In 2006, Kazutoshi Takahashi and Shinya Yamanaka demonstrated the ability of four transcription factors; Oct4, Sox2, Klf4 and c-Myc to 'reprogram' differentiated somatic cells to a pluripotent state. This technology holds huge potential in the field of regenerative medicine, but reprogramming also a model system by which to the common regulators of all forced cell identity changes, for example, transdifferentiation. Despite this, the mechanism underlying reprogramming remains poorly understood and the efficiency of induced pluripotent stem cell (iPSC) generation, inefficient. One powerful method for elucidating the gene components influencing a biological process, such as reprogramming, is screening for a phenotype of interest using genome-wide mutant libraries. Historically, large-scale knockout screens have been challenging to perform in diploid mammalian genomes, while other screening technologies such as RNAi can be disadvantaged by variable knockdown of target transcripts and off-target effects. Components of clustered regularly interspaced short palindromic repeats and associated Cas proteins (CRISPR-Cas) prokaryote adaptive immunity systems have recently been adapted to edit genomic sequences at high efficiency in mammalian systems. Furthermore, the application of CRISPR-Cas components to perform proofof- principle genome-wide KO screens has been successfully demonstrated. I have utilised the CRISPR-Cas9 system to perform genome-wide loss-of-function screening in the context of murine iPSC reprogramming, identifying 18 novel inhibitors of reprogramming, in addition to four known inhibitors, Trp53, Cdkn1a, Jun, Dot1l and Gtf2i. Understanding how these novel reprogramming roadblocks function to inhibit the reprogramming process will provide insight into the molecular mechanisms underpinning forced cell identity changes.

Bright/ARID3A was initially discovered for its role in immunoglobulin heavy chain transcription in the mouse. Bright has also been implicated as a target of p53 and as an E2F binding partner. We have previously shown that Bright is necessary for hematopoietic stem cell development in the embryo. In this work, we show that Bright has a much broader role in development than previously appreciated. Loss of Bright in mice usually results in embryonic lethality due to lack of hematopoietic stem cells. Rare survivor mice initially appear smaller in size than either wildtype or heterozygous littermates, but as they age, these differences diminish. We show that adult Bright null mice have age-dependent kidney defects. Previous work in the adult mouse has not indicated a role for Bright in kidney function. We observed an increase in cellular proliferation in Bright null kidneys, indicating a possible mechanism behind our observation. Loss of Bright has recently been implicated in causing developmental plasticity in somatic cells. Our data indicate that loss of Bright is sufficient to fully reprogram mouse embryonic fibroblasts (MEFs) back to a pluripotent state. We term these cells Bright repression induced pluripotent stem cells (BriPS). BriPS derived from Bright knockout MEFs can be stably maintained in standard embryonic stem cell culture conditions: they express pluripotency markers and can form teratomas in vivo. We further viii show that Bright is active during embryonic stem cell differentiation. Bright represses key pluripotency genes, suggesting the mechanism of reprogramming may be Bright’s direct repression of key pluripotency factors in somatic cells. Recent advances in inducing pluripotency in somatic cells (iPS cells) have created a new field of disease modeling, increased our knowledge of how pluripotency is regulated, and introduced the hope that they can be adapted to treat disease. However, current methods for producing iPS involve overexpression of potentially oncogenic transcription factors, leaving a large gap between the lab and the clinic. Our results mark the first demonstration of an alternative method to reprograming somatic cells that is not mediated by overexpression of pluripotency factors. / text

