative treatment but it can not be proposed to all the patients. In addition, gene therapy is not possible up to now. So there is a real lack of new therapies for this disease. However, to develop new therapeutic approaches, relevant physiopathological models must be available. Actually, existing models are imperfect or missing. Thus, the goal of our work is to produce cellular and animal models of CGD to develop a new proteoliposome-based therapy.Induced pluripotent stem cells (iPSCs) are a powerful tool for physiopathologic modeling due to their pluripotency and self-renewal properties. Using CGD patient-specific iPSCs regrogrammed from fibroblasts, we developped an efficient protocol for in vitro hematopoietic differentiation into neutrophils and macrophages. We showed that the phagocytic cells produced are mature and reproduce the ROS-deficient phenotype found in CGD patients. Thus, we obtained relevant cellular models for three genetic forms of CGD: X-linked CGD and the two autosomal recessive forms AR22CGD and AR47CGD.Then, we demonstrated the proof-of-concept of the efficacy of therapeutic proteoliposomes on X-CGD iPS-derived macrophages. Indeed, X-CGD is the main form of the disease (70% of cases) and is caused by the absence of the membrane cytochrome b558 (NOX2/p22phox). Thanks to a collaboration with the start-up Synthelis SAS, liposomes integrating the cytochrome b558 into lipid bilayers were produced in an E. coli-based cell-free protein expression system. These NOX2/p22phox liposomes were able to reconstitute a functional NADPH oxidase enzyme in vitro and to deliver the cytochrome b558 at the plasma membrane of X-CGD macrophages, leading to restore the NADPH oxidase activity with a ROS production.Finally, we proposed to generate « humanized » mice models with a human immune system after transplantation of CD34+ hematopoietic stem cells able to engraft and reconstitute long-term hematopoiesis in immunodeficient mice. Using healthy iPSCs, we successfuly produced CD34+ hematopoietic cells with in vitro hematopoietic potential. However, no in vivo engraftment was really confirmed yet.In conclusion, during this project, we produced cellular models of three genetic forms of CGD using patient-specific iPSCs. Then, X-CGD macrophages were used to demonstrate in vitro the efficacy of a new therapy. This « liposomal replacement enzymotherapy » could, in the future, represents a curative alternative against life-threatening lung infections refractory to conventional antibiotic and antifungal therapy.

O estudo de mutações genéticas como causa das cardiomiopatias teve início com a descoberta de mutações em proteínas sarcoméricas que levavam à Cardiomiopatia Hipertrófica, desde então, alterações em diversos genes, de proteínas contráteis ou não, foram descobertas e listadas como a responsável pelo desenvolvimento de diferentes cardiomiopatias. Estudar o efeito destas mutações nos cardiomiócitos destes pacientes permanecia um desafio devido ao difícil acesso às células cardíacas. Em 2007, a técnica de reprogramação de células somáticas em células-tronco pluripotentes foi descoberta. Pelo fato das células-tronco pluripotentes serem capazes de ser diferenciadas em cardiomiócitos, surgiu-se a possibilidade de se estudar essas células de indivíduos portadores das mutações genéticas. Esta tese teve como objetivo a criação de um modelo celular para estudar a Cardiomiopatia Hipertrófica causada por mutações genéticas. Inicialmente foi estabelecido um protocolo de reprogramação celular para se estabelecer linhagens celulares das células-tronco induzidas de um paciente com mutação no gene MYH7. Tendo as células caracterizadas, elas foram diferenciadas em cardiomiócitos através de um protocolo adaptado de protocolos de diferenciação direta em cardiomiócitos. Os cardiomiócitos gerados apresentaram características moleculares e funcionais semelhantes à cardiomiócitos primários humanos e foi visualizado, através de microscopia eletrônica de transmissão, que os cardiomiócitos do paciente com alteração genética possuíam grande proporção de sarcômeros desorganizados em comparação a cardiomiócitos de indivíduos saudáveis. Em conclusão, o modelo celular desenvolvido sugere ser possível o estudo do efeito de mutações genéticas em Cardiomiopatia Hipertrófica. / The study of genetic mutations as the cause of cardiomyopathies initiates with the discovery of mutations in sarcomeric proteins genes that lead to Hypertrophic Cardiomyopathy. Since then, mutations in several genes, coding to sarcomeric proteins or not, were discovered and listed as the reason to the cardiomyopathies. To study the effect of these mutations was a challenge due the difficulty to accesses cardiac cells. In 2007, the technique of reprogramming somatic cells into pluripotent stem cells was discovered. The fact that the pluripotent stem cells are capable of differentiating into cardiomyocytes opened the opportunity to study these cells from individuals with genetic mutations. This thesis aimed to create a cellular model to study Hypertrophic Cardiomyopathy caused by genetic mutations. Initially we established a cell reprogramming protocol to establish induced stem cells lines from a patient with mutation in MYH7 gene. Having characterized the cells, they were differentiated into cardiomyocytes using an adapted protocol from direct differentiation protocols. Cardiomyocytes generated showed molecular and functional characteristics similar to human primary cardiomyocytes and were visualized by means of transmission electron microscopy. The patient\'s cardiomyocytes had a large proportion of disorganized sarcomeres compared to cardiomyocytes from healthy individuals. In conclusion, the cell model developed suggests that it is possible to study the effect of genetic mutation in Hypertrophic Cardiomyopathy using induced pluripotent stem cells derived cardiomyocytes.

