Muitos subtipos de receptores são ativados pelo mesmo ligante, mas estão acoplados a diferentes mensageiros secundários podendo produzir sinalização divergente em uma célula, enquanto receptores ativados por diferentes ligantes, mas que compartilham o mesmo mensageiro secundário, podem produzir sinalização convergente. Para examinar as bases mecanísticas que influenciam a proliferação e a diferenciação celular determinamos as funções de liberação intracelular de Ca2+ e a excitabilidade celular mediada pelos receptores purinérgicos e colinérgicos utilizando imageamento de cálcio por microscopia confocal. Para tanto, caracterizamos a participação dos subtipos P2X1-7 e P2Y1,2,4,6 de receptores purinérgicos aos níveis dos transcritos de mRNA e de expressão protéica, assim como pela atividade de induzir os transientes de [Ca2+]i, aumento na concentração livre de cálcio intracelular, durante a diferenciação neuronal de células P19 de carcinoma embrionário, que foram utilizadas como modelo in vitro para o desenvolvimento neuronal precoce. Em células embriônicas os receptores P2Y1,2, P2X4 ou os heteromultímeros de P2X com farmacologia semelhante ao do receptor P2X4 foram os responsáveis pelos transientes de [Ca2+]i induzidos pelo ATP e seus análogos. Ao término da diferenciação neuronal, os receptores P2Y2,6 e P2X2 foram os principais mediadores das respostas de [Ca2+]i. Obtivemos evidências do envolvimento destes receptores na indução da proliferação tanto de células embriônicas como de progenitores neuronais, por ensaios de incorporação de BrdU, e da indução da diferenciação neuronal das células progenitoras, na presença de vários agonistas e antagonistas de receptores purinérgicos. Como resultado desses estudos, a regulação da proliferação e diferenciação celular foi principalmente devida aos subtipos de receptores P2Y1 e P2Y2, já que estes efeitos foram eliminados após a depleção dos depósitos intracelulares de cálcio e pela demonstração de que estes eram os possíveis receptores funcionais. Entre os receptores colinérgicos, fornecemos evidências para a expressão de receptores nicotínicos (nAChRs) e muscarínicos (mAChRs) funcionais durante a diferenciação de células P19. Detectamos a expressão e a atividade dos subtipos de nAChRs formados pelos subtipos α2-α7, β2, β4 e M1-M3 e M5 de mAChRs durante a diferenciação neuronal. As respostas de [Ca2+]i induzidas pelos agonistas dos nAChRs foram observadas em células P19 embriônicas e neuronais. As respostas de [Ca2+]i mediadas pelos receptores muscarínicos, em níveis próximos aos basais em células embriônicas, aumentaram durante a diferenciação. As elevações na [Ca2+]i induzidas pelos nAChRs em células indiferenciadas foram devidas ao influxo de Ca2+ do meio extracelular. Em células diferenciadas em neurônios, os aumentos de transientes de [Ca2+]i induzidos pelos nAChRs foram parcialmente inibidas após o pré-tratamento das células com a rianodina, enquanto as respostas de [Ca2+]i mediadas pelos mAChR não foram afetadas na presença deste composto, sugerindo uma contribuição da liberação de Ca2+ a partir dos depósitos de Ca2+ sensíveis à rianodina para as elevações mediadas pelos nAChRs. Demonstramos também, que a nicotina, agindo através dos nAChRs, inibiu a proliferação em células embriônicas, porém, a induziu em células progenitoras neuronais pela mobilização de Ca2+ dos depósitos intracelulares. A muscarina induziu em células embriônicas o aumento na proliferação via mAChRs acoplados às proteínas Gαq/11, e promoveu a diferenciação neuronal via M2 mAChRs em células precursoras neuronais. Estes dados sugeriram que a acetilcolina agindo via mAChR funciona como um mitógeno que ativa as proteínas quinases de trifosfato de inositol (IP3) e que poderia estar envolvida na síntese de DNA durante os estágios iniciais da neurogênese. Nós ainda provemos evidências que as oscilações de [Ca2+]i são características para cada estágio da diferenciação e são iniciadas pela liberação de Ca2+ mediada pelo IP3. As análises da determinação do fenótipo neuronal na presença de vários inibidores da transdução do sinal induzido pelo cálcio residem na liberação de Ca2+ induzida pelo IP3 é necessária para o progresso da diferenciação