The regulated differentiation and polarisation of macrophages are essential for successful wound healing process. During wound repair, macrophages are involved in the early inflammatory process of healing, as well in later regenerative phases by producing cytokines and growth factors relevant for each stage. Their plasticity made macrophages able to change their phenotype from M1 inflammatory during the inflammatory phase of healing to M2 reparative during regenerative phases of healing. Diabetes affects the ability of macrophages to mature from the bone marrow and on their ability to polarise to different phenotypic subsets. Whereas the non-diabetic macrophages can mature normally to M2 macrophages during mid-stages of healing, diabetic wound continues o display immature proinflammatory macrophages resulting in mixed M1/M2 macrophages in the wound that remain until late stages of healing. We previously showed that sustained expression of Hoxa3 reduced the-the excessive number of leukocytes recruited to the wound, suggesting an anti-inflammatory effect of Hoxa3 upon all leukocytes population. Hoxa3 protein transduction also promoted the differentiation of HSC/P into pro-angiogenic Gr1+CD11b+ myeloid cells. Here we showed that Hoxa3 promoted the differentiation of macrophages and upregulated the transcriptional machinery controlling macrophage differentiation, in THP-1 monocytes and primary macrophages from non-diabetic and diabetic mice. Using qRT-PCR and protein analysis of bone marrow derived macrophages from diabetic mice, we showed that Hoxa3 upregulated the master regulator of macrophages differentiation, Pu.1 transcriptionally and post- transcriptionally and that Hoxa3 protein interacted with Pu.1 protein in vitro and in vivo within macrophages proposing a mechanism of their regulation. Hoxa3 also inhibited proinflammatory markers in classically activated macrophages and augmented pro-healing markers in alternatively activated macrophages. Investigating the IL-4/Stat6 pathway of M2 macrophage activation revealed that Hoxa3 upregulated Stat6 and increased Stat6 phosphorylation, a novel effect of Hoxa3 on the signaling pathway of alternative macrophage activation. In vivo analysis of Hoxa3's effect on wound derived macrophages in diabetic mice, confirmed that Hoxa3 promoted the generation of pro-healing macrophages and showed reduced Nos2+ (M1) cells and increased Arg1+ (M2) cells suggesting that Hoxa3 can rescue the phenotype of diabetic macrophages in the wound. Altogether, this work has delineated the specific role of Hoxa3 in rescuing maturation and phenotype of diabetic macrophages thereby providing a better understanding of the therapeutic role of this transcription factor for myeloid cells dysregulation in diabetes.

Les lymphomes T périphériques (ou PTCL) sont des lymphomes malins non Hodgkiniens ayant pour cellules d’origine des lymphocytes T (LT) ou Natural Killer matures. Ces lymphomes sont rares, hétérogènes et méconnus. Des arguments issus de la littérature suggérant l’implication de la stimulation chronique du récepteur T à l’antigène (TCR) dans la transformation des LT, nous ont conduits à développer un modèle murin basé sur la stimulation chronique du TCR pour adresser spécifiquement cette question. Dans ce modèle, le transfert de LT p53-/- dans des souris CD3e-/- entraine l’apparition de lymphomes T périphériques (PTCL) clonaux dans 60% des cas avec une médiane de survenue de 230 jours alors que les souris transférées avec des LT wt ne développent pas de lymphomes. Ces PTCL présentent un phénotype T effecteur-mémoire CD62LLo-CD44hi-CD122lo-CD25lo ainsi qu’une profonde downrégulation de l’expression des gènes impliqués dans la voie du TCR illustrant l’impact de la stimulation chronique dans la lymphomagénèse. L’étude de ces lymphomes a révélé qu’ils ne dépendent plus, pour la plupart, de l’engagement du TCR pour leur survie et qu’ils acquièrent des caractéristiques « innate-like » avec notamment l’expression de récepteurs NK inhibiteurs (NKiR) et de récepteurs NK activateurs (NKaR) ainsi que des protéines adaptatrices DAP12 et FceRIg. Cette expression est associée à celle de Syk et PLC?2, impliquées dans la signalisation des NKaR. Nous montrons que les NKaR et leurs voies de signalisation associées sont fonctionnelles et participent à la survie des cellules lymphomateuses, le blocage de certains NKaR retardant notamment le développement lymphomateux in vivo. Nous avons par la suite exploré l’expression des NKR, de Syk et de PLCg2 au sein des PTCL humains et nous montrons ou confirmons que certaines entités expriment des panels variés de NKR ainsi que les effecteurs Syk et PLCg2 suggérant l’existence de mécanismes de lymphomagénèse similaires à ceux identifiés dans notre modèle au sein d’un certain nombres de PTCL humains / Peripheral T-cell lymphomas (PTCL) are rare non Hodgkin malignant lymphomas emerging from mature T or NK cells. PTCL are highly heterogeneous and mainly misunderstood. As several evidences pointed the potential role of TCR chronic stimulation in human T-cell lymphomagenesis, we developed a murine model based on chronic TCR stimulation to address this question. In this model, transfer of p53-/- T-cells into T-cell deficient mice (CD3e-/-) triggered PTCL development in 60% of cases with a median survival of 230 days while transfer of wt T-cells in CD3e-/- mice did not lead to PTCL development. These PTCL exhibited an effector-memory phenotype CD62LLo-CD44hi-CD122lo-CD25lo associated with a dramatic downregulation of TCR pathway genes expression consistent with a chronic TCR stimulation highlighting it’s implication in lymphomagenesis. The analysis of these PTCL revealed that a large majority of cases (80%) do not depend anymore on TCR stimulation for their growth and survival and that they acquire innate-like features with expression of inhibitory NKR (NKiR) and activating NK receptors (NKaR) as well as the adaptor proteins DAP12 or FceRIg. Expression of these receptors is associated with the expression of SYK and PLC?2, which are classical key effectors downstream of NKaR. We show that these NKaR are functional and can mediate TCR-independent activation in mPTCL and that this signaling is involved in cell survival/proliferation as in vivo blockade of NKG2D and NKp46 delays PTCL development in PTCL transplantation experiments. In parallel, we studied NKR, Syk and PLCg2 expression in human PTCL and found that some entities express a large range of these receptors as well as Syk and PLCg2, suggesting similar lymphomagenesis mechanisms in some human PTCL

