Like with many cancers, a single ovarian tumor can display remarkable diversity in genetics, epigenetics, expression profiles, microenvironment and cell differentiation and plasticity. This so-called intratumoral heterogeneity (ITH) is thought to greatly increase mortality by enabling tumors to adapt quickly to therapy, metastasize, and recur, thus the study of ITH holds great clinical significance. Clonal evolution and cancer stem cell (CSC) theory are two models for the initiation and propagation of a tumor, which offer differing views on the way that ITH is developed and maintained. In the clonal evolution model, cancer arises from a single cell and, through genetic instability, proliferates into a diverse population of daughter cells, which develop additional mutations and undergo Darwinian selection under the influence of the tumor microenvironment. Each cell of the clonal evolution model may be capable of initiating a tumor independently. In CSC theory, cancer arises from the transformation of a stem cell that has the capacity to self-renew and differentiate into a diverse population of daughter cells. Each cell is NOT capable of tumorigenesis as most are terminally differentiated and do not harbor self-renewing capabilities. According to CSC theory, small, rare subpopulations of CSCs persist throughout chemotherapy and are responsible for repopulating the heterogeneous tumor post-treatment. The hypothesis that CSCs may play a role in ovarian cancer progression is the subject of this thesis. Many studies have detected the presence of stem cell markers and dysregulated stem cell signaling pathways in ovarian cancer, but doubts remain as to the existence of ovarian CSCs; critics have pointed out inherent flaws in experimental designs meant to identify and characterize CSCs. For example, the presence of cancer cells which express the stem cell marker CD133 has been correlated to both positive and negative impacts on prognosis. Further challenging the study of ovarian CSCs is the lack of consensus on the true cell of origin for ovarian cancer - whether it be from the fallopian tube epithelium or ovarian surface epithelium, or elsewhere in the peritoneal cavity - this will have important implications for the identification and characterization of tumorigenic ovarian CSCs. Advocates of clonal evolution theory have put forth incredible effort to reveal the extent of inter and intra-tumoral heterogeneity in ovarian cancer, and from these data there has arisen a general consensus that cancer cell populations do evolve in a step-wise fashion, accumulating additional mutations over time. The involvement of cancer stem cells in this progression and how exactly they fit in (as a cell of origin or arising from genetic mutations), as well as their significance for different cancer types, is a question worth answering. Despite the challenges facing the study of ovarian CSCs, the clinical impact of cells with stem-like properties has been repeatedly demonstrated, especially with regard to metastatic processes and chemoresistance. Moreover, new drugs which target stem cell pathways have proven effective in the treatment of ovarian cancer. The existence of a rare subset of cells that have enhanced tumor-initiating properties is apparent in ovarian cancer, and more work is needed to characterize the unique identifiers and behavior of these cells in vivo. Future experiments involving lineage tracing promise to deepen our understanding of the nature of ovarian CSCs and address whether normal stem cells might serve as the cell of origin.

O avanço da ciência regenerativa está se comprometendo com a busca de novas opções terapêuticas no combate as diversas doenças e disfunções orgânicas. Nessas avançadas linhas de pesquisas, as células-tronco são um símbolo dessa evolução. A variedade da aplicação deste novo e experimental método de tratamento está sendo utilizado, pela bioengenharia, para reparar tecidos e órgãos lesados. Defeitos ósseos extensos ocorrem após diversos tipos de injúrias ao esqueleto facial, como traumas faciais, ressecções por lesões agressivas e malformações congênitas. Essas sequelas são tratadas, preferencialmente, através da reconstrução utilizando enxertos ósseos de origem autógena. Entretanto, às desvantagens proporcionadas pela obtenção do tecido ósseo autógeno, lançaram um dos maiores desafios, da bioengenharia que é a busca pelo aprimoramento dos substitutos ósseos. Nesse caminho do aprimoramento dos biomateriais a adição de células-tronco mesenquimais representam a possibilidade da criação de um sinergismo, entre as células e o arcabouço, para otimizar a osteogênese. Nessa linha, esse estudo propõe-se a avaliar o reparo ósseo em estudo experimental, em modelo animal, através do tratamento de defeito ósseos criados em fêmures de ratos. A amostra dessa pesquisa foi composta de 96 ratos albinos da espécie Rattus novergicus albinus, linhagem SHR (Spontaneously Hypertensive Rats), isogênicos. Os animais foram divididos aleatoriamente em quatro grupos de acordo com o tipo de tratamento (Grupo I: α – TCP + ADSCs; Grupo II: α – TCP + ADSCs/ENDO; Grupo III: α – TCP; Grupo IV). A cultura de células tronco tiveram como tecido de origem o tecido adiposo da região abdominal e as células endoteliais formam coletadas da medula óssea. A peças foram avaliadas em 03 períodos de tempo diferentes (07, 14 e 21 dias). A histomorfometria avaliou a área de neoformação óssea dos defeitos bem como a imunohistoquímica com marcação para a proteína VEGF avaliou a eficácia da adição das células endoteliais. Os