neuronal. Assim, os sinais espontâneos de [Ca2+]i são propriedades intrínsecas das células em diferenciação. A modulação de sua freqüência e amplitudes especifica a aquisição de um fenótipo de célula neuronal. / Various receptors subtypes are activated by the same ligand although coupled to different second messengers. These receptors act either by inducing divergent signaling in one cell, whereas in another cell different receptors may stimulate the very same pathways producing convergent signaling. We have characterized intracellular Ca2+- release and -influx mediated by purinergic and cholinergic receptors using calcium imaging by confocal microscopy to evaluate the mechanistic bases which influence cell proliferation and differentiation We have characterized the participation of purinergic subtypes P2X1-7 and P2Y1,2,4,6 receptor subtypes at mRNA transcription and protein expression levels as well as receptor-induced changes in free intracellular calcium concentration ([Ca2+]i) during differentiation of P19 embryonal carcinoma cells as an in vitro model for early neuronal development. The participation of individual P2X and P2Y receptor subtypes in the differentiation process was studied by employing different available purinergic receptor agonists and antagonists. In embryonic cells, P2Y1,2, P2X4 receptors, or P2X-heteromultimers with similar P2X4 pharmacology were responsible for ATP and ATP-analog-induced [Ca2+]i transients. Following completion of neuronal differentiation, P2Y2,6 receptors and P2X2 subtypes were the major mediators of the [Ca2+]i-response. Regulation of cell proliferation and differentiation of P19 embryonic and progenitor cells was mostly due to P2Y1 and P2Y2 receptor activation, as these effects were abolished following depletion of intracellular calcium stores, and they are probably the unique functional P2Y receptors at these stages of differentiation. We also provide evidence for expression of functional nicotinic (nAChRs) and muscarinic acetylcholine receptors (mAChRs) during neuronal differentiation of P19 cells. We have detected expression and activity of nAChRs formed by the subunits α2-α7, β2, β4, and M1-M3 and M5 mAChR subtypes along the differentiation process. Receptor response in terms of nicotinic agonist-evoked Ca2+ flux was observed in embryonic and neuronal-differentiated cells. However, mAChRs-induced calcium responses, merely present in undifferentiated P19 cells, increased during neuronal differentiation. The nAChR-induced [Ca2+]i response in undifferentiated cells was due to Ca2+ influx. However, in differentiated P19 neurons the nAChR-induced [Ca2+]i response was partially inhibited following pretreatment of the cells with ryanodine, while the mAChR-induced response remained unaffected, suggesting the contribution of Ca2+ release from ryanodine-sensitive stores to nAChR- but not mAChR-mediated Ca2+ responses. The presence of functional nAChRs in embryonic cells suggests that these receptors are involved in triggering Ca2+ waves during initial neuronal differentiation. In the present study we have also shown that nicotine, acting via nAChRs, inhibited proliferation in embryonic cells, but induced cell division of progenitor cells by Ca2+ mobilization from internal stores. Stimulation of progenitor cells by muscarine led to an increase in DNA synthesis mainly resulting from activation of Gαq/11-coupled mAChRs. Muscarine as well promoted differentiation of neural precursor cells by activation of M2 mAChRs subtypes. These data suggest that acetylcholine, acting via mAChRs, functions as a mitogen during early neurogenesis. We also provide evidence that oscillations of [Ca2+]i as characteristics for the respective stage of differentiation are initiated by triphosphate inositol (IP3)-mediated Ca2+-release. Neuronal cell fate determination analysis in the presence of various inhibitors of calcium-induced signal transduction underlined that IP3-mediated Ca2+-release is necessary for neuronal differentiation progress. Thus, spontaneous Ca2+-signals are an intrinsic property of differentiating neural precursor cells. Modulation of their frequency and amplitude is believed to direct the acquisition of a defined neuronal phenotype.

