Dissertations / Theses on the topic 'Functional reprogramming'

Yin Yang 1 (YY1) is a ubiquitous zinc finger transcription factor (TF) that occupies active enhancers and promoters contributing to physical interactions between these regions via DNA looping. Increasing evidence shows that disruption of non-coding regions such as enhancers is prevalent across different neurodevelopmental disorders (NDDs) with intellectual disability (ID) features. Indeed, YY1 haploinsufficiency causes a NDD with ID, named Gabriele-de Vries syndrome (GADEVS). Although it is known that YY1 controls the expression of a dazzling list of genes and influences various cellular processes in numerous cell types, the impact of this TF in the neurodevelopment of the human cortex is yet to be unraveled. By taking advantage of disease-modeling as a tool to investigate the pathogenesis of GADEVS across different time points and tissues we gathered new insights about how YY1 haploinsufficiency exerts such a dramatic phenotype in individuals carrying mutations. We reprogrammed patient-derived and healthy somatic cells into induced-pluripotent stem cells (iPSCs) and observed, already at the pluripotent stage, a major transcriptional dysregulation. Moreover, since YY1-mutated patients exhibit ID features, we differentiated our cohort of iPSCs into cortical neurons as well organoids and were able to capture stage-specific striking features, not only at the transcriptomic level, but also structural and compartmentalization impairments. Of note, YY1-mutated neurons displayed synaptic disparities, sufficient to induce astrogliosis-like features in surrounding astrocytes, both shown to be critical for proper brain function and plasticity forms in the CNS. Instead, in cortical organoids we recapitulated features of abnormal ventricle formation, pathological hallmarks observed in GADEVS patients and mice models followed by ID and developmental delay. This study showed, for the first time, the molecular signatures that possibly lead to cognitive defects in human patients and provide the first solid foundation for the development of therapeutic strategies and drug screening in the future.

The metabolic state of an immune cell directly influences its ability to function and differentiate, ultimately affecting immunity, inflammation and tolerance. Different immune cell subsets have differing metabolic requirements. Macrophages, as the frontline, tissue-resident cells of the innate immune system, undergo profound metabolic reprogramming in response to environmental stimuli. To date, there has been little consideration how macrophage metabolism might be affected by humoral immunity. IgG antibodies are the soluble effector molecules of the adaptive humoral immune system. Fcγ receptors (FcγRs) mediate the cellular functions of IgG antibodies and are expressed on most immune cells including macrophages. FcγR cross-linking induced by IgG immune complexes (ICs) is important for defence against some infections but can also play a pathogenic role in autoimmunity. Here, I studied the metabolic reprogramming induced in macrophages by IgG IC ligation of FcγRs. I first investigated how FcγRs cross-linking might impact glucose metabolism. We show that macrophages undergo a switch to glycolysis in response to IgG IC stimulation. FcγR-associated glycolysis was dependent on the mammalian target of rapamycin (mTOR) and hypoxia-inducible factor (HIF)1α. Moreover, this glycolytic switch was required to generate a number of pro-inflammatory mediators and cytokines. Inhibition of glycolysis, or genetic depletion of HIF1α in macrophages resulted in the attenuation of IL1β and other inflammatory mediators produced in response to IgG IC in vitro. To determine the relevance of these observations to responses to IgG IC in vivo and, in particular, to IC-associated tissue inflammation in autoimmune diseases such as system lupus erythematosus (SLE), I developed three models to interrogate tissue macrophages. Following administration of IC to peritoneal macrophages, I observed IL1β-associated neutrophil recruitment that was abrogated by inhibiting glycolysis, or in the presence of HIF-1a deficiency. Similarly, following administration of intravenous IC, or nephrotoxic serum, kidney macrophage activation was abrogated by glycolysis inhibition or by myeloid HIF-1a deficiency. Together my data reveal the cellular molecular mechanisms required for FcγR-mediated metabolic reprogramming in macrophages and define a novel therapeutic strategy in autoantibody-induced inflammation. In the final part of the thesis I identified additional metabolic pathways that were altered by FcγR ligation, including cholesterol biosynthesis and fatty acid biosynthesis. This has important implications for protective immune responses and autoimmune susceptibility, since a number of intermediates in these pathways can directly regulate and contribute to immune responses. In summary, I have demonstrated the metabolic alterations triggered by FcγR ligation, reveal the cellular molecular mechanisms required for FcγR-mediated cellular respiration reprogramming in macrophages and define a potential therapeutic target in autoimmunity.

