In addition to the well-characterized Induced Pluripotent Stem cells (iPSCs) that closely resemble Embryonic Stem cells (ESCs), a recent study has proven the existence of a stable state, resembling partially reprogrammed cells, termed F-class iPSCs. To study these distinct iPSC states, a reprogramming dataset has been generated, featuring the parallel analysis of multiple molecular platforms. MicroRNAs (miRNAs) are small RNA regulators of gene expression whose critical role in reprogramming is now being realized. In the present study, small RNA deep sequencing data from this novel reprogramming dataset was used to identify miRNAs that are likely to enhance reprogramming by detecting significantly up-regulated miRNAs in ESC-like iPSCs versus F-class iPSCs. These candidate miRNAs were cloned and overexpressed in reprogramming mouse embryonic fibroblasts and their effect on reprogramming efficiency was measured. miR-214 was discovered to increase iPSC generation efficiency, marking the first reprogramming-related role for this microRNA.

In addition to the well-characterized Induced Pluripotent Stem cells (iPSCs) that closely resemble Embryonic Stem cells (ESCs), a recent study has proven the existence of a stable state, resembling partially reprogrammed cells, termed F-class iPSCs. To study these distinct iPSC states, a reprogramming dataset has been generated, featuring the parallel analysis of multiple molecular platforms. MicroRNAs (miRNAs) are small RNA regulators of gene expression whose critical role in reprogramming is now being realized. In the present study, small RNA deep sequencing data from this novel reprogramming dataset was used to identify miRNAs that are likely to enhance reprogramming by detecting significantly up-regulated miRNAs in ESC-like iPSCs versus F-class iPSCs. These candidate miRNAs were cloned and overexpressed in reprogramming mouse embryonic fibroblasts and their effect on reprogramming efficiency was measured. miR-214 was discovered to increase iPSC generation efficiency, marking the first reprogramming-related role for this microRNA.

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.

Glioblastoma multiforme (GBM) is one of the most common and aggressive primary brain tumours, with a median patient survival time of 6-12 months in adults. It has been recently suggested that a typically small sub-population of brain tumour cells, in possession of certain defining properties of stem cells, is responsible for initiating and maintaining the tumour. More recent experiments have studied the interactions between this subpopulation of brain cancer cells and tumour microenvironmental factors such as hypoxia and high acidity. In this thesis a computational approach (based on Gillespie’s algorithm and cellular automata) is proposed to investigate the tumour heterogeneities that develop when exposed to various microenvironmental conditions of the cancerous tissue. The results suggest that microenvironmental conditions highly affect the characterization of cancer cells, including the self-renewal, differentiation and dedifferentiation properties of cancer cells.

The epigenetic contribution to phenotype is now well established. Studies over the past decade have shown that proteins that are able to establish and propagate epigenetic modifications are essential for mammalian development. Some of the genes involved in these processes have been identified, but the roles of many remain unknown. The mutagenesis screens for modifiers of position effect variegation in Drosophila suggest that there are over 200 genes that are able to modify epigenetic variegation. We emulated this screen in the mouse to identify mammalian modifiers of a variegating transgene. The screen aimed to identify novel genes involved in epigenetic reprogramming, and to generate mouse models to study the impact of disruption to the epigenome. Inbred male mice carrying a variegating GFP transgene expressed in erythrocytes were mutagenised with ENU. Offspring were screened by flow cytometry and in the initial rounds of mutagenesis, 11 dominant mutant lines were identified. These lines were called MommeDs (Modifiers of murine metastable epialleles, dominant). This thesis describes the mapping and phenotypic characterisation of three Momme lines: MommeD7, MommeD8 and MommeD9. The MommeD9 mutation enhances variegation and was mapped to a 3.4 Mb interval on Chromosome 7. A mutation in a 5? splice site was found in the Trim28 gene. Analysis of Trim28 mRNA and protein in heterozygotes showed that the mutant allele was null. Homozygotes die before mid-gestation. Heterozygotes are viable but display variable and complex phenotypes, including infertility, obesity, behavioural abnormalities and premature death. Obese MommeD9 mice have liver steatosis, impaired glucose tolerance and other indicators of metabolic syndrome. This phenotype has not previously been reported for mice haploinsufficient for Trim28. There is considerable variability of phenotypes among inbred MommeD9 heterozygotes, which suggests a role for epigenetics in phenotypic noise or “intangible variation”. MommeD8 is a semi-dominant enhancer of variegation. Some homozygotes are viable but some die around birth. Viable homozygotes weigh less than wildtype littermates and have increased CpG methylation at the GFP transgene enhancer element. The mutation was mapped to a 4 Mb interval on chromosome 4. Extensive candidate gene sequencing failed to find a mutation and so DNA from mutant and wildtype individuals were sequenced across the entire linked interval by 454 Sequencing technology. MommeD8 individuals carry two point mutations, one is intergenic and the other lies in an intron of the Ppie gene. Analysis of Ppie mRNA in heterozygotes and homozygotes shows that mutants have reduced transcript levels, suggesting that a deficiency in Ppie causes the increased silencing of GFP. The Ppie gene has not been reported to be involved in epigenetic reprogramming and little is known about its function. Mice heterozygous for MommeD7 have a marked increase in expression of GFP. Heterozygotes have a range of hematopoietic abnormalities including splenomegaly, anaemia and reticulocytosis. Homozygotes die at birth and appear pale. The increased GFP in the peripheral blood appears to be the consequence of an increase in reticulocytes. The mutation is linked to a 1.5 Mb interval on Chromosome 7. MommeD7 mice appear to have hematopoietic abnormalities that affect the expression of the erythroid-specific GFP reporter transgene. MommeD7 mice serve as a reminder that, as well as discovering bona fide modifiers of epigenetic reprogramming, the ENU screen can also identify hematopoietic mutants.