Introdução: O carcinoma de células renais metastático (CCRm) é um tumor altamente agressivo e resistente. Seu tratamento é baseado em terapia alvo molecular e citocinas. Avaliamos os efeitos antineoplásicos do Amblyomin-X, sobre CCR. Métodos: Avaliadas culturas de CCR RENCA e fibroblastos normais NIH/3T3 tratadas ou não com Amblyomin-X. Realizados ensaios de viabilidade celular por MTT e determinação, por citometria de fluxo, da proporção de células em apoptose/necrose; expressão da P-gp; Bad; Bax; Bcl-2; ciclina D1; caspase 3; Ki-67; p53; VEGFR1; citocromo c; análise das fase do ciclo celular; e atividade do proteassoma. Analisamos as populações celulares por microscopia eletrônica de varredura. Empregados testes T e One-way ANOVA para análise estatística. Resultados: O Amblyomin-X demonstrou citotoxicidade em células RENCA por indução de apoptose, diminuição de proliferação celular, inibição do proteassoma e modulação do ciclo celular em G0/G1. Em fibroblastos normais não houve citotoxicidade Conclusão: O Amblyomin-X apresentou efeito antineoplásico em CCR e não exerceu efeito citotóxico em células normais, demonstrando um possível potencial terapêutico no tratamento do CCRm. Estudos futuros deverão confirmar estes resultados. / Introduction: Metastatic renal cell carcinoma (mRCC) is a highly agressive and resistant tumour. Its treatment is based on targeted therapies and cytokines. We have evaluated the antineoplasic effects of Amblyomin-X on RCC. Methods: RCC (RENCA) and fibroblasts (NIH/3T3) cell cultures treated or not with Amblyomin-X were evaluated. MTT assay was performed to determine cell viability. Apoptosis/necrosis ratio; expression of P-gp; Bad; Bax; Bcl-2; cyclin D1; caspase-3; Ki-67; p53; VEGFR1; cytochrome c; cell cycle analysis and proteasome activity were obtained by flow cytometry. Cellular populations were analised by Scanning Electron Microscopy. Statistical analyses was performed using T-Tests and One-way ANOVA. Results: Amblyomin-X showed cytotoxic activity on RENCA tumor cells. It has induced apoptosis, decreased tumor cell proliferation, targeted the ubiquitinproteasome system and modulated genes related to cell cycle in G0/G1. There was no toxicity on fibroblasts. Conclusion: Amblyomin-X showed antineoplasic effects on RCC cells preserved normal fibroblast cells. There is a potential role of its therapeutic use in mRCC treatment. Future studies should confirm our initial results.

Células tronco hematopoéticas (CTH) de sangue de cordão umbilical (SCU) possuem grande potencial em terapia celular. Mesmo sendo bem caracterizadas quanto às suas propriedades funcionais e fenotípicas, a regulação da auto-renovação de CTH e os genes envolvidos são pouco conhecidos. Investigou-se através da curva de crescimento, ensaio clonogênico e citometria de fluxo, a expansão e diferenciação de CTH cultivadas sem e com suplementação dos mitógenos estradiol e LiCl. A expressão da subunidade da telomerase teve aumento significativo em todas as condições, bem como a expressão de Nanog e Oct4 relacionados a pluripotência e auto-renovação. Observou-se também a expressão de Nanog, Oct4, Sox2 e FoxD3 em células CD133, células CD3 de sangue periférico e células de colônias hematopoéticas. Concluiu-se que o meio sem suplementação já é suficiente na expansão de CTH, mantendo suas características, relacionadas à proliferação, auto-renovação e pluripotência celular. / Hematopoietic stem cells (HSC) from umbilical cord blood (UCB) have a great potencial for hematopoietic reconstitution. Although these stem cells have been well characterized by their functional and fenotipics properties, self-renewal regulation and genes involved are still unknown. Analyses of cell growth, clonogenic assay and flow cytometry revealed the expansion and differentiation of HSC grown in medium with or without suplementation of the mitogens estradiol and LiCl. Expression of the subunit of telomerase increased in all treatments. As well as the expression of Nanog and Oct4, related to plutipotency and self-renewal. Nanog, Oct4, Sox2 and FoxD3 expression was also high in CD133 cells, in CD3 cells from peripherical blood and in clonogenic assay derived cells. Conclusion: medium without the suplementation is sufficient for expansion of HSC, keeping their characteristcs, realted to proliferation, self-renewal and pluripotency.