Le métabolisme des cellules cancéreuses est fortement perturbé et dérégulé dans les cancers. Plusieurs exemples illustrent ce phénomène, notamment la reprogrammation métabolique décrite dans l’effet Warburg, les dérégulations fonctionnelles de certaines voies métaboliques telles que l’augmentation de la production de ROS dans les cellules cancéreuses, ou la mise en évidence d’oncométabolites liés à des mutations acquises telles que les mutations IDH1/2 qui entraînent la production d’un métabolite directement associé au processus leucémique dans les LAM. Afin de caractériser les reprogrammations métaboliques associées au processus leucémique, nous avons analysé par une approche HRMAS les métabolites produits par différentes lignées cellulaires leucémiques représentant différents sous types de LAM (génotype et phénotype différents). Dans ce modèle, nous avons montré que chaque type de lignée présentait un métabolisme particulier à l’état basal, témoin d’une signature métabolique différente selon la nature de la lignée. En situation de stress métabolique (culture en milieu sans sérum), toutes ces lignées développent des mécanismes d’adaptation de leur métabolisme à la carence en nutriments. En particulier, il existe une signature commune caractérisée par la surexpression de métabolites de la voie des phospholipides et de régulation du stress oxydant au bout de 24h de culture en milieu sans sérum. Grâce à ces mécanismes d’adaptation les cellules leucémiques retrouvent au bout de 48h, une viabilité supérieure à 95% et un profil métabolique quasi-identique aux conditions normales. Ces résultats montrent que les cellules leucémiques développent des mécanismes communs de survie, impliquant notamment des dérégulations du métabolisme des lipides, qui leur permettent de continuer à proliférer en situation de stress métabolique. D’autres conditions expérimentales ont été testées, notamment en condition de carence en glucose afin d’explorer la piste de la dérégulation de certains acides aminés comme l’alanine dans ces lignées. De plus, l’étude quantitative et qualitative des acides gras dans les LAM via une approche lipidomique révèle une adaptation similaire des profils lipidomiques des lignées dans les mêmes conditions de privation en sérum précédemment testées. En parallèle, dans une étude sur 54 patients au diagnostic de LAM, nous avons confirmé par l’approche HRMAS qu’il existait chez les patients LAM des différences de profil métabolique en fonction du sous-type de LAM. Nous avons également montré que ces signatures métaboliques étaient significativement corrélées aux sous-groupes pronostiques cytogénétiques, à la réponse au traitement par chimiothérapie et à la survie des patients. Nous montrons notamment que les métabolites surexprimés chez les patients de mauvais pronostic sont retrouvés surexprimés également chez les patients réfractaires au traitement. L’analyse de ces métabolites montrent le rôle particulier de plusieurs voies métaboliques dans le pronostic des LAM : i) la dérégulation de la synthèse de 2-hydroxyglutarate associée aux mutations de l’enzyme IDH1/2, ii) la dérégulation du métabolisme des phospholipides, retrouvant une surexpression de phospholipides dans les plasmas de patients de pronostic défavorable, et iii) la surexpression de la synthèse de certains acides aminés chez les patients chimiorésistants, suggérant une implication de la voie de signalisation LKB1/AMPK
Cells metabolism is strongly disturbed and deregulated in cancers. Several examples reflect this phenomenon, including metabolic reprogramming described in the Warburg effect, functional deregulations of particular metabolic pathways such as the increase of the ROS production in cancer cells, or the identification of oncometabolites linked to acquired mutations such as IDH1/2 mutations, which lead to the production of a metabolite directly linked to the leukemic process in AML. In order to characterize the metabolic reprogramming associated with the leukemic process, we analyzed by an HRMAS approach the metabolites produced by different leukemic cell lines representing different subtypes of AML (different genotype and phenotype). In this model, we have shown that each type of cell line exhibited a particular metabolism in the basal state, witnessing a different metabolic signature depending on the nature of the cell line. In condition of metabolic stress (culture in a serum-free environment), all these cell lines develop mechanisms to adapt their metabolism to nutrient deficiency. Particularly, there is a common signature characterized by the overexpression of metabolites of the phospholipid pathway and of regulation of oxidative stress after 24 hours of culture in a medium without serum. Thanks to these adaptation mechanisms, the leukemic cells find after 48 hours a viability higher than 95% and a metabolic profile almost identical to normal conditions. These results show that leukemic cells develop common survival mechanisms, notably involving deregulations of lipid metabolism, which allow them to continue to proliferate in condition of metabolic stress. Other experimental conditions have been tested, in particular in glucose deficiency conditions in order to explore the path of deregulation of some amino acids such as alanine in these cell lines. Moreover, the quantitative and qualitative study of fatty acids in AMLs through a lipidomic approach reveals a similar adaptation of the lipidomic profiles of the cell lines in the same serum-free conditions previously tested. In parallel, in a study on 54 patients diagnosed with AML, we confirmed by the HRMAS approach that there were differences in metabolic profile in AML patients according to the AML subtype. We also showed that these metabolic signatures were significantly correlated with cytogenetic prognostic subgroups, response to chemotherapy treatment and patient survival. We show in particular that the metabolites overexpressed in patients with poor prognosis are found overexpressed also in patients refractory to treatment. The analysis of these metabolites shows the particular role of several metabolic pathways in the prognosis of AML: i) deregulation of the synthesis of 2-hydroxyglutarate associated with mutations in the IDH1/2 enzyme, ii) deregulation of the metabolism of phospholipids, showing an overexpression of phospholipids in adverse prognosis patients plasmas, and iii) overexpression of the synthesis of some amino acids in chemoresistant patients, suggesting an involvement of the LKB1/AMPK signaling pathway

Les macrophages associés aux tumeurs (TAMs) sont d'importants composants cellulaires du microenvironnement tumoral, qui présentent des fonctions immunosuppressives et sont associés à un mauvais pronostic dans la plupart des cancers. La reprogrammation fonctionnelle de ces macrophages aux propriétés pro-tumorales vers un phénotype pro-inflammatoire aux propriétés anti-tumorales favorise le développement d'une réponse anti-tumorale. Notre équipe a récemment étudié la capacité des radiations ionisantes à reprogrammer les TAMs vers un phénotype pro-inflammatoire. L'augmentation de la capacité des rayonnements ionisants à reprogrammer les TAMs en macrophages pro-inflammatoires est un objectif à atteindre pour améliorer l'efficacité des traitements du cancer.C'est dans ce contexte que mes travaux de thèse ont permis (i) de poursuivre la caractérisation des mécanismes moléculaires impliqués dans la reprogrammation radio-induite des macrophages, (ii) d'identifier le rôle du récepteur purinergique P2Y2 comme un modulateur négatif de la reprogrammation pro-inflammatoire des macrophages ; (ii) de caractériser les bases moléculaires de ce processus biologique et (iii) de proposer d'inhiber l'activité biologique du récepteur purinergique P2Y2, afin d'augmenter la capacité des rayonnements ionisants à déclencher l'activation pro-inflammatoire des macrophages
Tumor-associated macrophages (TAMs) are key components of the tumor microenvironment that display immunosuppressive functions and are associated with poor prognosis in most cancers. The functional reprogramming of these macrophages with pro-tumor properties towards a proinflammatory phenotype with anti-tumor properties promotes the development of an anti-tumor response. Our team recently studied how ionizing radiation modulates macrophage reprogramming towards a proinflammatory phenotype. Increasing the ability of ionizing radiation to reprogram TAMs into proinflammatory macrophages is a key objective to improve the effectiveness of cancer treatments.In this context, my thesis work enabled (i) to further characterize the molecular mechanisms involved in the radiation-induced macrophage reprogramming, (ii) to identify the role of the purinergic receptor P2Y2 as a negative modulator of the proinflammatory reprogramming of macrophages; (ii) to characterize the molecular bases of this biological process, and (iii) to propose the inhibition of the biological activity of P2Y2 receptor, to increase the ability of ionizing radiation, triggering the pro-inflammatory activation of macrophages

La pluripotence est la capacité d'une cellule à s'auto-renouveler et à donner toutes les cellules somatiques ainsi que les cellules germinales. Les cellules pluripotentes peuvent être aussi reprogrammées à partir de cellules somatiques, ouvrant ainsi de nouvelles opportunités pour l'utilisation thérapeutique des cellules souches dans le traitement des maladies dégénératives. La connaissance des mécanismes moléculaires, en particulier des voix de signalisation qui contrôlent la pluripotence, est cruciale pour l'amélioration de notre compréhension de l'embryogenèse précoce et l'utilisation des iPSC (cellules souches pluripotentes induites) dans la médicine régénérative. Ici, je donne la première description de la Nétrine-1 en tant que régulateur de la reprogrammation et de la pluripotence. La Nétrine-1 et ses récepteurs ont été initialement caractérisés dans le système neuronal, mais il a aussi été montré qu'ils étaient exprimés dans différents types cellulaires et impliqués dans divers processus. Dans la première partie, j'ai contribué à explorer comment Nétrine-1 empêche l'apoptose médiée par son récepteur à dépendance DCC (Deleted in Colon Carcinoma) pendant la reprogrammation. Dans la deuxième partie, j'ai disséqué les fonctions et la régulation de cette voie dans le maintien de la pluripotence et dans l'engagement des lignages
Pluripotency is the ability of embryonic epiblast cells to self-renew and to give rise to all somatic cells as well as germ cells. Somatic cells can also be reprogrammed toward pluripotency, opening new avenues for stem cell based therapies in the treatment of degenerative diseases. Deciphering the molecular mechanisms, and in particular signaling pathways that control pluripotency is crucial to improve our understanding of early embryogenesis and the use of iPSC (inducible Pluripotent Stem Cell) in regenerative medicine.Herein, I provide the first description of Netrin-1 as a regulator of reprogramming and pluripotency. Netrin-1 and its receptors are present in many cell types and are engaged in a variety of cellular processes beyond its initial characterization in the neuronal system. In the first part, I contributed to explore how Netrin-1 prevents apoptosis mediated by its dependence receptor DCC (Deleted in Colon Carcinoma) during reprogramming. In the second part, I dissected the functions and regulation of this pathway in pluripotency maintenance and in lineage commitment