Os vírus são eficazes na transferência de genes em células devido aos seus mecanismos especializados. No entanto, vírus como veículos de entrega de genes podem acarretar em problemas, particularmente quando proposto para reprogramar células somáticas em células-tronco pluripotentes induzidas (iPS) visando utilização terapêutica. No presente estudo, procurou-se desenvolver um sistema alternativo para entregar diretamente proteínas nucleares (Oct4, Sox2, KLF4, e c-Myc) fusionadas com o domínio de transdução de proteína TAT, para promover a reprogramação de fibroblastos embrionários de camundongos (MEF) ou células mesenquimais derivadas de tecido adiposo humano (hASC) em células iPS. Primeiramente o PTD TAT ou TAT- foi fundido a proteína verde fluorescente (GFP) como modelo para prova de princípio e padronização detalhada. Inesperadamente, TAT-GFP produzido e secretado pelas células NIH-3T3 produtora não foi capaz de ser detectado no meio de cultura por verificação quantitativa fluorimétrica, nem foi capaz de ser detectada em células-alvo, por citometria de fluxo, depois de co-cultura em transwells. Essa observação pode ser explicada por: (1) ineficiência desse tipo de célula em secretar proteínas e (2) falta de resistência à clivagem por endoproteases furinas. Para contornar esses fatores limitantes usou-se citometria de fluxo para avaliar as melhores condições para a transfecção por seis diferentes tipos de células (CHO, NIH-3T3, HT1080, HEK-293A, HEK-293t e COS-7) com TAT (modificada para ser resistente à furinas) fundido a GFP. Células 293t-TAT-GFP exibiram a maior eficiência de transfecção e também de secreção. O mesmo pôde ser observado para as seis linhagens celulares expressando fatores de transcrição nucleares TAT, determinados por ELISA. Em seguida, diferentes estratégias de entrega foram testadas. A primeira foi baseada na co-cultura de uma mistura de células produtoras com MEF ou hASC. No entanto, não foi possível observar a reprogramação devido à morte celular. A segunda foi baseada na concentração de meio condicionado de cultura de células por centrifugação usando colunas Amicon, trocando o meio a cada 24h, em quatro ciclos. No entanto, apesar da presença de algumas colônias após 20-30 dias, nenhuma colônia verdadeira iPS foi obtida. Na sequência, as células foram tratadas com cada proteína de forma independente, e as demais foram substituídas pelo retrovírus correspondente, trocando meio a cada 72h, em quatro ciclos. Essa estratégia, apesar de permitir verificar a função de cada proteína, também não resultou em reprogramação. Este achado pode ser explicado pela diferenciação celular induzida por BCS, que também é concentrado no processo. Assim, passou-se a adaptação de \"células produtoras\" em condições de cultura livre de soro, para enriquecer a produção dos fatores nucleares individuais, necessários para a reprogramação. A otimização sistematizada deste processo está sendo realizada em parceria com o IPT e deve resultar em quantidades de proteína de fusão suficientes para o teste final da hipótese proposta. Em conjunto, são apresentados os dados da geração de linhagens celulares expressando estavelmente os vários fatores de transcrição e estratégias para melhorar a eficiência necessária para a produção iPS. Esta nova estratégia garante uma produção eficiente de TAT fundida a fatores nucleares de reprogramação e sua eficácia para promover a reprogramação de células somáticas de maneira livre de vírus merece ser investigado futuramente / Viruses are effective at transferring genes into cells by its specialized mechanisms. However, viruses as gene delivery vehicles entail problems, particularly when proposed to reprogram somatic cells into induced pluripotent stem cells (iPS) for therapeutic uses. In the present study, we aimed to develop an alternative system for directly delivering nuclear proteins (Oct4, Sox2, Klf4, and c-Myc) fused with TAT protein transduction domain to promote reprogramming of mouse embryonic fibroblasts (MEF) or human adipose tissue derived mesenchymal cells (hASC) into iPS cells. First TAT- or TAT- PTD was fused to green fluorescent protein (GFP) as a proof of principle model and for detailed standardization. Unexpectedly, TAT-GFP produced and secreted by NIH-3T3 producer cells was not detected in the culture medium by quantitative fluorimetric verification, nor detected on target cells, by flow cytometry, after