Cette thèse est consacrée à l’élaboration et l’étude théorique de modèles de transport décrivant les dynamiques cellulaires et la communication intercellulaire dans les tissus épithéliaux. Nous nous intéressons d’abord à l’influence du transport de microARNs (miRNAs) sur la dynamique spatiotemporelle de réseaux de régulation génétique. Ces courtes séquences d’ARN régulent la synthèse des protéines en bloquant l’activité des ARN messagers et leur sécrétion via des vesicules extracellulaires en font des agents de communication intercellulaire. Différents modèles faisant intervenir des miRNAs extracellulaires ont été construits et étudiés numériquement. Les premiers sont des modèles génériques destinés à mettre en évidence l'effet d'une cellule ayant une production de miRNAs anormale sur l'expression génétique dans les cellules voisines. Nous abordons ensuite des modèles plus complexes et réalistes dans lesquels des oscillations (liées à des rythmes biologiques) et de la bistabilité (liée à une différenciation cellulaire) sont observées. Ces modèles permettent d’étudier des dynamiques de communication complexes observées en biologie, comme la synchronisation de cellules couplées ou la propagation d'un changement de phénotype. Nous mettons également en évidence le rôle de défauts, tels que des mutations génétiques ou encore des variations de densité cellulaire dans les tissus, sur ces phénomènes de propagation. La deuxième partie de la thèse est dédiée à la construction de modèles de réaction-diffusion dans lesquels la dynamique des cellules dépend de leur état interne. Sur base d’études expérimentales montrant l’influence de protéines et de miRNAs sur la mobilité et la prolifération des cellules, nous établissons un modèle multi-échelle dans lequel la dynamique intracellulaire et le mouvement des cellules interagissent. En effet, certaines protéines sont responsables de l’adhésion cellulaire ou régulent la vitesse de prolifération. Dans notre modèle, chaque cellule synthétise ces espèces d’intérêt et les processus cellulaires (migration, prolifération) dépendent de la concentration de ces espèces biochimiques. Ce modèle permet de reproduire des expériences de migration cellulaire et de prédire, notamment, l'influence d'E-cadherin, une protéine clé dans l'adhesion cellulaire, sur la dynamique de régénération d'un tissu. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

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La progression du cancer du sein repose principalement sur la signalisation des œstrogènes et de la progestérone, et les traitements modulant l’action des œstrogènes ont amélioré la survie des patientes atteintes d’un cancer à récepteurs œstrogéniques (ERα). Des études récentes convergent sur le concept selon lequel, dans les cancers du sein ER+, PR (Progesterone Receptor) peut inhiber les fonctions favorisant la croissance induite par l'œstrogène en reprogrammant directement la liaison d'ERα sur de nouveaux gènes cibles. Les données cliniques montrent que cette signature génique est associée à un bon pronostic dans une cohorte de 1.959 patientes atteintes de cancer du sein et qu’un agoniste de la progestérone améliore l'activité antiproliférative des thérapies anti-oestrogéniques1. Ainsi, ces données démontrent qu’ER n’est pas le seul acteur de la tumorigénèse mammaire et qu'il existe une interférence fonctionnelle entre ces deux voies hormonales, soulignant le besoin d’une meilleure compréhension de la signalisation de PR. D’un point de vue mécanistique, l’activité de PR est étroitement liée à l’interaction avec les nucléosomes. En effet, PR fonctionne comme un facteur « pionnier » et se lie à la chromatine au sein de complexes protéiques, régulant son activité transcriptionnelle. Sans progestérone, PR forme un complexe répressif associé à des enzymes modificatrices de la chromatine comme LSD1, HDAC1/2 et la protéine de l'hétérochromatine HP1γ2. En réponse au traitement hormonal, ce complexe est déplacé, ce qui permet de recruter des coactivateurs et des cofacteurs associés, qui modifient la structure de la chromatine locale et entraînent l'activation ou la répression des gènes cibles de PR. Nous avons identifié un nouveau régulateur de la signalisation de la progestérone, l'arginine méthyltransférase PRMT1, enzyme souvent surexprimée dans les cancers mammaires3,4. Par diverses approches in vitro et in vivo, nous avons montré une interaction directe entre PR et PRMT1, dans le noyau des cellules tumorales mammaires, et à la fois en absence d’hormone et après 1h de stimulation