resultados demonstraram, através dos testes estatísticos que houve uma diferença estatisticamente significante, no reparo ósseo, favorecendo os tratamentos dos defeitos que utilizaram a terapia celular e a vascularização foi também otimizada no grupo que foi tratado com a adição das células endoteliais. Dessa forma conclui-se que nesse modelo de estudo a utilização das ADSCS foram capazes de otimizar o reparo ósseo. / The regenerative medicine has been searching for a new therapeutic options to manage diseases and also organic dysfunctions. The advanced research fields, has enrolled the stem cells to achieve the upgrade in the new treatment objectives. The bioengineering is application of this new and experimental, treatment method to repair damaged tissues and organs using the stem cells, includes the possibility to acelerating and improving the bone repair process. The autogenous bone graft has been considered the gold standart graft material to reconstruction the bone defects, fundamentally because of the osteogenic potential. However, the harvest disadvantages autogenous bone tissue leads to the search for bone substitutes improvement. In this field a promising alternative has been proposed by tissue engineering. The totipotent cells, also called mesenchymal stem cells, which has the cellular plasticity ability, is associated with biomaterials, creating a synergy, between these cells and the scaffold, to optimize the osteogenesis. The tissue engineering application to tissue repair has been extensively researched with the aim of proposing more reliable and more efficient clinical methods. Although the effects of the use of adult stem cells are well known in bone marrow transplants, in some areas, such as bone repair, there is still lack of scientific data. This research was conducted, in animal model, to assessment bone repair in created femural bone defects treated with mesenchymal stem cells. 96 animals (Rattus novergicus albinus - Spontaneously Hypertensive Rats) were randomly divided into four groups (Group I: α - TCP + ADSCs, Group II: α - TCP + ADSCs/Endo, and Group III: α - TCP; (07, 14 and 21 days). The histological sections were stained in H&E and the histomorphometry was used to evaluated the new bone formation area in the defects and also the immunohistochemical expression of VEGF was analysed. Our results suggest that the combination of ADSCs and the scaffold was able to enhance the bone repair in this study model.

L’amaurose congénitale de Leber (ACL) est une maladie génétique touchant la rétine. Les premiers symptômes apparaissent dès les premiers mois de la vie et mènent en quelques années à la cécité. A ce jour, des mutations dans 18 gènes ont été associées à la maladie. Cette hétérogénéité génétique rend difficile l’étude des mécanismes conduisant aux différents symptômes. Les modèles animaux utilisés en laboratoire, notamment les rongeurs, permettent d’étudier certains de ces mécanismes mais présentent des limites liées à l’espèce. Les cellules souches pluripotentes induites (iPSCs), qui proviennent de la reprogrammation de cellules somatiques issues de patients, constituent un nouvel outil pour étudier une maladie génétique dans un contexte humain naturel. Elles permettent d’obtenir tous les phénotypes cellulaires désirés sans limite quantitative ce qui ouvre la porte à des approches d’analyse à large échelle telle que l’analyse transcriptomique qui vise à explorer de manière systématique la modulation des gènes dans une maladie. L’objectif de mon projet de recherche a été de développer un modèle cellulaire humain naturellement porteur de l’ACL. Après avoir produit les iPSCs à partir de fibroblastes de patients, mes travaux ont consisté à les différencier en populations cellulaires homogènes et facilement amplifiables, les cellules souches neurales et les cellules de l’épithélium pigmentaire rétinien. Ces populations ont servi à mener des analyses transcriptomiques à large échelle qui ont permis d’identifier plusieurs gènes candidats, potentiellement impliqués dans le développement de la pathologie, parmi lesquels GSTT1 qui pourrait avoir un rôle dans le stress oxydatif. / Leber congenital amaurosis (LCA) is a genetic disease affecting the retina. The first symptoms appear in the first months of life and lead in few years to blindness. To date, mutations in 18 genes have been associated with the disease. This genetic heterogeneity makes it difficult to study mechanisms leading to different symptoms. Animal models, including rodents, are used to study some of these mechanisms but have limitations mostly related to the species. The induced pluripotent stem cells (iPSCs), which are reprogrammed somatic cells of patients, constitute a new tool for studying genetic diseases in a natural human context. They achieve all desired cell phenotypes without quantitative limits which opens the door to large-scale analysis approaches such as transcriptomic analysis that aims to systematically explore the modulation of genes in a disease. The aim of my research project was to develop a human cell model naturally carries the LCA. After producing the iPSCs from fibroblasts of patients, my work had consisted to differentiate them into homogeneous and easily amplifiable cell populations, neural stem cells and retinal pigment epithelial cells. These populations have served to conduct large-scale transcriptomic analyzes which have identified several candidate genes potentially involved in the development of the disease, including GSTT1 which might have a role in oxidative stress.