being co-cultured using transwells. This observation maybe explained by: (1) inefficiency of this cell type to be transfected and to secrete proteins and (2) lack of resistance to furin endoproteases cleavage on Golgi of TAT sequence. To circumvent these limiting factors we used flow cytometer to assess the best conditions for transfection in six different cell types (CHO, NIH-3T3, HT1080, HEK-293A, HEK-293t and COS-7) with TAT- (a modified PTD to be resistant to furin endoproteases) fused to GFP. 293t-TAT-GFP cells displayed the highest transfection efficiency and secretion levels. The same could be observed for the six cell lineages expressing TAT- nuclear transcription factors, determined by ELISA.Next, different delivery strategies were tested for TAT- nuclear transcription factor system. Co-culturing a mix of producer cells with MEF or hASC resulted in not reprogramming and this was associated with cell death. The second was based on the use of microconcentrated conditioned cell culture medium, changed every 24h, in four cycles. However, despite the presence of some emerging colonies after 20-30 days, no true iPS colonies were obtained. Then, cells were treated with each protein independently, and the others were replaced by the corresponding retrovirus, changing cell medium every 72h, in four cycles. We verified the reprogramming potential of each protein, but no true colonies were obtained.One possibility for this finding is that BCS is also concentrated by centrifugation and may induce cell differentiation. To circumvent these problems, we have started the adaptation of producer cells in a serum-free culture condition to enrich the production of the individual factors required for reprogramming. This optimization process is taking place in collaboration with the IPT and shall result in large amounts of the fusion protein to finally test the proposed hypothesis. Altogether, we presented the generation of several cell lines stably expressing the transcription factors and strategies to improve the efficiency required for iPS production. This novel strategy guarantees efficient production of TAT-fused reprogramming nuclear factors and its efficacy to promote somatic cells reprogramming in a virus-free manner deserves to be further investigated

A reprogramação nuclear de uma célula somática a um estado embrionário tem diversas aplicações, como pesquisas básicas na biologia do desenvolvimento, terapia celular, melhoramento genético em animais de produção e conservação de espécies. As principais técnicas utilizadas para a reprogramação nuclear são a transferência nuclear de células somáticas (TNCS) e a geração de células tronco pluripotente induzidas (iPS). Muitos trabalhos têm mostrado uma baixa eficiência no processo de reprogramação nuclear nas duas técnicas, além disso, modificações epigenéticas tem sido apontada como a principal barreira para uma reprogramação nuclear eficiente. Por esse motivo, medidas como a utilização de células menos diferenciadas e/ou alteração do perfil epigenético das células somáticas podem aumentar a eficiência destas técnicas. Por isso, o objetivo deste trabalho foi investigar a influência de marcas epigenéticas em células bovinas utilizadas na reprogramação nuclear mediada por TNCS ou superexpressão de genes relacionados a pluripotêcia (iPS). Para isso, utilizamos 3 abordagens. Primeiro, analisamos marcações epigenéticas relacionadas ao desenvolvimento embrionário e pluripotência (H3K9me2, H3K9me3, H3K9ac, 5mC e 5hmC) em diferentes tipos celulares, analisamos a expressão gênica de genes responsáveis por essas marcações em células de diferentes tecidos (ex. células tronco mesenquimais (MSC) e fibroblastos) e as utilizamos como doadoras de núcleo na TNCS. Na segunda e a terceira abordagem, utilizamos células com menores níveis de H3K9me2 para a geração de iPS e na TNCS, respectivamente. Além disso, por se mostrar eficiente na TNCS, analisamos o efeito da sincronização do ciclo celular por privação de soro fetal bovino (SFB) na geração de células iPS. Com o intuito de diminuir os níveis de H3K9me2, as células foram tratadas com UNC0638, um inibidor especifico das metiltransferases de histona G9a/GLP. Nossos resultados do primeiro experimento mostraram que as MSC podem ser utilizadas como doadoras de núcleo na TNCS, no entanto, mesmo com algumas diferenças na expressão gênica em relação aos fibroblastos, a produção de