à la progestérone. De plus, PRMT1 apparaît comme un nouveau membre du complexe répressif sur la chromatine, associé à PR et à ses partenaires, dans un sous-ensemble de gènes inductibles par la progestérone. Nos résultats indiquent également que l’expression de PRMT1 affecte l’activité transcriptionnelle de PR et que son inhibition perturbe l’activation rapide de la voie de la protéine kinase après une stimulation progestative. Nous montrons pour la première fois que PR est méthylé sur un résidu arginine, conservé parmi les récepteurs nucléaires (R637), localisé dans son domaine de liaison à l'ADN. La production d’un anticorps dirigé contre la forme méthylée de PR nous a permis de préciser qu’elle se localisait dans le noyau des cellules et n’était retrouvée qu’après traitement progestatif. En outre, la mutation de R637 de PR entraine une diminution de l’expression d'un sous-ensemble de cibles de PR, ce qui entraine un retard de croissance cellulaire. En conclusion, ces résultats confirment l'implication de PRMT1 et de son activité méthyltransférase dans la signalisation de PR et plus particulièrement dans son activité transcriptionnelle. Nous démontrons donc que la méthylation sur résidus d'arginine est un nouveau mécanisme de contrôle lors de la réponse à la progestérone dans les cellules tumorales mammaires / Breast cancer progression is mainly driven by estrogen and progesterone signalling and therapies modulating oestrogen‘s action have improved the survival of ER+ cancer patients. As progesterone receptor (PR) is an ER target gene, its expression in breast cancer was considered as a predictive marker of ER functionality. However, recent studies are converging on the concept that PR can directly affect ER functions in breast cancer cells1. Activated PR can redirect ER to novel chromatin binding sites associated with cell differentiation and apoptosis, leading to a potential improvement of the tumour response to anti-oestrogen therapies. In considering the differential effects of progesterone in breast cancer, it is important to define the variable might influence progesterone pathway and the downstream mediators involved in this signalling. Recently, Beato and al reported that, in breast cancer cells, the unliganded form of PR (non-activated with progesterone) bind to genomic sites and target a repressive complex containing enzyme modifying chromatin as the demethylase LSD1 or the Heterochromatin Protein 1 (HP1γ)2. Under hormonal treatment, this complex is displaced, which makes it possible to recruit coactivators and associated cofactors, which modify the structure of the local chromatin and cause the activation or repression of the target genes of PR. In addition, cellular response to progesterone is also regulated by receptor post-translational modifications that may affect its stability, its subcellular localization and its interactions with regulators. In our study, we demonstrated for the first time that PR is methylated on arginine residues, by the arginine methyltransferase PRMT1. We identified as target the arginine 637 (R637), a conserved arginine among nuclear receptor superfamily, located in the DNA-binding domain of the receptor. By in vitro and in vivo approaches, we are studying the impact of PRMT1 on PR signalling pathways. In T47D breast cancer cells, we demonstrated that PR interacts with PRMT1, mainly in the nucleus. Of interest, PRMT1 interacts with PR in the nucleus in absence of hormone stimulation and it appears as a new member of the repressive complex on a subset of progesterone inducible genes. Our results also indicate that PRMT1 expression affects PR transcriptional activity and PRMT1 knockdown disrupts the rapid activation of protein kinase pathway after progestin stimulation. The production of an antibody directed against the methylated form of PR allowed us to specify that methylated-PR is localized in the nucleus of cells and was found only after progesterone treatment. Furthermore, PRMT1 depletion and mutation of R637 resulted in an inhibition of a subset of PR-regulated genes which led to retarded cell growth.Our data reveal the impact of PRMT1 expression on PR pathways and provide evidence for the asymmetric arginine dimethylation of PR. We therefore demonstrate that methylation on arginine residues could be a novel control mechanism in the response to progesterone in mammary tumor cells