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.

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.

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.

The repetitive sequence composes nearly half of human and mouse genome, most of which are scattered repeats of transposable elements (TEs). The non-LTR retrotransposons are the most accumulated TEs in the mammalian genome and L1s are the most active and abundant autonomous retrotransposons. L1s are highly activated during the epigenetic reprogramming of early mammalian embryos and have the highest level of expression among all retrotransposons throughout the preimplantation state. Moreover, the reprogramming of somatic cells into iPSCs is associated with an increase in L1 expression. The transcription of L1 during the early embryogenesis is necessary to regulate developmental genes and prevent heterochromatin formation to maintain cellular pluripotency state, that guarantying an appropriate future differentiation. However, the role of L1 reactivation during the somatic cell reprogramming remains unclear. Therefore, aim of this work is to study the impact of L1 transcription during the reprogramming process of the iPSCs. We used CRISPR-mediated gene activation (CRISPRa) system that fuse a deactivated Cas9 (dCas9) with transactivation domains (VPR). We confirm the ability to overexpress L1 in Human Embryonic Kidney cells (HEK293) and Human Dermal Fibroblasts (HDFs) by utilizing CRISPR activation system and this will provide a good opportunity to study the role of L1 transcripts during the reprogramming of HDFs into iPSCs. Furthermore, we established stable HDFs that able to express combinations of “Yamanaka” reprogramming factors. The model system will allow to investigate the effect of overexpressing L1 with reprogramming factors to answer the question of whether L1 can trigger or facilitate the reprogramming processes and its underlying mechanism.

Myeloid precursor cell reprogramming into a myeloid-derived suppressor cell (MDSC) contributes to high mortality rates in mouse and human sepsis. S100A9 mRNA and intracellular protein levels increase during early sepsis and remain elevated in Gr1+CD11b+ MDSCs after pro-inflammatory sepsis transitions to the later chronic anti-inflammatory and immunosuppressive phenotype. The purpose of this study was to determine whether intracellular S100A9 protein might sustain Gr1+CD11b+ MDSC repressor cell reprogramming during sepsis. We used a chronic model of sepsis in mice to show that S100A9 release from MDSCs and circulating phagocytes decreases after early sepsis and that targeting the S100a9 gene improves survival. Surprisingly, we find that intracellular S100A9 protein translocates from the cytosol to nucleus in Gr1+CD11b+ MDSCs during late sepsis and promotes expression of miR-21 and miR-181b immune repressor mediators. We further provide support of this immunosuppression pathway in human sepsis. This study may inform a new therapeutic target for improving sepsis outcome.

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