blastocistos não foi diferente entre as duas células. No segundo experimento, as células privadas de SFB geraram mais colônias que as células controle, enquanto que as células tratadas não apresentaram diferença. Por último, as células tratadas com o UNC0638 apresentaram um menor nível de metilação no DNA em zigotos em relação às células controle. Os resultados encontrados neste trabalho podem contribuir para o melhor entendimento dos mecanismos epigenéticos envolvidos na reprogramação nuclear de bovinos / Nuclear reprogramming of somatic cells to embryonic state has several aplications, such as basic research on developmental biology, cell therapy, genetic improvement in livestock animals and preservation of endangered species. The principal techniques utilized to achieve nuclear reprogramming are Somatic Cell Nuclear Transfer (SCNT) and induced pluripotency. Several works has reported low efficiency rates of nuclear reprogramming when these techniques are used to reprogram somatic cells. Moreover, epigenetic modifications acquired during development act as epigenetic barrier to the complete reprogramming process. For this reason, strategies such as use of less differentiated cells and/or modification of epigenetic profile of somatic cells might increase the efficiency these techniques. The objective of this work was investigate the influence of epigenetic marks in bovine cells utilized on nuclear reprogramming experiments mediated by SCNT or induced pluripotency. To investigate it, we used three approaches. First, we analyzed the epigenetic marks related to the embryonic development and pluripotency (e.g H3K9me2, H3K9me3, H3K9ac, 5mC and 5hmC), gene expression of genes involved in these epigenetic marks in different tissues (i.e. mesenchymal stem cells (MSC) and fibroblasts) and their use as nuclear donor cells on SCNT procedure. Regarding the second and the third approach, we utilized cells with reduced levels of H3K9me2 to generate iPS cells and cloned embryos, respectively. Furthermore, since serum starvation has been demonstrated increase SCNT developmental rates, we assessed the effect of cell cycle synchronization mediated by serum starvation on nuclear reprogramming using iPS cells. Aiming decrease the levels of H3K9me2, cells were treated with UNC0638, a chemical probe that works as a specific inhibitor of the histone methyltransferases G9a and its counterpartner GLP. Our results showed that MSC are suitable to be used as nuclear donors on SCNT procedures, however, in spite of differences on gene expression comparing with fibroblasts, the embryonic developmental rates were not improved. On the second experiment, cells privated of fetal calf serum produced more iPS cells colonies than control cells, whereas cells treated with UNC did not show differences when compared with untreated cells. Lastly, UNC treated donor cells treated produced cloned zygotes with lower levels of DNA methylation compared to zygotes derivated from untreated cells. The results presented here will contribute to the better understanding of the epigenetic mechanisms involved on bovine nuclear reprogramming

La sclérose latérale amyotrophique (SLA) est une maladie neurodégénérative incurable de l’adulte qui affecte principalement les motoneurones. Les mécanismes conduisant à la mort des motoneurones restent mal connus, notamment du fait de l'hétérogénéité de la maladie et du manque d'accès aux neurones humains affectés. La technologie des cellules souches pluripotentes induites humaines (iPSc) est un outil prometteur pour la modélisation de la SLA, car elle offre la possibilité unique d'obtenir et d’étudier des motoneurones humains.Des clones d’iPSc de deux sujets témoins ont été générés et nous avons comparé plusieurs protocoles afin de mettre au point un protocole efficace de différenciation des iPSc en motoneurones. Les cultures obtenues étaient hétérogènes et contenaient différents types de neurones et des précurseurs neuraux. Afin de pouvoir étudier des mécanismes intrinsèques aux motoneurones dans la SLA, nous avons développé une nouvelle technique pour purifier les motoneurones. Cette technique a consisté à trier les motoneurones par FACS en combinant l'utilisation d'un vecteur lentiviral rapporteur exprimant une protéine fluorescente sous le contrôle d'un promoteur spécifique des motoneurones, et d'un anticorps monoclonal dirigé contre le récepteur aux neurotrophines