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<p>As the human population increases and the demand for aquaculture increases, aquaculturists are coming up with new ways to mitigate stress in fish to increase their production. Cannabidiol (CBD) is an up and coming nutraceutical that may have potential to reduce stress in not only humans but also other vertebrates such as fish. In this project the effect of CBD on the stress physiology and immunology of Nile tilapia was evaluated both <i>in-vitro</i> and <i>in-vivo</i>. In the <i>in-vitro</i> study, spleen cell proliferation was conducted to observe the effect of CBD on fish T and B cells and were compared to mouse T and B cell proliferation. In the <i>in-vivo</i> study, the fish were reared in a recirculating aquaculture system. The effect of CBD on the stress physiology of the fish in short term and long term were evaluated. Based on the short-term acute study, a longer chronic study was designed where tilapia were fed with and without CBD (0.001% of feed weight) and with and without hydrocortisone stress hormone (0.01% of body weight) every day for four weeks. This experiment compared the various growth and feed utilization parameters as well as physiological and immunological parameters such as, plasma cortisol, blood glucose and protein levels, liver and spleen somatic indices (HSI and SSI, respectively), and lysozyme activity of the fish. From our current research, CBD shows potential in stress modulation and in immune modulation. It may have different effects based on the species, whether they need to enhance their immune response or reduce inflammation to be healthy. It also seems to have had different effect on different parts of the immune system. Hematological parameters were not significantly affected by acute stress. CBD did not make any substantial difference in growth. However, in the presence of stress, CBD was able to lower lysozyme activity down to the normal control levels. By administering the proper dosage of CBD on a case by case basis, health benefits can be achieved. Further investigation into the matter may not just be useful in stress mediation in aquatic organisms but may also have implications in human medicine as well.</p>

Cell turnover is a common feature of many organs in all animals and is required to maintain organ structure and function. It is achieved by a tightly regulated balance between cell death and cell division, which can be re-adjusted in response to injury and nutrient availability. How the balance between dying and dividing cells is coordinated has however remained unclear. Planarians represent an important model for studying cell turnover in adult animals, because all tissues undergo continuous cell turnover and a single stem cell type – the neoblast – is the exclusive source of all new cells. Moreover, planarians change their body size proportionally and reversibly depending on the nutritional status: feeding induces rapid and transient neoblast proliferation that results in animal growth, while starvation increases the rate of cell death, leading to de-growth. Importantly, also during starvation neoblasts keep proliferating at a basal-level. The hypothesis I addressed with my thesis research is that planarian energy metabolism might be a central mediator of cell turnover, particularly proliferation control and growth. I approached this hypothesis at several levels, including the characterization of the planarian energy metabolism and energy stores, the dependency of proliferation on the diet, and genetic requirements of proliferation control during starvation and feeding. I found that planarians have orthologs of key enzymes of most animal metabolic pathways, but, surprisingly, seem to lack fatty acid synthase. This suggests that planarians are likely not only auxotrophic for cholesterol, but also for fatty acids. I described that planarians store energy as triacylglycerols (TAGs, stored in lipid droplets) and glycogen, with the intestine as the main storage organ. Interestingly, the amount of TAGs and glycogen changes with size and is higher for larger animals, suggesting a regulatory interplay with the known size-dependency of growth/degrowth rates. Further, we demonstrated that the energy stores are the physiological basis of Kleiber’s law that describes the near-universal scaling between metabolic rate and body mass. I further showed that proliferation occurs in three different modes, one during starvation when proliferation is maintained at basal levels and two after feeding, an initial proliferation mode (at three hours after feeding), which is diet independent and a later proliferation (at 24 hours after feeding), which is diet dependent. The two