<p>The success of gene medicine ultimately depends on the efficient intracellular delivery and sustained expression of nucleic acid therapeutics, yet nonviral gene delivery performed with cationic polymer carriers has been chronically hindered by the slow release of nucleic acid payloads at their targets, as well as the transient nature of exogenous transgene expression. Polymer-nucleic acid nanocomplexes made with passive gene carriers using traditional bulk methods have proven inadequate for most translational applications. The objective of this work is to improve nonviral gene delivery through the selection, formulation, and application of improved nanoparticles. </p><p> After screening a number of number of cationic polymer delivery systems ranging from natural to synthetic, high molecular weight to low, binary and ternary, we identified a bioreducible linear poly(amido amine) able to give sustained, robust expression of both DNA and RNA through serial dosing. We next turned our attention to the process of nanocomplex assembly. Traditional assembly via bulk mixing is poorly controlled, and the poor quality of these nanocomplexes is a significant impediment to both the establishment of robust structure-function relationships and the advancement of nonviral gene delivery. So, we developed an emulsion-based microfluidic nanomanufacturing platform to better control the self-assembly process, and thus the physical properties of nanocomplexes. Confined mixing within picoliter droplets generates self-assembled nanocomplexes that are more uniform and more effective. This microfluidic nanomanufacturing approach possesses broad utility in the production of polymer-nucleic acid nanocomplexes; we demonstrated that its benefits extend to multiple gene carriers, a range of nucleic acid payloads, and translationally relevant cell types. Then, we applied the improved nanomanufactured particles to begin to address an unmet clinical need, namely the lack of a safe and ethical source of cells to treat neurodegenerative diseases. Nonviral cellular reprogramming strategies eliminate the integration of viral DNA sequences and represent a potentially safer alternative to viral transdifferentiation methods to generate therapeutic cells. Using nanomanufactured polymer-nucleic acid nanocomplexes, we improved the efficiency of the nonviral cellular reprogramming of fibroblasts directly to functional induced neuronal cells. </p><p> Nonviral gene therapy will continue to demand more sophisticated delivery systems to continue to progress. Microfluidic nanomanufacturing represents a reproducible and scalable platform to synthesize more uniform and effective nanocomplexes that not only improves their functional performance, but may also help establish clearer structure-function relationships that will inform future gene carrier design. Complementing the innovative chemical and biological approaches to create multifunctional nanoparticles, this study indicates that microfluidic nanomanufacturing can serve as a parallel physical strategy to both optimize the properties of polymer-nucleic acid nanocomplexes and improve their performance in applications with important clinical implications.</p> / Dissertation