p75. Cette double sélection a permis l'isolement efficace de motoneurones purs. En parallèle, la technologie iPSc a été utilisée pour établir des modèles cellulaires de la SLA. Des clones de cellules iPS ont été générés à partir d’un patient avec une forme familiale de la SLA présentant une mutation dans le gène TARDBP (codant pour une protéine de liaison à l’ADN, TDP-43) et un patient atteint d’une forme sporadique de SLA. Afin de valider nos modèles, nous avons recherché des phénotypes caractéristiques de la maladie au cours de la différenciation des iPSc : i) la formation d’agrégats cytoplasmiques, ii) des altérations de génération et de survie des motoneurones, iii) des défauts de croissance neuritique. / Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset neurodegenerative disorder primarily affecting motor neurons. Mechanisms leading to motor neuron death in ALS are poorly understood mostly because of disease heterogeneity and lack of access to affected cells. The induced pluripotent stem cell (iPSc) technology provides the opportunity to obtain and study human motor neurons and is therefore a promising tool for ALS modeling.IPSc clones from control subjects were generated, and we compared several protocols in order to set up an efficient protocol for iPSc differentiation into motor neurons. The obtained cultures were heterogenous, comprising different neuron subtypes and neural precursors. To allow investigation of intrinsic disease mechanisms in ALS motor neurons, we developed a new technique to purify motor neurons by FACS sorting. By combining the use of a lentiviral vector expressing a fluorescent protein under control of a motoneuron-specific promoter and of a monoclonal antibody directed against the p75 neurotrophin receptor, isolation of exquisitely pure motor neurons was achieved. In parallel, iPSc technology was used to establish cellular models of ALS. IPSc were generated from one patient with familial ALS carrying a mutation in the TARDBP gene (encoding a DNA-binding protein, TDP-43) and one patient with sporadic ALS. To validate our models, we investigated characteristic disease phenotypes during iPSc differentiation, including i) cytoplasmic aggregate formation, ii) motor neuron generation and survival defects, iii) neurite growth alterations.

Protein misfolding and aggregation underlie several neurodegenerative proteinopathies including amyotrophic lateral sclerosis (ALS). Superoxide dismutase 1 (SOD1) was the first gene found to be associated with familial ALS. Overexpression of human mutant or wild type SOD1 in transgenic mouse models induces motor neuron (MN) degeneration and an ALS-like phenotype. SOD1 mutations, leading to the destabilization of the SOD1 protein is associated with ALS pathogenesis. However, how misfolded SOD1 toxicity specifically affects human MNs is not clear. The aim of this thesis was to develop patient-derived, cellular models of ALS to help understand the pathogenic mechanisms underlying SOD1. To understand which cellular pathways impact on the level of misfolded SOD1 in human cells, we established a model using patient-derived fibroblasts and quantified misfolded SOD1 in relation to disturbances in several ALS-related cellular pathways. Misfolded SOD1 levels did not change following reduction in autophagy, inhibition of the mitochondrial respiratory chain, or induction of endoplasmic reticulum (ER)-stress. However, inhibition of the ubiquitin-proteasome system (UPS) lead to a dramatic increase in misfolded SOD1 levels. Hence, an age-related decline in proteasome activity might underlie the late-life onset that is typically seen in SOD1 ALS. To address whether or not SOD1 misfolding is enhanced in human MNs, we used mixed MN/astrocyte cultures (MNCs) generated in vitro from patient-specific induced pluripotent stem cells (iPSCs). Levels of soluble misfolded SOD1 were increased in MNCs as well as in pure iPSC-derived astrocytes compared to other cell types, including sensory neuron cultures. Interestingly, this was the case for both mutant and wild type human SOD1, although the increase was enhanced in SOD1 FALS MNCs. Misfolded SOD1 was also found to exist in the same form as in mouse SOD1 overexpression models and was identified as a substrate for 20S proteasome degradation. Hence, the vulnerability of motor areas to ALS could be explained by increased SOD1 misfolding, specifically in MNs and astrocytes. To