Les leucémies aiguës myéloblastiques (LAM) sont un groupe hétérogène de pathologies malignes représentant environ 70% des leucémies aiguës. Il existe une prolifération, dans le cadre des LAM de cellules immatures appartenant à la lignée myéloïde appelées myéloblastes ou communément blastes. Les traitements actuels reposent essentiellement sur la chimiothérapie antimitotique. L’homéostasie du fer est une cible dans le traitement des LAM en induisant la différentiation des blastes. Le mécanisme implique la modulation des ROS. Leur action est synergique avec celle de la Vitamine D (VD) au travers de l’activation de la voie des MAPK. Cette association a été utilisée chez plusieurs patients avec succès permettant un doublement de leur espérance de vie. Nous avons ensuite montré que l’expression du récepteur expression vitamine D (VD) est altérée dans les états indifférenciés / immatures sous-types de LAM et que la diminution de l'expression du VDR et de ces gènes cibles est corrélée à un mauvais pronostic chez les patients. Le mécanisme moléculaire entraînant le blocage de l'expression VDR implique la méthylation de son promoteur. Les souris invalidées pour le VDR ont une expansion du compartiment des cellules souches hématopoïétiques demeurant à un état quiescent ainsi qu’une diminution des niveaux du stress oxydatif en leur sein. En outre, la transformation maligne des cellules déficientes en VDR a abouti à une différenciation myéloïde limitée, à l'augmentation du nombre de progéniteurs hématopoïétiques précoces et ces cellules présentaient un potentiel d'auto-renouvellement accru et étaient résistantes aux inhibiteurs de la méthyltransférase et à la chimiothérapie. Enfin, l'induction de l'expression du VDR dans les modèles de LAM par un traitement combinant des agents de déméthylation et les agonistes de VDR a permis de diminuer la séminalité, de promouvoir la différenciation cellulaire, de bloquer la croissance tumorale et de restaurer la sensibilité à la chimiothérapie. Par conséquent, nous proposons que le VDR est un gène maître contrôlant la séminalité et la prolifération / différenciation cellulaire des cellules souches hématopoïétiques normales et leucémiques. Ainsi, la combinaison d'agents déméthylants et d’agonistes de VDR pourrait à l’avenir être proposée en thérapeutique pour traiter les LAM. / Acute myeloid leukemia (AML) is a heterogeneous group of malignancies representing approximately 70% of acute leukemias. There is a proliferation of immature cells belonging to the myeloid lineage commonly called myeloblasts or blasts. Current treatments are mainly based on antimitotic chemotherapy. Iron homeostasis is a target for the treatment of AML blasts inducing cell differentiation. The mechanism involves the modulation of ROS. Their action is synergistic with that of Vitamin D (VD) through the activation of MAPK. This association has been used successfully in several patients for a doubling of life expectancy. Then, we show that Vitamin D receptor (VDR) expression was impaired in undifferentiated/immature AML subtypes and that decreased expression of VDR and VDR-targeted genes was correlated with a negative prognosis of patients. Molecular mechanism resulting in the blockade of VDR expression involved VDR promoter methylation. VDR-deficient mice showed an expansion of the hematopoietic stem cell compartment which presented an improved quiescent status and decreased ROS levels that have been shown to be involved in both AML differentiation and stem cells longevity. Moreover, malignant transformation of VDR-deficient cells resulted in limited myeloid differentiation, increased numbers of early hematopoietic progenitors and those cells presented an enhanced self-renewal potential and were resistant to DNA methyltransferase inhibitors and to chemotherapy. Finally, induction of VDR expression in AML models by combined treatment of demethylating agents and VDR agonists decreased stemness, promoted cell differentiation, blocked tumor propagation and restored sensitivity to chemotherapy. Therefore, we propose that VDR is a master gene controlling stemness and proliferation/cell differentiation of normal hematopoietic stem cells and leukemic cells. Thus, combination of demethylation agents and VDR agonists may be used therapeutically to treat AML.