Caloric restriction (CR) extends lifespan from yeast to mammals. In budding yeast, inhibition of the conserved TOR and/or PKA pathways has been shown to mediate lifespan extension by CR partly through the activation of stress response. However, how the stress response is regulated at the systems level is poorly understood. In this study, by using fluorescent reporters whose expression is dependent on the transcription factors Msn2/4 and Gis1, two separate screenings were conducted to reveal novel regulators of the stress response induced by starvation. A 'focused' screening on the 272 'signalling' mutants revealed that, apart from the previously identified Rim15, Yak1 and Mck1 kinases, the SNF1/AMPK complex, the cell wall integrity (CWI) pathway and a number of cell cycle regulators are necessary to elicit appropriate stress response. The chronological lifespan (CLS) of these signalling mutants correlates well with the amount of accumulated storage carbohydrates but poorly with transition-phase cell cycle status. Subsequent analyses reveal that the levels of intracellular reactive oxygen species are controlled by Rim15, Yak1 and Mck1. Furthermore, CLS extension enabled by tor1 deletion is dependent on the above three kinases. These data suggest that the signalling pathways (SNF1 and CWI) and the kinases downstream of TOR/PKA (Rim15, Yak1 and Mck1) coordinate the metabolic reprogramming (to accumulate storage carbohydrates) and the activation of anti-oxidant defence systems (to control ROS levels) to extend chronological lifespan. A 'genome-wide' screening of a haploid deletion library indicates that less than 10% of the non-essential genes are implicated in the regulation of starvation-induced stress response. Gene ontology analysis suggests that they can be grouped into major clusters including mitochondrial function, r-RNA processing, DNA damage and repair, transcription from RNA polymerase and cell cycle regulation. Further phenotypic assays confirm the previous observation that CLS extension is mostly correlated with the accumulation of storage carbohydrates. Compromised expression of stress response reporters is confirmed by FACS in a variety of mitochondrial mutants, suggesting that mitochondrial respiration also plays a key role in the activation of stress response. Put together, the above findings indicate that stress response and metabolic reprogramming induced by glucose starvation are coordinated by multiple signalling pathways and the activation of mitochondrial respiration is essential to both cellular processes and to CLS extension.

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.

Apolipoprotéines L est une famille nouvellement caractérisée en humain sans une fonction patho- physiologique définitive. Ces protéines sont classiquement considérées être impliquées dans le transport et métabolisme des lipides, principalement due à l'association de son premier membre de la famille sécrétée l’apolipoprotéine L1 aux particules des lipoprotéines de haute densité. Néanmoins, le reste des membres sont des protéines intracellulaires (absence de domaine de peptide signal). Apolipoprotéine L1 a été initialement identifiée comme l'élément clé du facteur trypanolytique dans le sérum humain. L'exploration de la séquence des différents apolipoprotéines L a révélé un domaine distinct «B cell lymphoma-2 homology domain 3» ayant des similitudes structurelles et fonctionnelles avec le domaine B cell lymphoma-2 homology domain 3 des protéines de la famille B cell lymphoma-2. Ainsi la découverte de ce domaine peut contribuer à la compréhension de la fonction et rôle des apoLs dans différents mécanismes et processus tels que la mort cellulaire programmée, la prolifération cellulaire, le métabolisme cellulaire .Notre étude visait à caractériser les fonctions de patho- physiologique du premier membre de la famille «apolipoprotéine L1 ». L’expression de l’apolipoprotéine L1 ARNm, à partir de 48 carcinomes papillaires de la thyroïde, a été évaluée par des études à haut débit et normalisée à un pool de tissus normal de la thyroïde. Une confirmation de PCR en temps réel valide ainsi la surexpression d’apoL1 dans 91,67 % des cas testés. Le niveau élevé de l’apolipoprotéine L1 ARNm est en corrélation avec une expression protéique élevée dans les échantillons histologiques (70%), et détermine que les cellules folliculaires de la thyroïde dans la zone de la tumeur sont les cellules principales responsables de l’expression spécifique de l’apolipoprotéine L1. Nous avons étudié l'expression apolipoprotéine L1 dans le modèle de cancer pour approfondir notre compréhension des relations reliant cette expression distincte dans le cancer papillaire de la thyroïde et son rôle et fonction concernant le métabolisme du cancer (de reprogrammation métabolique :effet Warburg).7En outre, la localisation de l’apolipoprotéine L1 dans la mitochondrie des cellules cancéreuses de la thyroïde ainsi que dans la mitochondrie de levure, a été le point de départ de la recherche dans ce nouveau modèle, il nous a permis de révéler et d'introduire de nouvelles hypothèses pour expliquer l'effet inhibiteur de l’apolipoprotéine L1 en fonction des conditions métabolique variantes et l’effet pléotropiques de l’apolipoprotéine L1 sur la levure (dommages des mitochondries et vacuoles). Dans ce manuscrit, nous avons décrit nos efforts à mettre en évidence la spécificité d'expression de l’apolipoprotéine L1 dans le cancer papillaire thyroïdien notamment au niveau de la transcription ainsi que la localisation mitochondriale et l'interférence probable avec les voies métaboliques.
Option Biologie moléculaire du Doctorat en Sciences
info:eu-repo/semantics/nonPublished

Cancer cells require survival strategies to respond to microenviromental changes and out-compete their neighbours. They activate stress response mechanisms under extreme microenvironmental conditions, some of which are controlled by the amino acid-sensitive kinase complex, mechanistic Target of Rapamycin Complex 1 (mTORC1). Exosomes are secreted nanovesicles made inside intracellular endosomal compartments that mediate a specialised and complex form of intercellular signalling that can reprogramme target cells via the action of multiple active cargos. I investigated whether mTORC1 activity might modulate the type of exosome secreted in response to microenvironmental changes. Here I identify a new form of mTORC1-regulated exosome biogenesis and signalling involving recycling multivesicular endosomes (rMVEs), a previously unrecognised site for exosome biogenesis. Reduced activity of a specific form of glutamine-sensitive mTORC1 in HCT116 colorectal cancer cells results in an ‘exosome switch’ in which exosomes are preferentially released from these compartments instead of late endosomes. Importantly, RAB11a is found in association with at least a proportion of rMVEs that generate these alternative exosomes and is loaded on to some of their ILVs, providing a RAB signature of compartmental origin. I provide evidence that this exosome switch is conserved in other cancer cell types. My study also presents a proteomics analysis of extracellular vesicle (EV) preparations from normal and mTORC1-inhibited cells. I demonstrate that EV preparations isolated following exosome switching have enhanced pro-angiogenic properties and novel tumour growth-promoting activities. Activation of the receptor tyrosine kinase c-MET and its downstream mitogen-activated protein kinase (MAPK) ERK via phosphorylation is stimulated by these EVs, providing a potential explanation for their growth-promoting effects. Subsequent studies in the lab have demonstrated that several of these pro-tumorigenic activities are mediated by exosomes. I conclude that stress-induced mTORC1 inhibition allows tumour cells to initiate a novel exosome secretion pathway that potentially mediates a cancer cell survival plan that reverses microenvironmental change and supports tumour adaptation. In the future, blocking this response could improve patient outcome following treatment with mTORC1-inhibitory or anti-angiogenic drugs that have currently met with limited success in the clinic.