investigate factors that might promote SOD1 misfolding, we focussed on the stability of SOD1 mediated by a crucial, stabilizing C57-C146 disulphide bond and its redox status. Formation of disulphide bond is dependent on oxidation by O2 and catalysed by CCS. To investigate whether low O2 tension affects the stability of SOD1 in vitro we cultured fibroblasts and iPSC-derived MNCs under different oxygen tensions. Low oxygen tension promoted disulphide-reduction, SOD1 misfolding and aggregation. This response was much greater in MNCs compared to fibroblasts, suggesting that MNs may be especially sensitive to low oxygen tension and areas with low oxygen supply could serve as foci for ALS initiation. SOD1 truncation mutations often lack C146, and cannot adopt a native fold and are rapidly degraded. We characterized soluble misfolded and aggregated SOD1 in patient-derived cells carrying a novel SOD1 D96Mfs*8 mutation as well as in cells fom an unaffected mutation carrier. The truncated protein has a C-terminal fusion of seven non-native amino acids and was found to be extremely prone to aggregation in vitro. Since not all mutation carriers develop ALS, our results suggested this novel mutation is associated with reduced penetrance. In summary, patient derived cells are useful models to study factors affecting SOD1 misfolded and aggregation. We show for the first time that misfolding of a disordered and disease associated protein is enhanced in disease-related cell types. Showing that misfolded SOD1 exists in human cells in the same form as in transgenic mouse models strengthens the translatability of results obtained in the two species. Our results demonstrate disulphide-reduction and misfolding/aggregation of SOD1 and suggest that 20S proteasome could be an important therapeutic target for early stages of disease. This model provides a great opportunity to study pathogenic mechanisms of both familial and sporadic ALS in patient-derived models of ALS. / Varje år insjuknar omkring 5300 personer i världen i motorneuronsjukdomen Amyotrofisk lateralskleros (ALS). Sjukdomen kännetecknas av degeneration av motorneuron i hjärnan och ryggmärgen, de nervceller som styr kroppens muskler, vilket leder till musklerförtvining och gradvis förlamning. ALS-patienter avlider oftast till följd av andningssvikt när sjukdomen når andningsmuskulaturen. I de allra flesta fall uppkommer ALS sporadiskt (SALS), det vill säga utan känd genetisk orsak, medan ärftliga fall (FALS) drabbar omkring 10 % och beror på mutationer i ett antal kända gener. Upp till 6 % av alla ALS fall kan härledas till mutationer i genen superoxid dismutas 1 (SOD1). SOD1 är ett enzym som ansvarar för att omvandla och oskadliggöra fria syreradikaler som bildas vid normal ämnesomsättning. 206 olika SOD1 mutationer har identifierats, alla orsakar inte ALS men många leder till att den tredimensionella proteinstrukturen förändras, vilket ökar proteinets benägenhet att felveckas. Initialt trodde man att SOD1 mutationer förhindrade proteinets normalfunktion och följaktligen orsakade ALS. Studier har emellertid visat att den enzymatiska funktionen ofta bevaras, även hos muterade proteiner. Däremot kan små mängder felveckat SOD1 störa andra viktiga cellulära funktioner. Felveckat SOD1 har en benägenhet att klumpa ihop sig och bilda aggregat i det centrala nervsystemet (CNS). Dessa aggregat återfinns hos patienter med såväl FALS som SALS vilket tyder på att även vildtyps-SOD1 kan felveckas och vara involverat i sjukdomsutvecklingen. De flesta studier är baserade på transgena musmodeller som uttrycker extremt stora mängder av muterat humant SOD1. Det är dock oklart hur väl studier i möss överensstämmer med sjukdomsutvecklingen hos ALS-patienter, där mängden SOD1 är betydligt lägre. En central fråga som fortfarande står obesvarad är varför just motorneuron degenererar i ALS, trots att SOD1 uttrycks i alla kroppens celler. Det övergripande syftet med den här avhandlingen har varit att karakterisera felveckat SOD1 i patientceller för att studera dess roll i ALSrelaterade sjukdomsmekanismer med fysiologiskt relevanta nivåer av SOD1. Samtliga studier är gjorda in vitro med celler från friska donatorer med vildtyps-SOD1, celler från patienter med SOD1-FALS, FALS som bär andra ALS-associerade gener, samt SALS. I de allra flesta fallen har vi analyserat både lösligt felveckat SOD1 samt aggregerade former av SOD1 proteinet.