Orientador: Andrigo Barboza de Nardi / Banca: Jorge Luiz Costa Castro / Banca: Wilson Gómez Manrique / Resumo: Tradicionalmente um enxerto é realizado em um leito receptor coberto por tecido de granulação saudável, e na atualidade, as células tronco mesenquimais representam uma excelente alternativa para estimular a cicatrização de feridas agudas e crônicas. Sendo assim, este trabalho teve por objetivo avaliar os efeitos de células tronco mesenquimais xenógenas na cicatrização de auto-enxertos cutâneos em leito receptor sem tecido de granulação de coelhos. Foram utilizados 60 coelhos, divididos em três grupos de 20 pacientes. O primeiro grupo (GIL) e o segundo (GIV) receberam tratamento de 2 x106 células tronco, por vias intralesional e intravenosa respetivamente, enquanto o grupo controle (GC) recebeu apenas o auto-enxerto cutâneo. Na avaliação macroscópica, enxertos dos grupos GIL e GIV apresentaram melhor coloração comparado com o grupo controle, no entanto, na avaliação microscópica as variáveis inflamação, reepitelização, necrose e neovascularização não mostraram diferencia significativa entre os três grupos (p>0,05). Conclusão: a reepitelização dos auto-enxertos cutâneos em malha em leito receptor desprovido de tecido de granulação em coelhos (Oryctolagus cuniculus ) provavelmente não precisam de técnica adjuvante para estimular a cicatrização. A versatilidade da malha permite uma aderência estável ao leito receptor, e através das fendas criadas permite a eliminação da secreção inflamatória, evitando assim a presença de seroma e garantindo a nutrição e revascularização do enxer... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Traditionally, a graft is performed in a healthy granulation tissue bed, although nowadays, the mesenchymal stem cells represent an excellent alternative to stimulate wound healing in a recipient bed without a healthy granulation tissue bed. Thus, this study aimed to evaluate the effects of mesenchymal stem cells xenogeneic at the healing of skin autografts in a recipient bed without granulation tissue in rabbits. A total of 60 rabbits were divided into three groups of 20 patients. The first (GIL) and the second group (GIV) were treated with 2x106 intralesional and intravenous stem cells respectively; meanwhile the control group (CG) received only skin autograft. At the macroscopic examination, the grafts GIL and GIV groups showed better color than the control group, however, microscopic evaluation of the variables: inflammation, re-epithelization, necrosis and angiogenese showed no significant difference among the three groups (p>0,05). Conclusion: reepithelialization of meshed skin autografts from recipient bed without granulation tissue in rabbits (Oryctolagus cuniculus) probably does not need an adjuvant technique to stimulate wound healing. The versatility of the mesh allows a stable adherence to the recipient bed, and through the created slits allows the evacuation of inflammatory secretion, avoiding the presence of seroma and providing nutrition and graft revascularization / Mestre

Orientador: José Luiz Laus / Banca: Paola Castro Moraes / Banca: Luís Gustavo Gosuen Gonçalves Dias / Banca: João Antônio Tadeu Pigatto / Banca: Cláudia Valéria Seullner Brandão / Resumo: O omento, tecido gorduroso rico em células tronco mesenquimais, expressa fatores de crescimento, que atuam na regeneração tecidual, tornando-o atrativo para utilização em medicina regenerativa. Não há relatos, todavia, quanto à investigação sobre células do omento na cirurgia regenerativa em oftalmologia. Avaliaram-se os efeitos clínicos e à microscópia do omento com membrana amniótica na reparação da córnea de coelhos submetidos a ceratectomia. Duas modalidades foram avaliadas. Na primeira, o omento foi coberto com membrana amniótica imediatamente à sua implantação na córnea (grupo OM); na segunda, a membrana amniótica foi colonizada por células progenitoras extraídas do omento e transplantadas para a córnea (grupo OMC). Os efeitos clínicos foram avaliados a partir de dados coletados por até 60 dias de pós-operatório. A incidência e a prevalência de quemose foram maiores no grupo OM (p<0,05). O grupo OMC mostrou mais incidência de blefarospasmo (p<0,001). À microscopia de luz, encontrou-se reparação corneal gradativa em ambos os grupos, com reepitelização ao 100 de pós-operatótio. Aos 60 dias da avaliação, todas as córneas estavam reparadas, entretanto, no grupo OMC constatou-se vascularização. Proliferação