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2016-08-25T20:54:16Z No. of bitstreams: 1 MalekChouchane_TESE.pdf: 3043835 bytes, checksum: b90ef34a2d4072ef5abda48d216aebb4 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2016-08-26T21:40:31Z (GMT) No. of bitstreams: 1 MalekChouchane_TESE.pdf: 3043835 bytes, checksum: b90ef34a2d4072ef5abda48d216aebb4 (MD5) / Made available in DSpace on 2016-08-26T21:40:31Z (GMT). No. of bitstreams: 1 MalekChouchane_TESE.pdf: 3043835 bytes, checksum: b90ef34a2d4072ef5abda48d216aebb4 (MD5) Previous issue date: 2016-02-29 / Recently, the field of cellular reprogramming has been revolutionized by works showing the potential to directly lineage-reprogram somatic cells into neurons upon overexpression of specific transcription factors. This technique offers a promising strategy to study the molecular mechanisms of neuronal specification, identify potential therapeutic targets for neurological diseases and eventually repair the central nervous system damaged by neurological conditions. Notably, studies with cortical astroglia revealed the high potential of these cells to reprogram into neurons using a single neuronal transcription factor. However, it remains unknown whether astroglia isolated from different regions of the central nervous system have the same neurogenic potential and generate induced neurons (iN) with similar phenotypes. Similarly, little is known about the fate that iNs could adopt after transplantation in the brain of host animals. In this study we compare the potential to reprogram astroglial cells isolated from the postnatal cerebral cortex and cerebellum into iNs both in vitro and in vivo using the proneural transcription factors Neurogenin-2 (Neurog2) and Achaete scute homolog-1 (Ascl1). Our results indicate cerebellar astroglia can be reprogrammed into induced neurons (iNs) with similar efficiencies to cerebral cortex astroglia. Notably however, while iNs in vitro adopt fates reminiscent of cortical or cerebellar neurons depending on the astroglial population used for reprogramming, in situ, after transplantation in the postnatal and adult mouse brain, iNs adopt fates compatible with the region of integration. Thus, our data suggest that the origin of the astroglial population used for lineage-reprogramming affects the fate of iNs in vitro, but this imprinting can be overridden by environmental cues after grafting.

Le virus de l'hépatite B (HBV) infecte chroniquement plus de 250 millions de personnes. Des traitements existent permettant de contrôler la production de particules infectieuses. Cependant, aucun des traitements actuels ne permet d'éradiquer complètement l'infection. Il est donc nécessaire de développer de nouvelles stratégies thérapeutiques, incluant des approches immunothérapeutiques pour permettre un meilleur contrôle immunologique des infections HBV. Dans une étude récente menée au sein du laboratoire, il a été montré que l'IL-1ß est la cytokine ayant le plus fort effet antiviral contre la réplication d'HBV dans les hépatocytes. Dans le foie, la cytokine IL-1ß est principalement produite par les macrophages résidents (les cellules de Kupffer) ou infiltrant (monocytes inflammatoires différenciés en macrophages). De nombreuses études récentes ont montrés qu'HBV était capable de bloquer partiellement l'induction des réponses immunitaires innées. Il est donc important de déterminer si HBV est capable d'empêcher la production d'IL-1ß par les différents types de macrophages. L'objectif de cette thèse était d'étudier l'effet du virus sur le phénotype des macrophages et les implications de ces modifications phénotypiques sur l'établissement de l'infection dans les hépatocytes. Des monocytes du sang ou des macrophages du foie ont été purifiés, respectivement, du sang périphérique ou de résections hépatiques, et ont été exposés au virus pendant leur différentiation et/ou leur activation pour les monocytes, ou seulement pendant leur activation pour les macrophages hépatiques déjà différenciés. Il a été démontré que le virus de l'hépatite B est capable d'inhiber la sécrétion d'IL-6 et d'IL-1ß par les macrophages pro-inflammatoires. De plus, HBV est capable d'inhiber la sécrétion d'IL-1ß par les macrophages hépatiques stimulés par différents ligands. Finalement, les surnageants de macrophages pro-inflammatoires sont capables de bloquer l'établissement de l'infection, ce qui n'est pas le cas quand les macrophages ont été exposés au virus. Il apparait donc qu'HBV est capable de modifier le phénotype des macrophages pour favoriser l'établissement et la persistance de l'infection. La compréhension des mécanismes de subversion du phénotype des macrophages par le virus de l'hépatite B serait un premier pas vers le développement de nouvelles stratégies thérapeutiques / Hepatitis B virus (HBV) chronically infects over 250 million people worldwide. Several treatments can be used to prevent the formation of de novo particles. However, they do not allow the total eradication of the infection. Therefore, it is necessary to develop new therapeutic strategies, including immune-therapeutic ones, which would be more likely to lead to an immunological control of HBV infections. We have recently shown that IL-1ß is the most effective antiviral cytokine against the replication of HBV in vitro. In the liver, IL-1ß is mainly produced by resident macrophages (also called Kupffer cells) or infiltrating cells (inflammatory monocytes differentiated into macrophages). Recent studies have shown that HBV is able to partially inhibit the induction of innate immune responses. Hence, it was necessary to determine if HBV was also able to block the production of IL-1ß by the different types of macrophages.The objective of this thesis was to study the effect of HBV on macrophage phenotypes and the impact of those modifications on the establishment of HBV infection in hepatocytes.Blood monocytes and liver macrophages were purified, respectively, from peripheral blood or hepatic resections, and were exposed to HBV during their differentiation and/or activation for monocytes, or only during activation for liver macrophages which are already in a differentiated state. HBV was able to partially inhibit the secretion of IL-6 and IL-1ß by pro-inflammatory macrophages. Moreover, HBV was able to inhibit IL-1ß secretion by liver macrophages stimulated by different ligands and, conditioned medium of pro-inflammatory macrophages could inhibit the establishment of infection in hepatocytes. This effect was reverted when macrophages were exposed to HBV, concomitantly with a lower production of IL-6 and IL-1ß.In summary, HBV is able to modify macrophage phenotypes to favour the establishment and persistence of HBV infection. The full understanding of the mechanistic basis of how HBV phenotypically modifies macrophages will be a first step towards the development of new therapeutic strategies