Les dystrophies rétiniennes héréditaires (DRH) sont des maladies qui conduisent à une perte de la vision au cours de leur évolution. Les premiers essais cliniques utilisant la thérapie génique pour traiter ces maladies ont été réalisés et apportent des résultats encourageants. En amont de telles études, les essais précliniques s'effectuent le plus souvent sur modèle animal. Cependant, pour un certain nombre de DRH, il n'existe pas de modèle animal approprié ce qui compromet l'arrivée d'un traitement à un stade clinique. C'est le cas de la Choroïdérémie, qui représente 2% des DRH. La choroïdérémie est caractérisée par une perte de la vision nocturne dès la petite enfance et conduit à la cécité autour des 40-50 ans. Son diagnostic précoce et son évolution lente résultent en une grande fenêtre thérapeutique qui fait de la choroïdérémie une bonne candidate pour la thérapie génique. Sur le plan génétique, la maladie est causée par une mutation dans le gène CHM qui est localisé sur le chromosome X et code pour la Rab Escort Protein 1 (REP1). Cette protéine est impliquée dans le processus de prénylation de petites protéines GTPases, les protéines Rab. Afin de pallier au manque de modèle animal, nous avons généré au cours de ce travail de thèse, un modèle cellulaire humain de la choroïdérémie pour évaluer l'efficacité d'un protocole de thérapie génique sur le tissu réellement atteint in vivo. Pour cela, nous avons reprogrammé des fibroblastes de patient CHM-/y en cellules souches pluripotentes induites (iPS), que nous avons ensuite différenciées en Epithélium Pigmentaire Rétinien (EPR). Nous avons caractérisé cet EPR, montrant que c'est une couche monocellulaire polarisée possédant une morphologie et une expression de marqueurs caractéristiques. De plus, ce tissu est fonctionnel, sur le plan du transport de fluide et de la phagocytose, et possède le même phénotype biochimique que celui observé chez les patients. Dans un but de thérapie génique et afin d'évaluer le vecteur viral le plus efficace sur nos cellules, j'ai testé un panel de 5 sérotypes d'AAV et démontré que l'AAV2/5 est le plus efficient pour transduire un EPR dérivé de cellules iPS humaines. J'ai ensuite utilisé un AAV2/5-CAG-CHM afin d'évaluer l'efficacité fonctionnelle du vecteur et j'ai pu montrer qu'outre une expression correcte du transgène, le traitement de cellules de patients déficientes pour REP1 avec ce vecteur permet de restaurer une activité normale de prénylation. Nous avons donc démontré la supériorité d'efficacité de transduction de l'AAV2/5 dans des cellules d'EPR humain et soulignons le potentiel d'un modèle d'EPR pathologique dérivé de cellules iPS pour apporter une preuve de concept de thérapie génique en absence d'un modèle animal approprié. / Inherited retinal dystrophies (IRDs) lead to a progressive vision loss. The first clinical trials using gene transfer to treat such diseases have been performed with positive results. Prior to clinical trials, preclinical studies are usually performed on animal models. However, for many IRDs, appropriate animal models do not exist, which compromises their progress towards a clinical trial. An example of an IRD that lacks an appropriate model is choroideremia, which represents 2% of IRD patients. It is characterized by night blindness in childhood, followed by progressive loss of the visual field resulting in blindness by 40–50 years of age. Its early diagnosis and slow evolution result in a large therapeutic window making choroideremia a good candidate for gene therapy. Genetically, the disease is caused by a mutation in the CHM gene located on the X chromosome and encoding the Rab Escort Protein 1 (REP1). This protein is involved in the prenylation of small GTPases, the Rab proteins. To palliate the lack of an animal model, we generated a human cellular model of choroideremia in order to evaluate the efficacy of a gene therapy approach in the tissue that is affected in vivo.Towards this aim, we reprogrammed REP1-deficient fibroblasts from a CHM-/y patient into induced pluripotent stem cells (iPScs), which we differentiated into retinal pigment epithelium (RPE). We characterized the iPSc-derived RPE that is a polarized monolayer with a classic morphology, expresses characteristic markers, is functional for fluid transport and phagocytosis, and mimics the biochemical phenotype of patients. In terms of gene therapy and to evaluate the most efficient viral vector, I assayed a panel of 5 adeno-associated virus (AAV) vector serotypes and showed that AAV2/5 is the most efficient at transduce the iPSc-derived RPE. I then transduced the iPSc-derived RPE of a choroideremia patient with an AAV2/5-CAG-CHM and demonstrated that this vector is able to restore a normal prenylation function to the cells.To conclude, I demonstrated the superiority of the transduction efficiency of AAV2/5 in the iPSc-derived RPE and highlight the potential of a diseased RPE model derived from iPS cells to provide a proof of concept of gene therapy in the absence of a suitable animal model.