celular acentuada foi observada com o ki-67, nos estágios finais do processo de reparação de córneas que receberam MA contendo células do omento (grupo OMC). Somente no grupo OMC, a reparação corneal sem tranparência foi alcançada / Abstract: The omentum, a fatty tissue rich in mesenchymal stem cells, expresses growth factors that act in tissue regeneration, making it attractive for use in regenerative medicine. To our knowledge, reports that investigate the use of omentum cells in ophthalmology have yet to be published. The purpose of this study was to assess the clinical and microscopic effects of omentum associated with amniotic membrane on the repair of rabbit cornea submitted to keratectomy. Two methods were evaluated; in the first, the omentum was covered with amniotic membrane immediately following its implantation on the cornea (OM group), while in the second, the amniotic membrane was colonized by omental progenitor cells and transplanted to the cornea (OMC group). Clinical parameters were evaluated from data collected up to 60 days postoperatively. The incidence and prevalence of chemosis were higher in the OM group (p<0.05). The OMC group showed greater incidence of blepharospasm (p<0.001). Under light microscopy, the absence of epithelium in the lesioned area and stromal disorganization were observed in the early postoperative period. After 60 days of evaluation, all the corneas were repaired. Intense cell proliferation was detected with Ki-67, in the final stages of the repair process, where amniotic membrane containing omental progenitor cells was transplanted to the corneas. The use of omentum and its cells, in association with amniotic membrane, favored the repair of the cornea and did not induce complications / Doutor

Specific regions within the adult mammalian brain maintain the ability to generate neurons. The largest of these, the ventricular-subventricular zone (V-SVZ), comprises the entire lateral wall of the lateral ventricles. Here, a subset of glial fibrillary acid protein (GFAP)-positive astrocytes (B cells) gives rise to neurons and oligodendrocytes throughout life. This process of neurogenesis involves quiescent B cells becoming proliferative (epidermal growth factor receptor (EGFR)-positive) and giving rise to neuroblasts via transit amplifying precursors. The neuroblasts then migrate through the rostral migratory stream (RMS) to the olfactory bulbs (OBs), where they mature into neurons. Studying the stem cells in the V-SVZ has been hindered by the shortage of molecular markers to selectively target them. Using microarray and qPCR analysis of putative quiescent neural stem cells we determined that they were enriched for PDGFRβ mRNA. We used immunostaining to determine the in vivo identity of PDGFRβ+ cells, and discovered that only GFAP+ cells within the V-SVZ stem cell lineage express PDGFRβ. Moreover, these PDGFRβ+ B cells contact the ventricle at the center of ependymal pinwheel structures and the vast majority of them are EGFR-. Importantly, the V-SVZ/RMS/OBcore axis was highly enriched for PDGFRβ expression compared with other brain regions. Detailed morphological analyses of PDGFRβ+ B cells revealed primary cilia at their apical process in contact with the ventricle and long radial processes contacting blood vessels deep within the V-SVZ, both of which are characteristics of adult neural stem cells. When PDGFRβ+ cells were lineage traced in vivo they formed olfactory bulb neurons. Using fluorescence-activated cell sorting (FACS) to purify PDGFRβ+ astrocytes we discovered this receptor is expressed by all adult V-SVZ neural stem cells, including a novel population of EGFR+ PDGFRβ+ cells which correspond to the activated neural stem cells. RNA-sequencing analysis of the purified populations revealed that PDGFRβ+ EGFR+ cells possess a transcriptional profile intermediate between quiescent neural stem cells and actively proliferating GFAP- progenitor cells. Finally, when PDGFRβ is deleted in adult GFAP+ NSCs we observe a decrease in EGFR+ and Dcx+ progenitor cells, together with an increase in quiescent GFAP+ astrocytes. A larger proportion of these mutant cells come in contact with the ventricular lumen, suggesting that PDGFRβ is required for V-SVZ astrocytes to act as stem cells, possibly by mediating interactions with their niche. Taken together, these data identify PDGFRβ as a novel marker for adult V-SVZ neural stem cells that is an important regulator of their stem cell capabilities.