Mycobacterium tuberculosis, the causative agent for the disease Tuberculosis, is a serious public health problem that is responsible for 1.6 million deaths each year. The WHO’s recent report on Tuberculosis estimates that a third of the world’s population is latently infected with the bacteria, and, of those, 10% will progress to active disease. M. tuberculosis is a successful pathogen mainly due to its ability to adapt and survive in changing environments. It can survive a dormant state with limited metabolic activity during latent infection, while also being able to escape the macrophage and disseminate into active disease. Efforts to eradicate the disease must be based on understanding the biology of this organism, and the mechanisms it uses to infect, colonize, and evade the immune system. Understanding the behaviour of pathogenic mycobacteria in the macrophage is also important to the discovery of new drug targets. In this thesis, we employed state of the art mass spectrometry techniques, which allowed us to unpack the biology of this bacterium in different growth environments and expand our understanding of the mechanisms it employs to adapt and survive. We investigated protein regulation by the process of phosphorylation, through sensory kinases, which add a phosphate group to a protein of interest, thereby regulating its function. First, we interrogated the phosphoproteomic landscape between M. bovis BCG and M. smegmatis to explain how differential protein regulation results in the differences between slow and fast growth of mycobacteria. Second, we focused on Protein Kinase G (PknG), which plays an important role in bacterial survival by blocking phagosome/lysosome fusion. We identified the in vivo physiological substrates of this kinase in actively growing M.bovis BCG culture. Our results revealed that this kinase is a regulator of protein synthesis. We then examined the mechanisms of survival in murine RAW 246.7 macrophages mediated by PknG, using M. bovis BCG reference strain and PknG knock-out mutant. Our results indicated strong evidence that pathogenic mycobacteria disrupt the macrophagic cytoskeleton, through phosphorylation of proteins that are involved in cytoskeleton rearrangement. These results explain the strategies that pathogenic mycobacteria employ mediated by PknG to block phagosome-lysosome fusion and evade the host immune system and survive for prolonged periods in the macrophages. The findings of this thesis contribute to our understanding of the physiology of pathogenic mycobacteria and their interaction with the host.

Skeletal muscle is a highly compliant organ system that is composed of muscle fibres, nerves, sensory cells, blood vessels and connective tissue. A central concept of skeletal muscle biology is the existence of an inverse relationship between muscle fibre size and its oxidative capacity which has been used to explain why small fibres are oxidative and large fibres glycolytic. However, sturdiness of this relationship is unknown. In order to investigate the rigour of this relationship we made use of a genetic model that enhances oxidative metabolism, mediated by estrogen-related receptor gamma (Errγ) (a constitutively active orphan nuclear receptor belongs to the ERR subfamily), and the hypertrophic background of Myostatin (a member of the Transforming Growth Factor beta (TGF-β) superfamily that is negatively regulating skeletal muscle mass development) null (Mtn‐/‐) mice. We show that superimposition of Errγ on the Mtn‐/‐ background results in hypertrophic muscle that displays a high oxidative capacity (Mtn‐/‐/ErrγTg/+), thus violating the inverse relationship between muscle fibre cross-sectional area and its oxidative capacity. Thereafter, we examined the canonical view that there is a high number of satellite cells (skeletal muscle resident stem cells) in oxidative muscles. Surprisingly, I found that hypertrophic oxidative muscle fibres from Mtn‐/‐/ErrγTg/+ mice showed a deficit in the number of satellite cells. Unexpectedly, the lower population of satellite cells in the hypertrophic oxidative model is not associated with a lower regenerative capacity. We also examined the relationship between muscle fibre phenotype (size and metabolism) and components of its force transducer apparatus that consists of both extracellular matrix (ECM) and dystrophinglycoprotein complex (DGC). Interestingly, I showed that levels of ECM and DGC entities can be influenced by muscle fibre phenotype. Observations of this work firstly, challenge the notion of a constraint between skeletal muscle fiber size and oxidative capacity, secondly, indicate the important role of the microcirculation in the regenerative capacity of a muscle even with low population of satellite cells, and thirdly, show that the metabolic properties of a muscle fibre are a critical factor to regulate the levels of ECM and DGC proteins.

Embryonic stem (ES) cell pluripotency is sustained by a network of transcription factors centred on Oct4, Sox2 and Nanog. Whilst Oct4 and Sox2 expression is relatively uniform, ES cells fluctuate between states of high Nanog expression possessing high self-renewal efficiency, and low Nanog expression exhibiting increased differentiation propensity. Moreover, modulation in the level of Nanog expression determines the efficiency of ES cell self-renewal. To identify genes regulated by Nanog, genome-wide transcriptional profiling was performed on ES cells expressing different Nanog levels and Nanog-null ES cells expressing a Nanog-ERT2 fusion protein in which nuclear Nanog activity can be regulated by tamoxifen. Surprisingly, only a minor fraction of the genes to which Nanog binds showed significant changes in response to Nanog induction. Prominent amongst Nanog-responsive genes is Estrogen-related receptor b (Esrrb). Nanog binds directly to Esrrb, enhances binding and pause-release of RNAPolII from the Esrrb promoter and stimulates Esrrb transcription. Consistent with these findings, elevation of Nanog produces a cell population that expresses uniformly high Esrrb levels. Moreover, double fluorescent reporter lines show that Esrrb and Nanog levels are strongly correlated in individual cells. Loss of Nanog is required for downregulation of Esrrb, which coincides with commitment to differentiate. Esrrb overexpression results in LIF independent self-renewal, and blocks neural differentiation, even in the absence of Nanog. Cell fusion experiments between ES and neural stem (NS) cells show that elevated Esrrb levels allow the reprogramming of the NS cell genome in the absence of Nanog. Esrrb can rescue stalled reprogramming during the derivation of Nanog-/- induced pluripotent stem (iPS) cells. Moreover, targeted knock-in of Esrrb at the Nanog locus rescues the ability of Nanog null ES cells to maintain germ cell development beyond E12. Finally, Esrrb deletion abolishes the defining ability of Nanog to confer LIF-independent selfrenewal to ES cells. Together these data identify Esrrb as a critical downstream mediator of Nanog function.