feeding-induced proliferation modes differ not only in their diet-dependencies, but also in their gene expression profiles, as assessed by RNA-sequencing. To identify genes involved in proliferation regulation, I assessed the requirements of different candidate genes in all three proliferation modes in a small-scale RNA interference screen. This screen revealed that insulin signaling, TORC1 and FGFR are involved in regulating basal proliferation during starvation and – most interestingly –that AMP-activated protein kinase (AMPK)-depleted animals showed increased proliferation during starvation at levels characteristic of recently fed animals. This result uncovered AMPK as a modulator that adjusts the neoblast proliferative activity to the nutritional state, potentially independently of TOR. In sum, my work shows how energy metabolism and storage are coordinated with proliferation and growth in planarians and identified AMPK as a central modulator that adjust proliferation to cellular energy states. I discuss potential mechanisms by which AMPK modulates proliferation and putative links between AMPK and cell death, the second process of cell turnover. The energy state as the central mediator of cell turnover and the key players and mechanisms that my work revealed in planarians might also apply across different species:Chapter 1 1. Introduction 1 1.1 Cell turnover is a crucial process for tissue homeostasis 1 1.2 Cell division 2 1.2.1 Control mechanisms of cell division 2 1.2.1.1 Cell cycle machinery 2 1.2.1.2 Organization of the cell cycle control system – cell-cycle intrinsic regulation by Cdk-cyclin complexes 3 1.2.1.3 External control of cell cycle progression 4 1.2.1.4 Metabolic control of cell cycle progression 6 1.2.2 Metabolic requirements of proliferating cells 10 1.2.2.1 The energy stores 11 1.3 Cell death 13 1.4 Suggested mechanisms that coordinate cell death and division and their caveats 14 1.5 Planarians as a model to study cell turnover 16 1.6 Planarian body anatomy 18 1.7 Planarian stem cell system 19 1.7.1 Neoblasts form a heterogeneous population 19 1.7.2 Neoblast proliferative activity 21 1.7.3 Neoblast cell cycle machinery 22 1.7.4 Regulation of neoblast proliferative activity 22 1.8 Cell death in planarians 23 1.9 Mechanisms that coordinate the rate of dividing and dying cells in planarians still remain elusive 24 1.10 Scope of the thesis 24 Chapter 2 2. Planarian energy metabolism and the regulation of planarian growth dynamics 26 2.1 Introduction 26 2.2 Part 1: Planarian energy metabolism 27 2.2.1 The metabolic machinery of S. mediterranea 27 2.2.2 Planarian energy stores 30 2.2.2.1 Visualization of lipid and glycogen storage compartments in planarians 30 2.2.2.2 Investigation of feeding-dependent changes in lipid and glycogen stores 31 2.3 Part 2: Role of planarian organismal energy stores in regulating their growth and degrowth dynamics 36 2.3.1 Background information about known aspects of growth and degrowth dynamics in planarians 36 2.3.1.1 Growth and degrowth arise mainly from changes in cell number 36 2.3.1.2 Growth and degrowth rates are size dependent 37 2.3.2 Energy stores increase disproportionately with size and strongly contribute to the size-dependent dry mass increase 38 2.3.3 Metabolic rate and energy intake are unlikely causes of the size-dependency of the energy stores 41 2.4 Summary and Discussion 43 2.4.1 Part 1: First insights into planarian energy metabolism 43 2.4.1.1 Core planarian metabolic pathways 43 2.4.1.2 Characterization of planarian energy stores 44 2.4.2 Part 2: Implications of size-dependent behavior of planarian energy stores 44 2.4.2.1 Role of energy stores as the physiological origin of Kleiber’s law in planarians 44 2.5 Outlook 46 Chapter 3 3. Towards understanding a systems-level regulation of neoblast proliferative activity 48 3.1 Introduction 48 3.2 Assay development for quantitative determination of proliferating cells 50 3.3 Food quantity and quality affect the later proliferation phase, but not the initial response to feeding 53 3.4 Deep sequencing time course provides insights into gene-expression changes in response to feeding 56 3.5 Discussion 59 3.5.1 Evidence for feeding-induced neoblast regulation at the G0/G1-to-S transition 59 3.5.2 Three distinct modes of neoblast proliferation 59 3.5.3 Early and late proliferation modes show distinct transcriptional profiles 59 3.5.4 Implications from feeding and gene expression profiling experiments 60 3.5.4.1 Potential explanations for diet dependence of the late proliferation mode 