Quiescent neural stem cells support continuous, lifelong neurogenesis in specific regions of the adult mammalian brain. The largest adult neurogenic region is the ventricular-subventricular zone (V-SVZ), which lines the entire lateral wall of the lateral ventricles. Quiescent neural stem cells (qNSCs) enter the cell cycle (activate) and give rise to new neurons during homeostasis and regeneration, suggesting they can potentially be harnessed for regenerating the brain after neurodegenerative disease, stroke, and injury. Defining the signals that regulate NSC quiescence and activation is essential to unlock their potential for regenerative medicine. NSCs residing in specific regions of the V-SVZ give rise to distinct subtypes of olfactory bulb interneurons. It is unknown whether quiescence-regulating signals map onto the regional heterogeneity of NSCs, and might thereby underlie the production of distinct interneuron subtypes. A major limitation to our understanding of the regulation of NSC quiescence has been the lack of specific markers to identify qNSCs, and prospectively purify them from their in vivo niche. Using a novel fluorescence-activated cell sorting (FACS) strategy that allows the purification of qNSCs from the adult mouse V-SVZ niche for the first time, I performed in vitro screens for quiescence-regulating signals. Unexpectedly, neurotransmitters emerged as the main class of qNSC-activating signals, including dopamine, GABA, serotonin, acetylcholine, and opioids. Local and long-range neurons that use these neurotransmitters innervate the V-SVZ in unique regional patterns, suggesting these signals map onto the regional heterogeneity of NSCs. Consistent with this hypothesis, infusions of cholinergic agonist and antagonists into the lateral ventricle resulted in regional changes in NSC proliferation. Moreover, cholinergic antagonists blocked the activation of qNSCs during regeneration, providing evidence that neurotransmitter signaling activates qNSCs in vivo. I then showed that hypothalamic Pomc-expressing neurons innervate the anterior-ventral V-SVZ and promote the activation of Nkx2.1+ qNSCs. Ablation of Pomc+ neurons resulted in decreased proliferation of NSCs in the anterior-ventral, but not anterior-dorsal, V-SVZ. Moreover, both the activity of Pomc+ neurons, and the proliferation of Nkx2.1+ NSCs in the anterior-ventral V-SVZ decreased in fasted animals, suggesting that hunger and satiety states regulation the generation of a single olfactory bulb interneuron subtype. Indeed, ablation of Pomc+ neurons resulted in a loss of the subtype of olfactory bulb interneuron that is generated by Nkx2.1+ NSCs. Together, my findings suggest that both local and long-range neurons regionally innervate the V-SVZ and mediate neural stem cell activation from the quiescent state.