A nonsynonymous single polymorphism (SNP) in G protein-coupled receptor kinase 5 (GRK5) was discovered in 2008 that changes the amino acid at the position 41 from Glutamine (Q) into Leucine (L) (Liggett et al., 2008). The putative functions of the GRK5-L41 polymorphism were reported to be involved in faster desensitisation of both b1 and b2 adrenergic receptors in vitro and the cardiac protective functions by improving the survival rate of patients with heart failure conditions or transplantation in vivo. Nevertheless, the mechanisms underlying these processes are still poorly understood, which is the purpose of this thesis. This thesis presents the establishment of the first human cardiac models of GRK5- L41 polymorphism using human pluripotent stem cells (hPSCs) and their derived cardiomyocytes (CMs). The thesis is distributed into four main themes, containing (1) the formulation of monolayer cardiac differentiation protocols; (2) the establishment of human induced pluripotent stem cells from lymphoblastoid cell lines bearing the GRK5- L41 sequence; (3) the development of footprint-free and shortcut CRISRP/Nickase approach that allowed generating the gene-edited human embryonic stem cells expressing GRK5-Q41, GRK5-Q/L41, and GRK5-L41; and (4) the evaluation of GRK5- L41 functions using cardiac functional analysis assays. Relating to disease modelling and cardiovascular biomedical research, the ability to differentiate the hPSCs into CMs plays a critical role by providing an unlimited resource of human CMs for in vitro testing and experiments. Here, three main monolayer cardiac differentiation protocols, including E8-AB, mTeSR-AB, and mTeSR-CHIR, were described in details and proven to be highly consistent, efficient, robust, and reproducible. Additionally, these protocols have been ascertained to be effective in more than 27 hPSC lines routinely maintained in the lab regardless of the culture conditions (non-defined vs. defined culture conditions), cell types (human embryonic stem cells vs. human induced pluripotent stem cells), reprogramming methods, and somatic cell sources (in the case of induced pluripotent stem cells). Indeed, by using the E8-AB protocol, more than 1x107 CMs/line have been produced in this thesis, providing enough resource for functional assay analysis and mechanistic studies of GRK5-L41. Furthermore, two independent approaches were made to create the human cardiac model of GRK5-L41 polymorphism, involving the establishment of GRK5-L41 bearing hiPSCs from lymphoblastoid cell lines, and simultaneously introducing the GRK5-L41 sequence to the HUES7 genome to create the Q/L41, and L41 expressing HUES7 lines. In general, four hPSC lines were successfully generated in this thesis, including hiPSC-GRK5-L41, hiPSC-GRK5-Q41, HUES7-GRK5-Q/L41, and HUES7- GRK5-L41. The H-Fib-hiPSC, cell lines generated from HUES7-derived fibroblast, was the additional line obtained after testing the effectiveness of episomal plasmid. All cell lines were able to differentiate into CMs at high purity, approximately 85%, and were used for the development of functional assays. Four main functional experiments were developed focusing on the GRK5-related functions in the heart, consisting of contractility and hypertrophic response to catecholamine induction, especially during the chronic response. The effects of catecholamine, in this case, Isoprenaline (ISO), on the contractility of the CMs were measured by two assays, the CardioExcyte96 platform detecting the contraction rate and beating pattern in real-time, and the LANCE Ultra cAMP assay assessing the production of cAMP. These results indicated that extended culture of CMs in ISO (>30h) introduced the detrimental effects on the contractility and beating pattern of the CMs in vitro, generating the arrhythmias in GRK5-Q41 CMs. Interestingly, the GRK5-L41 CMs exhibited a high level of the beat rate in response to ISO and maintained it constantly during prolonged exposure to ISO similar to that of b-blocker treatments in GRK5-Q41 CMs. The Western Blot analysis of the cellular distribution of GRK5 spotted the localisation of GRK5 during ISO treatment for 72h. Further characterisation using immunofluorescence analysis of chronic exposure to ISO demonstrated the elevation of BNP level, a hypertrophic marker, indicating that ISO treatment duration (>30h) induced the hypertrophic response of hPSC-CMs in vitro. Taken together, the findings within this thesis has been the first step in a discovery process of the cardiac protective functions of GRK5-L41 polymorphism during heart failure. Despite the presence of limitation and difficulty, it manages to provide sufficient information to explore further the interrelationship between nuclear accumulation of GRK5, hypertrophic response, and contractility regulation mediated by either GRK5-Q41 and GRK5-L41 in hPSC-CMs in vitro.

Heart disease is one of the leading causes of mortality in developed countries. The associated pathology is typically characterized by the loss of cardiomyocytes that leads, eventually, to heart failure. Presently, there are many promising strategies for cardiac regeneration. Direct cardiac reprogramming is becoming known as a novel therapeutic approach to regenerate injured hearts. Direct cardiac reprogramming is a simple and quick process however, the molecular mechanisms of cardiac reprogramming and cardiomyocyte-like cells functional maturation remain to be understood. Direct cardiac reprogramming has great potential to become one of the main strategies for regenerative medicine in heart failure since fibroblasts, contrary to cardiomyocytes which do not divide, are easily available in the heart, they are a large population of cells in the heart, which become activated and turn to myofibroblasts, contributing to fibrosis after cardiac injury. Currently it is known that a specific combination of three transcription factors, Mef2c, Gata4 and Tbx5 (MGT), are enough to reprogram non-myocyte mouse heart cells into induced cardiomyocyte-like cells. Nevertheless, human fibroblasts when infected with MGT appeared to have a small percentage of conversion. With MGT retrovirus we successfully transfected: mouse adult fibroblasts (MAFs), Feeders and Gm 03348 (human fibroblasts with 10 years old). Through qPCR analysis we evaluated the expression of lncRNAs: Gm 15856, Mir22hg, Gm 027028 and Gm 28592. Our goal was to understand which lncRNAs are the best candidates to knockdown in order no enhance direct cardiac reprogramming. In addition, we studied how nutrient manipulation in cell culture media can influence direct cardiac reprogramming. It was found that media with higher levels of glucose and glutamine had larger rates of cellular survival and proliferation.
As doenças cardíacas são uma das principais causas de mortalidade nos países desenvolvidos. A patologia associada é tipicamente caracterizada pela perda de cardiomiócitos que leva, eventualmente, à insuficiência cardíaca. Atualmente, existem muitas estratégias promissoras para a regeneração cardíaca. A reprogramação cardíaca direta tem se tornado conhecida como uma nova abordagem terapêutica para regeneração cardíaca depois de uma lesão. A reprogramação cardíaca direta é um processo simples e rápido, no entanto os seus mecanismos moleculares e de maturação celular continuam maioritariamente desconhecidos. A reprogramação cardíaca direta é uma abordagem terapêutica com grande potencial para se tornar uma das principais estratégias da medicina regenerativa no combate à insuficiência cardíaca, uma vez que os fibroblastos estão facilmente disponíveis no coração e dividem-se facilmente ao contrário dos cardiomiócitos. Os fibroblastos cardíacos são uma população alargada no coração que, após uma lesão, tornam-se em miofibroblastos ativos contribuindo para a fibrose. Atualmente, sabe-se que uma combinação específica de três fatores de transcrição, Mef2c, Gata4 e Tbx5 (MGT), é suficiente para reprogramar fibroblastos cardíacos de ratinho em cardiomiócitos induzidos. Por outro lado, quando fibroblastos humanos são infetados com MGT apresentam uma pequena percentagem de conversão. Com o retrovírus MGT transfectamos com sucesso: fibroblastos adultos de ratinho (MAFs), Feeders e Gm 03348 (fibroblastos humanos com 10 anos de idade). Através da análise de qPCR, avaliamos a expressão dos lncRNAs: Gm 15856, Mir22hg, Gm 027028 e Gm 28592. O nosso objetivo foi estudar quais os lncRNAs são os melhores candidatos para knockdown, e assim melhorar a eficiência da reprogramação cardíaca direta. Para além disso, estudamos como a manipulação de nutrientes nos meios de cultura pode influenciar a reprogramação cardíaca direta. Verificou-se que meios com níveis mais altos de glucose e glutamina apresentaram maiores taxas de sobrevivência e proliferação celular.
Mestrado em Biologia Molecular e Celular