Dans ce travail, nous démontrons que l’expression ectopique de Pax4 dans les cellules glucagon+ adultes induit, indépendamment de l’âge, leur néogenèse et transformation en cellules «bêta-like», ce qui entraîne une hypertrophie des îlots et une néogenèse inattendue des îlots. Par l’utilisation de plusieurs approches de traçage, nous démontrons que la conversion des cellules alpha en cellules «bêta-like» médiée par l’expression de Pax4, induit également la mobilisation de précurseurs situés dans ou à proximité des canaux pancréatiques. Ces cellules ré-expriment le gène développemental Ngn3 et adoptent successivement une identité de cellules glucagon+ puis de cellules «bêta-like», suggérant le réveil des mécanismes embryonnaires. Il et à noter que ces processus sont capables de régénérer la totalité de la masse de cellules bêta après plusieurs séries d’induction chimique du diabète. Ces résultats offrent ainsi des perspectives prometteuses pour concevoir de nouvelles stratégies thérapeutiques et régénératrices dans le contexte du diabète du type I. Dans un deuxième chapitre, ce travail décrit nos résultats d'analyse par puce à ADN de pancréas transgénique d’animaux exprimant conditionnellement le gène Pax4 dans les cellules alpha adultes. Cette approche nous permis d'identifier de potentiels gènes cibles de Pax4, qui pourraient jouer un rôle important dans les processus de régénération de la masse de cellules bêta. L’analyse de la fonction de l’un de ces gènes, le facteur de croissance indépendante 1 (Gfi1) est décrite. / In this work we demonstrate that the inducible misexpression of Pax4 in glucagon+ cells age-independently provokes their conversion into beta-like cells and their glucagon shortage-mediated replacement, this process resulting in islet hypertrophy and in an unexpected islet neogenesis. Taking advantage of several lineage-tracing approaches, we show that, upon Pax4-mediated alpha-to-beta-like cell conversion, pancreatic duct-lining precursor cells are mobilized, re-express the developmental gene Ngn3, and successively adopt a glucagon+ and a beta-like cell identity through a mechanism involving the reawakening of the epithelial-to-mesenchymal transition (EMT). It is worth mentioning, that these processes can repeatedly regenerate the entire beta-cell mass, and thereby reverse several rounds of streptozotocin-mediated chemically-induced Type I diabetes. This approach thereby provides promising perspectives to design novel therapeutic regenerative strategies. Aiming to gain further insight into the molecular mechanisms underlying these regeneration and reprogramming processes, and thereby identify new putative targets of interest, a thorough micro array analysis was performed using pancreata from transgenic mice conditionally misexpressing Pax4 in adult alpha-cells. We thereby identified several promising candidate genes, whose gene expression was significantly altered in induces animals. Among these was Growth factor independent 1 (Gfi1): its expression pattern and putative function in the murine pancreas will be described in this work.