Regulation of hematopoiesis through the finite control of hematopoietic stem cell (HSC) quiescence and proliferation is critical to the health of the organism since disturbances in blood production can lead to clinical malignancies such as anemia or leukemia. Therefore, elucidating the processes that govern HSCs is vital to advance our understanding of hematological diseases. Interestingly, HSCs can be regulated through a variety of ways. Extrinsic cues from the niche provide signals that govern HSC quiescence, proliferation, self-renewal, and differentiation. These external signals are converted to internal messages through the use of signal transduction pathways that relay information from the cytoplasm to the nucleus. While many pathways contribute to HSC regulation, the PI3K/AKT/mTOR-pathway is especially pertinent because it has been implicated in cell survival, metabolism, proliferation, and death. Many groups have identified key players within PI3K/AKT/mTOR-signaling that regulate HSC quiescence; however, these studies are hindered by the redundancy of multiple isoforms and compensatory signaling mechanisms by other members within the pathway. PDK1 is considered to be a master regulator of PI3K-signaling because it is directly activated by PI3Ks and can govern activity of a variety of substrates within the PI3K-pathway. Because of this, it is an excellent candidate to fully delineate how PDK1-mediated PI3K-signaling functions to maintain HSC quiescence. In the current study, conditional deletion of PDK1 in HSCs was achieved through the use of a hematopoietic specific Vav-Cre line. The loss of PDK1 in HSCs led to reduced numbers and an inability to provide radioprotection in primary BMTs. Furthermore, PDK1-deficient HSCs exhibit impaired quiescence and increased cycling. Strikingly, PDK1-mutant HSCs have markedly high levels of reactive oxygen species (ROS), which led to increased proliferation, DNA damage, and apoptosis in progenitor cells. Administration of a ROS scavenger, N-acetyl cysteine (NAC) partially rescued the increased proliferation and differentiation of phenotype Pdk1Hem-KO cells both in vitro and in vivo, suggesting that abnormal HSC activity in PDK1-deficient cells was in part due to increased ROS. Furthermore, mechanistic studies identified a remarkable decrease in the levels of nuclear HIF1α in HSCs. Immunoblots and phosflow studies demonstrated reduced activation of p-p70S6K, a well defined positive regulator, and increased GSK3β, a key negative regulator of the HIF1α protein. These data suggested that ROS levels are unrestrained since HIF1α is not present in Pdk1Hem-KO HSCs to activate transcription of genes that moderate oxidative stress. In addition, Pdk1Hem-KO HSCs also show increased levels of Hif3α and IPAS mRNA, which are believed to inhibit the transcriptional activation of HIF1α. In essence, the results from these experiments are the first to implicate PDK1 as a critical regulator of HSC quiescence and as a moderator of PI3K-signaling to alleviate oxidative stress. In addition, this study is the first to suggest that HIF3α and IPAS may play a role in HSCs.

Primary cilia are sensory organelles that facilitate early skeletal development, as well as maintenance and adaptation of bone later in life. These solitary, immotile organelles are known to be involved in cell differentiation, proliferation, and mechanotransduction, a process by which cells sense and covert external physical stimuli into intracellular biochemical signals. Bone is a metabolically active tissue that continuously recruits osteogenic precursors and relies on osteocytes, the sensory cells of bone, to coordinate skeletal maintenance. Overall bone quality is dependent on the integrity of the initial structure formed, as well as this organ’s ability to adapt to physical loads. Proper differentiation and controlled proliferation of osteogenic progenitors are critical to the initial formation of the skeleton, while osteocyte mechanotransduction is essential for adaptation of developed bone. These phenomena rely on primary cilia, but little is known about the origin of osteogenic precursors and the ciliary mechanisms that promote osteogenesis. In this thesis, we first characterize an osteochondroprogenitor (OCP) population that rapidly and extensively populates skeletal tissues during juvenile skeletal development (Chapter 2). We also demonstrate that the primary cilium is critical for these cells to differentiate and contribute to skeletogenesis. We then show this OCP population is required for adult bone adaptation and is mechanoresponsive (Chapter 3). Again, we demonstrate that primary cilia are necessary for these OCPs to sense physical stimuli and differentiate into active bone-forming cells. Finally, we identify a novel link between ciliary calcium and cAMP dynamics in the osteocyte primary cilium (Chapter 4). Specifically, we show that a calcium channel (TRPV4) and adenylyl cyclases, which produce cAMP, bind calcium to mediate calcium entry and cAMP production, respectively, and these phenomena are critical to fluid flow-induced osteogenesis. Collectively, our results demonstrate that an easily extracted progenitor population is pre-programmed towards an osteogenic fate and extensively contributes to bone generation through primary cilium-mediated mechanisms at multiple stages of life. Furthermore, we identified ciliary proteins that are potentially unique to the osteocyte and can be manipulated to encourage osteogenesis by tuning calcium/ cAMP dynamics. For these reasons, we propose that this OCP population and their primary cilia, as well as osteocyte ciliary proteins that coordinate calcium/ cAMP dynamics, are attractive therapeutic targets to encourage bone regeneration.