60 3.5.4.2 Potential mechanisms of diet-independent early proliferation response 61 3.5.5 Summary and Outlook 61 Chapter 4 4. Towards identifying the mechanisms underlying the regulation of neoblast proliferation 63 4.1 Introduction 63 4.1.1 Chosen gene candidates and their known role in proliferation 64 4.2 RNAi-mediated depletion of candidate genes to test their regulatory role in proliferation 67 4.2.1 Assay design and optimization for the functional RNAi screen 67 4.2.2 Results of small-scale RNAi screen 69 4.3 AMPK - a potential integrator of neoblast proliferation to the nutritional state of the animal 73 4.3.1 AMPK and LKB1 knockdown increases proliferation during starvation 73 4.3.2 AMPK depletion-phenotype of increased proliferation during starvation seems to be TOR independent 73 4.4 Discussion 76 4.4.1 Evidence for a mechanism that regulates basal proliferation during starvation 76 4.4.2 AMPK integrates neoblast activity in response to feeding 77 4.4.2.1 Implications of my observations 77 4.4.2.2 Possible experiments to test the role of AMPK during the regulation of proliferation 78 4.4.3 AMPK potentially regulates proliferation independently of TOR 79 4.4.4 An evolutionarily conserved stem cell switch? 80 4.4.5 Summary and Outlook 80 Chapter 5 5. Discussion and Outlook 81 5.1 Cell-autonomous roles of AMPK in proliferation regulation 83 5.1.1 Independent regulation of ribosomal translation elongation as a potential modulator of neoblast proliferation 83 5.1.2 AMPK might regulate cell cycle progression directly 85 5.1.3 AMPK might regulate symmetric versus asymmetric cell division 85 5.2 Cell non-autonomous roles of AMPK in proliferation regulation 86 5.2.1 AMPK might modulate the release of lipid stores 86 5.3 Possible role of AMPK in regulation of autophagic cell death 87 5.4 AMPK as a potential modulator of cell turnover that couples cell proliferation and cell death to the animal’s energy state 88 5.5 Summary and Outlook 89 Materials and Methods 91 List of Figures 106 List of Tables 107 Acknowledgments 108 References 110

My thesis research began with a project in which we were trying to determine the function of embryonic stem cell (ESC)-specific miRNAs. Using luciferase constructs containing miRNA binding sites, luciferase expression was inhibited by endogenous miRNAs in ESCs, and by exogenous miRNAs in HeLa cells. Inhibition of luciferase expression by miRNAs was inhibited in HeLa cells using 2’O-methyl-oligonucleotides. In ESCs, 2’O-methyl-oligonucleotides were only effective in partially inhibiting miR290 function. Partial inhibition of miR290 did not result in any obvious phenotypic changes in mESCs. Later studies using 2’O-methyl-oligonucleotides in ESCs were also unsuccessful. The function of ESC-specific miRNAs has since been studied by re-introducing miRNAs into Dicer -/- cells which cannot make miRNAs. These studies have shown that ESC-specific miRNAs are involved in de novo DNA methylation, self-renewal, and cell-cycle regulation. Newly diagnosed glioblastoma (GBM) patients rarely survive more than two years even after surgery, radiotherapy, and chemotherapy using temozolomide (TMZ) or 1,3-bis(2-chloroethy)-1-nitrosourea (BCNU). Eventual regrowth of the tumor indicates that some tumor cells are resistant to therapy. GBM neurosphere-initiating cells (NICs) are thought to be similar to tumor-initiating cells in vivo, and will form invasive tumors in mice, making neurosphere cultures a good model system for studying GBMs. To test whether GBM NICs were resistant to chemotherapy, we used a neurosphere formation assay to measure the number of proliferating NICs in the presence of TMZ or BCNU. The concentrations of chemotherapy drugs required to inhibit neurosphere formation were much less than those required to inhibit bulk cell proliferation or to induce cell death in our neurosphere cultures. For some cultures, there was a robust recovery of neurosphere formation after chemotherapy treatment which appeared to be DNA damage independent. Some of the cultures that showed significant recovery of neurosphere formation underwent reversible cell cycle arrest, possibly reducing chemotoxicity in these cultures. Collectively, these results indicate that GBM neurosphere cultures can regrow after being treated with clinically relevant doses of chemotherapy drugs. Chemotherapy-treated neurosphere cultures remained viable, and formed tumors when injected into mice. Our experiments show that these in vitro assays may be useful in predicting in vivo responses to chemotherapeutic agents.