Tese de mestrado, Ciências Biomédicas, Universidade de Lisboa, Faculdade de Farmácia, 2016
Previous studies have accomplished direct lineage reprogramming of many cell types to different ones by using defined combinations of transcription factors. Vierbuchen et al. showed that the combined ectopic expression of Ascl1, Brn2 and MyT1L can efficiently reprogram mouse embryonic fibroblasts (MEFs) into induced neuronal (iN) cells. In another study, Ascl1 was characterized as the main driver of this process by its activity as a pioneer factor. Previous experiments with Ascl1 single-reprogramming showed that Ascl1 is capable of converting MEFs into iN cells, although the reprogrammed neurons show low levels of maturity. On the other hand, several reprogramming experiments associated MyT1L with a late function by promoting the maturation of iN cells, but not with the capacity to reprogram MEFs into iN cells like Ascl1. However, a mechanistic characterization of MyT1L still needed to be clarified. MyT1L is a member of the MYT1 family, also including MyT1 and MyT3, all zinc-finger transcription factors. Recent work from our laboratory showed that MyT1, a paralog of MyT1L, acts as a repressor of Notch targets, in neural stem/progenitor cells. One of those identified Notch targets was Hes1. In neurogenesis, the Notch pathway induces the activation of the Notch downstream effector Hes1. Hes1 functions as a repressor of proneural genes, such as Ascl1, as well as their target genes. Similar to the neurogenesis context, it is tempting to speculate that in Ascl1-dependent reprogramming Hes1 may be functioning as a repressor of Ascl1 targets in MEFs. The goal of this work is to investigate the role of MyT1L and the Notch signalling pathway in Ascl1-dependent reprogramming of MEFs into iN cells. To evaluate Notch activity in MEFs, I compared the expression levels of two Notch targets, Hes1 and Hes5, between MEFs and neural stem cells. I show that Hes1 expression in MEFs is similar to Hes1 expression in neural stem cells. Hes5 expression is substantially lower in MEFs than in neural stem cells. This suggests low Notch activity in MEFs as previous studies identify the Hes5 promoter as readout of Notch activation. Chemical inhibition of Notch signalling did not alter the Hes1 expression in MEFs. I show that the proximal promoter region of Hes1, that mediates regulation by Notch and MyT1 in neural stem/progenitor cells, is accessible to transcription factor binding in MEFs. Additionally, I show that the Notch effector transcription factor RBPJ binds to the Hes1 proximal promoter region. These results in conjunction with the high levels of Hes1 expression in MEFs suggest that the Notch pathway is not the main regulator of Hes1 expression in these cells. Work from our laboratory showed that, in transcriptional assays, MyT1 represses the Hes1 proximal promoter activity, after Notch activation. Here I show that Myt1L can counteract the Notch activation of the Hes1 promoter in a transcriptional assay. The Hes1 proximal promoter contains three consensus binding sites of the MYT1 family suggesting that MyT1L regulates the Hes1 promoter by direct DNA-binding to this region. Using chromatin immunoprecipitation assay against a tagged version of Myt1L, I show that MyT1L directly binds to the Hes1 promoter region two days after being ectopically expressed in MEFs. Finally I started the optimization of the Ascl1-depedent reprogramming protocol in MEFs. I did observe reprogrammed iN cells after single or combined expression of Ascl1 or Ascl1/MyT1L, respectively. However, the percentage of iN cells to total number of cells in culture revealed low reprogramming efficiency. Additionally, iN cells observed show low levels of maturity in single or combined expression of Ascl1 or Ascl1/MyT1L. Nonetheless, this protocol still needs further improvement. Overall, my findings indicate that MyT1L binds to DNA in MEFs at early stages of the Ascl1-dependent reprogramming protocol. The results suggest that MyT1L represses the expression of Hes1 in Ascl1-dependent reprogramming and this may lead to the activation of the Ascl1 targets that promote iN cell maturation.
Vários estudos têm vindo a demonstrar que a reprogramação directa de uma linha celular somática para outros tipos celulares pode ser alcançada através da expressão ectópica de factores de transcrição. De facto, trabalho desenvolvido por Yamanaka e Takahashi (Takahashi and Yamanaka, 2006) demonstrou que a adição de quatro factores de transcrição é suficiente para reprogramar fibroblastos em células estaminais pluripotentes. Este estudo estabeleceu uma mudança de paradigma na forma como olhamos para o programa de transcrição e a plasticidade do genoma da célula. A reprogramação de um tipo celular a partir de células estaminais ou somáticas oferece um enorme potencial de aplicações na medicina regenerativa e na terapia de doenças. A reprogramação de fibroblastos em células neuronais foi alcançada através da adição de três factores de transcrição, Brn2, Ascl1 e MyT1L (BAM) (Vierbuchen et al., 2010), em que o Ascl1 é o factor de transcrição principal, uma vez que, sozinho, é capaz de converter os fibroblastos em neurónios, apesar de apresentarem baixa complexidade morfológica e capacidade funcional (Chanda et al., 2014). Curiosamente, Ascl1 funciona como um factor pioneiro, sendo capaz de se associar às regiões genómicas, independentemente de se encontrarem em locais de cromatina acessível (Raposo et al., 2015; Wapinski et al., 2013). O Ascl1 é um factor de transcrição proneural que actua como um regulador da diferenciação neuronal no cérebro de mamíferos (Bertrand et al., 2002; Wilkinson et al., 2013). No processo de neurogénese, Ascl1 actua principalmente como um activador de transcrição sobre uma grande variedade de genes que controlam vários passos da neurogénese, como a proliferação das células estaminais neurais/progenitoras, migração celular e crescimento das neurites (Borromeo et al., 2014; Castro et al., 2011, 2006). Recentemente, Ascl1 foi identificado como um factor de transcrição capaz de modificar a cromatina dos seus genes alvos, durante a neurogénese, promovendo a acessibilidade da cromatina para Ascl1 (Raposo et al., 2015). Durante a neurogénese, o Ascl1 é regulado pela via de sinalização Notch. No desenvolvimento do sistema nervoso, a via de sinalização Notch é responsável pela manutenção da população de células estaminais neuronais/progenitoras, através da inibição da diferenciação neuronal. A proteína Notch activa a expressão de genes repressores da neurogénese, dos quais se incluem os genes Hes1 e Hes5. Os genes Hes1/5 actuam como repressores da transcrição, sendo um dos seus alvos Asc1. Adicionalmente, resultados anteriores do nosso laboratório demonstraram que Hes1 inibe a expressão dos genes alvos de Ascl1. Recentemente, estudos realizados no nosso laboratório revelaram que um alvo de Ascl1 durante a neurogénese, o factor MyT1, tem um papel importante em bloquear a expressão de genes alvo de Notch, em particular o Hes1. MyT1 é um factor de transcrição da família MYT1, que é composta por outros 2 factores de transcrição: MyT1L e MyT3. Os membros desta família são altamente homólogos, particularmente nos domínios proteicos zinc-fingers, responsáveis pela ligação ao ADN (Bellefroid et al., 1996; Kim et al., 1997). Todos os membros da família MYT1 são expressos no desenvolvimento do sistema nervoso central. Em particular, MyT1L é expresso exclusivamente em neurónios e é detectado tanto na neurogénese como na fase adulta do organismo (Matsushita et al., 2014). Na reprogramação neuronal, o MyT1L tem sido utilizado em vários protocolos para promover um aumento da complexidade morfológica e das propriedades electrofisiológicas das células neuronais (Ambasudhan et al., 2011; Pang et al., 2011; Vierbuchen et al., 2010; Yoo et al., 2011). Considerando os resultados do nosso laboratório em que se demonstrou que MyT1 é um repressor da expressão de Hes1, colocámos a hipótese de que MyT1L pudesse também actuar na reprogramação de células neuronais como um repressor da expressão de Hes1. O trabalho desta dissertação teve como objectivo investigar o papel do MyT1L e da via de sinalização Notch na reprogramação de fibroblastos em células neuronais promovida por Ascl1. Em primeiro lugar analisei a actividade da via de sinalização Notch nos fibroblastos através da análise de expressão de dois genes alvos de Notch, Hes1 e Hes5. A expressão destes genes foi comparada entre fibroblastos e células NS-5, uma linha de células estaminais neurais com elevada actividade da via Notch. Os resultados demonstraram que o nível de expressão de Hes1 em fibroblastos e em células NS-5 são semelhantes. No entanto, após inibição química da actividade de Notch não observei nenhuma alteração na expressão de Hes1, o que sugere que a via sinalização Notch não é a principal reguladora de Hes1 nos fibroblastos. Contrariamente a Hes1, a expressão de Hes5 é consideravelmente inferior nos fibroblastos em relação às células NS-5. Por outro lado, a inibição química da actividade de Notch levou a uma diminuição da actividade da expressão de Hes5, indicando que Hes5 é regulado por Notch em fibroblastos. Em segundo lugar, investiguei qual o possível papel de MyT1L na regulação da expressão de Hes1 em fibroblastos. Analisei que a região promotora de Hes1, onde anteriormente o nosso laboratório demonstrou haver associação de MyT1 em células neurais/progenitoras estaminais, se encontra com cromatina acessível à associação de factores de trancrição, em fibroblastos. Também analisei a actividade de MyT1L nessa região promotora de Hes1 através de um ensaio de transcrição com a co-expressão de MyT1L e receptor Notch1 activado. Esta análise revelou que o MyT1L é um repressor do promotor de Hes1, dependente da activação pela via Notch. Esta região promotora de Hes1 contém três sítios de ligação ao ADN comum à família MYT1. De facto, os resultados da imunoprecipitação da cromatina extraída de fibroblastos revelaram uma associação do MyT1L ectopicamente expresso na região promotora de Hes1. Hes1 é um factor repressor da expressão de Ascl1 e dos seus genes alvos. De facto, é possível que, no contexto da reprogramação promovida por Ascl1, os níveis de Hes1 endógeno possam estar a reprimir a expressão dos genes alvos de Ascl1. Esta repressão de Hes1 pode explicar o baixo nível de diferenciação das células neuronais observado na reprogramação com apenas sobre-expressão de Ascl1. De facto, MyT1L foi descrito como tendo um papel importante no desenvolvimento de características de neurónios morfologicamente complexos. Assim é possível que, o MyT1L promova indirectamente a expressão dos genes alvos de Ascl1, através da inibição da expressão de Hes1. Deste modo, o protocolo de reprogramação de fibroblastos em células neuronais mediado por Ascl1 foi optimizado, com o objectivo de investigar a interacção de MyT1L e Hes1, no contexto desta reprogramação. Infelizmente, o protocolo não foi estabelecido com sucesso, devido, a uma elevada taxa de morte célular. Apesar da elevada morte celular, consegui obter células neuronais, a partir de fibroblastos, com apenas a sobre-expressão de Ascl1 em co-expressão com MyT1L. As células neuronais obtidas com estas duas condições apresentavam baixos níveis de complexidade morfológica. Em conclusão, demonstrei que MyT1L encontra-se associado à região promotora de Hes1 quando expresso de modo ectópico em fibroblastos e que Myt1L actua como um repressor da actividade da região promotora de Hes1 promovida pela activação da via de sinalização Notch. A junção destes dois resultados sugere que a inibição da expressão de Hes1 se reflicta nos fibroblastos após sobre-expressão de Myt1L. A função de MyT1L pode incluir a repressão da expressão de Hes1, promovendo a activação de genes alvos de Ascl1 responsáveis pela maturação neuronal. Experiências futuras que demonstrem uma diminuição da expressão de Hes1 após a sobre-expressão de MyT1L em fibroblastos devem ser consideradas. Adicionalmente, futuras experiências devem também focar-se na descoberta de outros genes alvo de MyT1L em fibroblastos que possam ter um papel importante na reprogramação promovida por Ascl1 de fibroblastos em neurónios.
The studies presented in this thesis were carried out at the Instituto Gulbenkian Ciência (IGC) at the Molecular Neurobiology Laboratory, Oeiras. The present study was supported by Fundação Calouste Gulbenkian.

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, in fulfillment of the requirements for the degree of Masters of Science 2016
This study focused on reprogramming of energy metabolism of cancer cells, since most cancer and proliferating cells have been shown to display a metabolic shift by displaying increased dependence on glycolysis and reduced oxidative phosphorylation (OXPHOS) for energy. Dichloroacetate (DCA) and Methyl pyruvate (MP) were used to attempt the reversal of the metabolic program of THP-1 cells. Flow cytometry was used to determine the mode of cell death and to analyse the changes in cell cycle. In this study, an overexpression of TLR4 was observed in THP-1 cells treated with 5ng/ml of lipopolysaccharides (LPS). Further analysis of cell death showed that MP and DCA-treated cells resulted to minimal induction of apoptotic cell death. This suggests that the 2 drugs (MP and DCA) cause cell death via apoptosis. Furthermore, LPS treated cells (infected cancer cells) showed an increase in glycolysis (Warburg effect). This study has shown that indeed treatment with drugs such as MP and DCA was effective in reversing the glycolytic phenotype of THP-1 cells, resulting in cell death via apoptosis by boosting OXPHOS.
MT 2018

Thesis (M.Sc.)--University of the Witwatersrand, Faculty of Science, School of Molecular and Cell Biology, 2018.
ABSTRACT The Retinoblastoma binding protein 6 is dysregulated in most cancers, indicating it may play a role in metabolic reprograming- a hallmark of carcinogenesis. Its human isoforms have been shown to play diverse roles in apoptosis. This study aimed to elucidate biochemical roles of RBBP6 isoforms in metabolic reprogramming during carcinogenesis. Drosophila melanogaster wild type and p53 null mutants were treated with drug permutations of irinotecan (DNA damaging agent) and exogenous pyruvate to perturb metabolism. Moreover, using RT-PCR and Western blot expression profiles of SNAMA (Drosophila Orthologue of RBBP6) isoforms were shown followed by survival studies to investigate the effects of these drugs. Furthermore, using bioinformatics the domains of RBBP6 isoforms in various species were shown. Results indicate that RBBP6 isoforms show contrasting expression patterns. Furthermore, exogenous pyruvate protects the wild type flies from irinotecan toxicity while killing p53 null mutants. RBBP6 proves to be a potential druggable target for chemotherapy.
EM2018

Dissertação de Mestrado em Biologia Celular e Molecular apresentada à Faculdade de Ciências e Tecnologia
The development of T cells, essential mediators of adaptive immune responses towards infectious agents and cancer cells, is orchestrated in the thymus, a process known as thymopoiesis. Importantly, thymopoiesis is not a cell-autonomous process. In this regard, the thymus holds an epithelial framework composed by cortical (cTEC) and medullary (mTEC) thymic epithelial cells that educate each step of the differentiation of T-cell progenitors into functionally competent and self-tolerant T cells. Despite their non-redundant roles in the thymopoietic process and distinct anatomical position within the thymus, cTECs and mTECs arise from the same bipotent thymic epithelial progenitor cells (TEPCs). Bipotent TEPCs are the main accountable for the patterning of the thymus into cortical and medullary domains during embryogenesis and renew the thymic epithelium throughout ontogeny. Yet, the nature and kinetics of embryonic and postnatal and adult TEPCs are poorly understood. Through the establishment of clonogenic assays that selectively support the growth and expansion of epithelial stem cells, our group has reported that mouse TECs with clonogenic potential preferentially reside in the cTEC compartment at a postnatal stage. Interestingly, cells that emerged from these clonogenic TECs, referred to as clonoTECs, lacked cTEC/mTEC traits, but expressed epithelial stem cell markers. Furthermore, clonoTECs lose the expression of Forkhead box N1 (FoxN1), a transcription factor indispensable for the entry of TEPCs into the TEC differentiation program and their downstream maturation. Thus, we postulate that clonoTECs revert to a TEC progenitor stage due to a failure in activating and maintaining FoxN1 expression. In this thesis, by integrating TEC clonogenic assays and a conditional FoxN1 knock-in mouse model (iFoxN1), we demonstrated that enforced constitutive expression of FoxN1 in iFoxN1-derived clonoTECs, termed iclonoTECs, promoted their differentiation into the cTEC lineage. Although this was not transversal to all iclonoTEC, depriving iclonoTECs of hydrocortisone, cholera toxin and epidermal growth factor, commonly employed to improve the colony forming efficiency and expansion of epithelial stem cells, augmented the representation of reprogrammed iclonoTECs by selectively impairing the homeostasis of non-reprogrammed iclonoTECs. Additionally, iclonoTEC reprogramming was further potentiated through the parallel activation of the Wnt, but not FGF, BMP or RA signalling pathways. Lastly, we illustrated the functional competence of reprogrammed clonoTECs in promoting early stages of T-cell development in vitro, namely progression of double negative thymocytes to the double positive stage. Collectively, our findings demonstrate that FoxN1-induced clonoTECs could represent a reliable platform to promote future studies on the early events of TEC differentiation and T-cell development.
O desenvolvimento de células T, mediadores essenciais de respostas imunes adaptativas contra agentes infeciosos e células tumorais, é orquestrado no timo. Este processo, vulgarmente referido como timopoiese, não é uma propriedade autónoma. Neste sentido, o timo possui uma matriz composta por células epiteliais do cortéx (cTECs) e medula (mTECs) que educam cada estágio da diferenciação de progenitores hematopoiéticos em células T funcionais e auto-tolerantes. Apesar de possuírem papeis distintos no processo timopoiético e da diferente localização anatómica dentro do timo, tanto cTECs como mTECs derivam dos mesmos progenitores bipotentes de células epiteliais do timo (TEPCs). Estes progenitores são os principais responsáveis pela organização do timo em domínios corticais e medulares, e rejuvenescem o epitélio tímico ao longo da ontogenia. Contudo, a identidade de e dinâmica de TEPCs no estágio de desenvolvimento embrionário e adulto estão pouco caraterizadas. Através de ensaios clonogénicos que suportam a expansão de células epiteliais estaminais, o nosso grupo descreveu recentemente que TECs com propriedades clonogénicas residem preferencialmente no córtex tímico durante o desenvolvimento pós-natal. Células que emergiram de TECs com potencial clonogénico, denominadas de clonoTECs, eram desprovidas de características que definem cTECs e mTECs, mas expressavam marcadores de células estaminais epiteliais. Além disso, clonoTECs perderam progressivamente a expressão da proteína Forkhead box N1 (FoxN1), um fator de transcrição indispensável para a diferenciação de TECs a partir de TEPCs. Consequentemente, hipotetizámos que clonoTECs reverteram a um estágio de desenvolvimento primordial devido a uma incapacidade de ativar e/ou manter a expressão de FoxN1.Nesta tese, através da combinação de ensaios clonogénicos com um modelo transgénico condicional de murganho para o FoxN1, nós demonstrámos que forçar a expressão de FoxN1 em clonoTECs derivadas do iFoxN1 (iclonoTECs) promoveu a sua entrada num programa de diferenciação enviesado para a linhagem cortical. Apesar de isto não ter sido transversal a todas as iclonoTECs, a remoção de hidrocortisona, da toxina da cólera e do fator de crescimento epitelial, comummente suplementados para amplificar a eficiência de formação de colónias e a expansão de células epiteliais estaminais, aumentou a representatividade de iclonoTECs reprogramadas ao inibir seletivamente a homeostase de iclonoTECs não reprogramadas. Adicionalmente, a reprogramação de iclonoTECs foi potenciada através da ativação paralela de vias de sinalização do Wnt, mas não FGF, BMP ou RA. Por último, ilustrámos a competência de clonoTECs diferenciadas em promover eventos iniciais do desenvolvimento de células T in vitro, nomeadamente a progressão de timócitos duplos negativos para o estágio duplo positivo. Coletivamente, nós estabelecemos clonoTECs induzidas com FoxN1 como uma plataforma que poderá permitir realizar futuros estudos nos eventos iniciais da diferenciação de TECs e do desenvolvimento de células T.
Outro - This work was supported by a starting grant (637843) from the European Research Council (ERC) and by Portuguese funds through FCT—Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior (projects PTDC/MED-IMU/29129/2017 and PTDC/MED-IMU/1416/2020).