Pendant les derniers stades de la spermatogenèse, les cellules germinales mâles post-méiotiques subissent une réorganisation dramatique de l'architecture de leur chromatine, impliquant notamment le remplacement presque total des histones par les protamines, créant des noyaux fortement condensés que l'on trouve dans le sperme mature. Au cours de ce processus, un événement précoce clé est la vague d'hyperacetylation des histones, qui précède leur remplacement. Notre équipe a précédemment identifié le facteur d'expression testiculaire de la famille BET, Brdt (BRomoDomain Testis), qui se lie aux histones acétylées via ses deux bromodomaines, comme essentiel au cours de ce processus. Cependant, les mécanismes aboutissant à l'hyperacétylation des histones à l'échelle génomique sont encore inconnus, ce qui reste l'une des questions majeures dans le domaine. La protéine NUclear in Testis (NUT) est un facteur spécifique testiculaire dont la fonction physiologique dans les cellules germinales mâles était inconnue. Cette protéine se trouve exprimée de manière ectopique dans un cancer rare mais très agressif, le carcinome de la ligne médiane (NUT Midline Carcinoma), en fusion avec BRD4, produisant ainsi une protéine de fusion hautement oncogène. Dans les cellules cancéreuses NUT est capable de recruter et d'activer l'histone acétyltransférase p300, contribuant ainsi à l'activité oncogénique de la protéine de fusion BRD4-NUT. Mon projet de doctorat est d'explorer la fonction physiologique de NUT, en étudiant des souris knock-out pour NUT qui ont été générées par notre équipe en collaboration avec Mathieu Gérard (Saclay). L'absence de NUT provoque une stérilité mâle associée à un arrêt de la spermatogenèse lors de l'allongement et de la condensation des spermatides, au stade où normalement les histones sont remplacées. D'autres expériences suggèrent que NUT pourrait agir sur la régulation de marques épigénétiques, y compris l'hyperacétylation des histones. Les mécanismes par lesquels NUT interfère avec la vague d'acétylation et les facteurs en interaction, y compris Brdt, sont explorées. Au total, cette étude démontre la contribution essentielle du NUT à la régulation épigénétique et au remplacement des histones au cours de la maturation post-méiotique des cellules germinales mâles. / During the late stages of spermatogenesis, post-meiotic male germ cells undergo a dramatic reorganization of their chromatin architecture involving the almost genome wide replacement of histones by protamines, creating highly condensed nuclei that are found in the mature sperm. During this process a key early event is known to be the wave of histone hyperacetylation, which precedes their replacement. Our team previously reported that the testis specific BET factor BRDT (BRomoDomain Testis specific), which binds acetylated histones, is essential during this process. However, how this genome wide hyperacetylation occurs has remained one of the major questions in the field. NUclear protein in Testis (NUT) is a testis specific factor whose physiological function in male germ cells was unknown. It has been found ectopically expressed in NUT Midline Carcinoma, a rare but highly aggressive cancer, in fusion with BRD4, resulting in a highly oncogenic fusion protein. In cancer cells, NUT is able to recruit and activate the histone acetyltransferase p300, hence contributing to the oncogenic activity of the BRD4-NUT fusion protein. My Ph.D. project investigates the original function of NUT by using NUT knockout mice that were generated by our team in collaboration with Mathieu Gerard (Saclay). The absence of NUT causes male sterility associated with a spermatogenic arrest during spermatids elongation/condensation, at a stage when histone replacement normally takes place. Additional experiments suggest that NUT could act through the regulation of epigenetic marks, including histone hyperacetylation. The mechanisms by which NUT interferes with the hyperacetylation wave and interacting factors, including Brdt, are explored. Altogether this study demonstrates the essential contribution of NUT to the epigenetic regulation and histone replacement during the post-meiotic maturation of male germ cells.