The aim of this dissertation is an analysis of the regulatory framework for the procurement and distribution of stem cells in South Africa. This research includes aspects of the law of obligations, medical law and human rights law as found in the Bill of Rights. More specifically however, this dissertation attempts to bring to attention the shortcomings of chapter 8 of the National Health Act. An examination is undertaken according to the multilayered approach and therefore the proposed regulatory framework is examined within a constitutional framework, an ethical framework, the framework as established by common law, in this case the doctrine of informed consent and lastly within the national legislation framework as found in the National Health Act of 2003 and the regulations made in terms of the Act. This dissertation further entails a brief comparative study of the regulatory mechanisms of the United Kingdom as entrenched in the Human Fertilisation and Embryology Act of 2008 and the Human Tissue Act of 2004 and as practiced by the Human Fertilisation and Embryology Authority and the Human Tissue Authority. The analysis in this dissertation firstly provides an overview of the clinical manifestations and science of stem cell technology. Secondly, the impact of the Constitution of the Republic of South Africa is discussed with particular reference to the Bill of Rights on stem cell research and therapy. The most noteworthy conclusion to be made in this regard is that the embryo is not the bearer of constitutional rights. The ethical guidelines which act as regulatory tools in this field are then discussed with attention to general ethical principles as provided for by the Health Professions Council of South Africa as well as the Medical research Council. The doctrine of informed consent further enjoys attention as it is discussed in context of medical research and key issues are addressed regarding the process of obtaining consent in context of stem cell technologies. Certain recommendations are then made pertaining to the minimum scope required for lawful consent. Lastly a critical analysis is made of chapter 8 of the National Health Act. The findings which are made here lead to further recommendations regarding the regulation of stem cells. / Dissertation (LLM)--University of Pretoria, 2011. / Public Law / unrestricted

Les réactions immunitaires de la peau sont initiées par les cellules dendritiques cutanées (dendritic cells, DCs). L'effet potentiellement sensibilisateur d'un composé peut être prédit in vitro en utilisant des monocytes humains différenciés en DCs (MonoDCs). Cependant, ces modèles simplistes restent imprécis car l'activation des DCs cutanés par les sensibilisateurs peut être déclenchée ou modulée par des interactions microenvironnementales avec de multiples types de cellules non immunitaires. Notre objectif est de développer une peau immunocompétente qui combinera des MonoDCs avec tous les éléments structurels et fonctionnels de la peau, c'est-à-dire une barrière épidermique posée sur un derme contenant une pseudo-vascularisation et des neurones nociceptifs. Une matrice de collagène a été ensemencée avec des fibroblastes et des cellules endothéliales, puis avec des précurseurs de fibres nerveuses dérivées soit de l'iPSC humaine, soit de la DRG embryonnaire murins. Enfin, nous avons introduit les MonoDC et les kératinocytes. Nous avons observé que les neurones différenciés in situ innervent l'épiderme comme observé habituellement dans la peau humaine normale. De plus, les neurones dérivées d’iPSCs, expriment neuropeptides et canaux calcique spécifiques des fibres nociceptives. Enfin, les Mono-DC intégrés au modèle restent stable pendant toute la durée nécessaire à la formation de l’épiderme et peuvent être stimulé. Le modèle sera utilisé pour prédire le potentiel irritant des composés chimiques et l'impact de l’innervation nociceptive sur l'activation des DCs. / Immune reactions in the skin are initiated by the cutaneous dendritic cells (DCs). The potential sensitizing effect of a compound can be predicted in vitro using human monocytes differentiated into DCs (Mono-DCs). However, these simplistic models remain inaccurate because the activation of cutaneous DCs by sensitizers may be triggered or modulated by microenvironmental interactions with multiple types of non-immune cells. Our goal is to develop an immunocompetent human tissue-engineered skin that will combine DCs with all structural and functional element of the skin, i.e. an epidermal barrier laid upon a dermis containing a pseudo-vascularization and nociceptive neurons. Collagen matrix was seeded with fibroblasts and endothelial cells, then with precursors of nerve fibers derived from either human iPSC or murine embryonic DRG. Finally, we introduced Mono-DCs and keratinocytes. We observed that in situ differentiated neurons grow axons towards the epidermis as usually observed in normal human skin. What's more, the neurons derive from iPSC, express neuropeptides and calcium channel as normal nociceptive fibers. Moreover, Mono-DCs settled as expected beneath the epidermis and remained sessile to stimulation for several weeks. The model will be used to predict the irritant potential of chemical compounds, and the impact of nerves on DC activation.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第22379号 / 医科博第109号 / 新制||医科||7(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 遊佐 宏介, 教授 武田 俊一, 教授 近藤 玄 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM