Actin cytoskeletal regulation is of critical importance for a number of diverse cellular functions, including cell motility, endocytosis, cell division and transcription. Tight regulation of actin is critical for many aspects of cancer biology and in particular invasion and metastasis. ADF/cofilins are among the most important actin regulatory proteins. Mammals have three highly conserved members, ADF, CFL1 and CFL2, which regulate actin dynamics by severing and depolymerizing actin filaments. Despite a huge literature on the roles of ADF/cofilins in actin treadmilling and cell migration in vitro and in cancer cell behavior during invasion, very little is known about their collective roles in tissue homeostasis. By employing genetic knock-outs of ADF, in conjunction with conditional depletion of CFL1 using a Cre-LoxP system under the control of the keratin 14 promoter, we were able to study the effects of ADF/CFL1 loss in vivo in the mouse epidermis. Furthermore, by generating ADF-null squamous cell carcinoma (SCC) cell lines and by transiently downregulating CFL1 with RNAi, we were able to investigate further the cellular responses after ADF/CFL1 depletion in vitro. Co-depletion of ADF and CFL1 from the mouse epidermis triggered loss of tissue homeostasis characterized by abnormal thickening of the tissue, actin filament accumulation and nuclear deformation. Loss of ADF/CFL1 in cultured malignant keratinocytes also led to aberrant cell morphology accompanied by unrestrained accumulation of actin stress fibers tethered to enlarged focal adhesions. Enhanced SRF/MAL-mediated transcription fuels this uncontrolled actin polymerization which is also mediated by Arp3. Furthermore, these actin filaments are decorated with phospho-myosin light chain, which indicates their contractile nature. As a consequence, the increased intracellular acto-myosin tension results in nuclear deformation, which is promoted by the deregulated actin filaments tethered to the nuclear envelope via the linker of nucleoskeleton and cytoskeleton (LINC) complex. Overall, we describe new conceptual insight into the cellular functions of ADF/cofilins. We show that their activities are essential for the dynamic regulation of contractile actin filaments that, if left unchecked, lead to loss of cellular homeostasis and cell death promoted by loss of nuclear integrity. Additionally, the critical roles of nuclear actin and actin-associated proteins have recently started being appreciated. Thus, for the first time we set out to investigate new functions of cofilins in the nucleus using proteomics, and identify new cofilin binding partners that implicate them in novel cellular pathways, expanding our knowledge on these small actin-binding proteins.

Objetivo:Caracterizar o padrão de homeostase (proliferação celular e apoptose) de tecido endometrial eutópico e ectópico de coelhas submetidas à indução de lesões de endometriose por modelo experimental já conhecido, quatro e oito semanas após o procedimento de implantação endometrial. Material e Métodos:Estudo experimental animal sendo utilizado 20 coelhas adultas Nova Zelândia, fêmeas e virgens, submetidas à laparotomia para indução da lesão de endometriose, através da ressecção de um corno uterino e fixação no peritônio pélvico de fragmento de 5mm. As coelhas foram divididas em dois grupos de 10 animais, sendo os animais do grupo 1, sacrificados após 4 semanas da indução da lesão endometrial ectópica e os do grupo 2 após 8 semanas. A lesão foi excisada para análise histológica juntamentecom o corno uterino contralateral, comprovando a presença de tecido endometrial glandular e estromal. Reações de imunohistoquímica foram realizadas, no tecido endometrial eutópico e ectópico, para proliferação celular através do PCNA e para apoptose através do fas, na glândula e estroma, sendo obtido o índice de proliferação celular (IPC) e de apoptose (IA) através do número de células marcadas por 1000 contadas, e o índice dehomeostase tecidual através do coeficiente entre o IPC e IA. Resultados:Observou-se maior índice de proliferação no tecido ectópico, tanto glandular como estromal, quando comparado com o endométrio eutópico, com 4 e 8 semanas após a indução da lesão. Contudo, quando as lesões ectópicas foram comparadas entre si, com 4 e 8 semanas, não foi observada diferença significativa. Quando comparamos o índice de apoptose, observamos que não houve diferença entre o tecido ectópico e o eutópico, tanto glandular como estromal nas lesões induzidas e analisadas com 4 semanas, porém no tecido glandular das lesões analisadas com 8 semanas houve diferença significativa entre a lesão ectópica e o tecido endometrialeutópico 0,0819 ± 0,0213 e 0,0995 ± 0,01336, respectivamente (p=0,04). A homeostase tecidual foi calculada e observou-se uma tendência destes tecidos a proliferação, sempre com índicesde homeostase tecidual (IPC/IA) acima de 1. Conclusão:As lesões ectópicas parecem ter uma proliferação celular maior que o endométrio eutópico levando a uma tendência ao crescimento tecidual descontrolado nas lesões de endometriose induzidas. / Objective: To characterize the pattern of tissue homeostasis (cell proliferation and apoptosis) of eutopic and ectopic endometrium in rabbitsfour and eight weeks after endometrium implantation for induction of endometriotic lesions by experimental standardized method. Material and methods: Animal experimental study with 20 female, virgins and adult New Zeland rabbits, submitted to laparotomy for endometriosis induction by resection of one uterine horn, isolation of the correspondent endometrium and fixation of a 5mm tissue segment to the pelvic peritoneum. The animals were divided in two groups of 10, group 1 was sacrificed after 4 weeks of endometriosis induction and group 2 eight weeks after the procedure. The lesion was excised for later histological analysis together with the opposite uterine horn, just to verify the presence of endometrial gland and stroma. Immunohistochemistry reactions were performed in order to study cell proliferation (by PCNA technique) and apoptosis (by fastechnique) in the eutopic and ectopic endometrium, and a Cell Proliferation Index (CPI) and an Apoptotic Index (AI) werecalculated based on these findings, considering the number of marked cellsper 1000 counted cells. The Homeostatic Tissue Index (HTI) was considered to be the relation between CPI and AI (CPI/AI). Gland and stroma were analyzed separately. Results: There was an increase in the CPI of the ectopic tissue, considering gland and stroma, after four and eight weeks of endometriosis induction when comparing to the eutopic one. However, when comparing the ectopic lesions, there was no difference between four and eight weeks of induction. Analyzing the only the AI, there was no difference between the eutopic and the ectopic endometrium with four weeks,nevertheless there was a significant difference in the glandular tissue of the lesions with eight weeks when comparing eutopic and ectopic tissues (0.0819 ± 0.0213 e 0.0995 ± 0.01336, respectively (p=0,04)). Based on HTI there was a tendency to cell proliferation on these tissues, always with and HTI (CPI/AI) higher than 1. Conclusions: Ectopic lesions seem to have a higher cell proliferation than the eutopic endometrium, tending to present an uncontrolled tissue growth in the endometriotic inducted lesions.

Objetivo:Caracterizar o padrão de homeostase (proliferação celular e apoptose) de tecido endometrial eutópico e ectópico de coelhas submetidas à indução de lesões de endometriose por modelo experimental já conhecido, quatro e oito semanas após o procedimento de implantação endometrial. Material e Métodos:Estudo experimental animal sendo utilizado 20 coelhas adultas Nova Zelândia, fêmeas e virgens, submetidas à laparotomia para indução da lesão de endometriose, através da ressecção de um corno uterino e fixação no peritônio pélvico de fragmento de 5mm. As coelhas foram divididas em dois grupos de 10 animais, sendo os animais do grupo 1, sacrificados após 4 semanas da indução da lesão endometrial ectópica e os do grupo 2 após 8 semanas. A lesão foi excisada para análise histológica juntamentecom o corno uterino contralateral, comprovando a presença de tecido endometrial glandular e estromal. Reações de imunohistoquímica foram realizadas, no tecido endometrial eutópico e ectópico, para proliferação celular através do PCNA e para apoptose através do fas, na glândula e estroma, sendo obtido o índice de proliferação celular (IPC) e de apoptose (IA) através do número de células marcadas por 1000 contadas, e o índice dehomeostase tecidual através do coeficiente entre o IPC e IA. Resultados:Observou-se maior índice de proliferação no tecido ectópico, tanto glandular como estromal, quando comparado com o endométrio eutópico, com 4 e 8 semanas após a indução da lesão. Contudo, quando as lesões ectópicas foram comparadas entre si, com 4 e 8 semanas, não foi observada diferença significativa. Quando comparamos o índice de apoptose, observamos que não houve diferença entre o tecido ectópico e o eutópico, tanto glandular como estromal nas lesões induzidas e analisadas com 4 semanas, porém no tecido glandular das lesões analisadas com 8 semanas houve diferença significativa entre a lesão ectópica e o tecido endometrialeutópico 0,0819 ± 0,0213 e 0,0995 ± 0,01336, respectivamente (p=0,04). A homeostase tecidual foi calculada e observou-se uma tendência destes tecidos a proliferação, sempre com índicesde homeostase tecidual (IPC/IA) acima de 1. Conclusão:As lesões ectópicas parecem ter uma proliferação celular maior que o endométrio eutópico levando a uma tendência ao crescimento tecidual descontrolado nas lesões de endometriose induzidas. / Objective: To characterize the pattern of tissue homeostasis (cell proliferation and apoptosis) of eutopic and ectopic endometrium in rabbitsfour and eight weeks after endometrium implantation for induction of endometriotic lesions by experimental standardized method. Material and methods: Animal experimental study with 20 female, virgins and adult New Zeland rabbits, submitted to laparotomy for endometriosis induction by resection of one uterine horn, isolation of the correspondent endometrium and fixation of a 5mm tissue segment to the pelvic peritoneum. The animals were divided in two groups of 10, group 1 was sacrificed after 4 weeks of endometriosis induction and group 2 eight weeks after the procedure. The lesion was excised for later histological analysis together with the opposite uterine horn, just to verify the presence of endometrial gland and stroma. Immunohistochemistry reactions were performed in order to study cell proliferation (by PCNA technique) and apoptosis (by fastechnique) in the eutopic and ectopic endometrium, and a Cell Proliferation Index (CPI) and an Apoptotic Index (AI) werecalculated based on these findings, considering the number of marked cellsper 1000 counted cells. The Homeostatic Tissue Index (HTI) was considered to be the relation between CPI and AI (CPI/AI). Gland and stroma were analyzed separately. Results: There was an increase in the CPI of the ectopic tissue, considering gland and stroma, after four and eight weeks of endometriosis induction when comparing to the eutopic one. However, when comparing the ectopic lesions, there was no difference between four and eight weeks of induction. Analyzing the only the AI, there was no difference between the eutopic and the ectopic endometrium with four weeks,nevertheless there was a significant difference in the glandular tissue of the lesions with eight weeks when comparing eutopic and ectopic tissues (0.0819 ± 0.0213 e 0.0995 ± 0.01336, respectively (p=0,04)). Based on HTI there was a tendency to cell proliferation on these tissues, always with and HTI (CPI/AI) higher than 1. Conclusions: Ectopic lesions seem to have a higher cell proliferation than the eutopic endometrium, tending to present an uncontrolled tissue growth in the endometriotic inducted lesions.

L’homéostasie des tissus et la formation d’organes fonctionnels au cours du développement mettent en jeu un équilibre entre prolifération, croissance, mort et différenciation des cellules. Cycline G de D. melanogaster est une protéine impliquée dans plusieurs de ces processus de maintien de l’homéostasie tissulaire. Au cours de ma thèse, j’ai montré que Cycline G est impliquée dans le maintien épigénétique des identités cellulaires au cours du développement. En effet, CycG interagit avec des gènes codant des facteurs chromatiniens des groupes Polycomb (PcG) et Trithorax (TrxG), impliqués respectivement dans le maintien d’un état réprimé ou activé des gènes au cours des divisions cellulaires. Ces interactions corrèlent avec une modification du profil d'expression des gènes homéotiques Scr et Ubx, qui suggère un rôle de Cycline G dans la répression de ces gènes. Je me suis également intéressée au rôle de CycG dans la stabilité du développement (SD), qui participe au maintien de l’homéostasie en contrôlant les variations stochastiques des processus développementaux. En effet, la surexpression de CycG induit une asymétrie visible de la taille des ailes des mouches, indiquant que la SD est altérée. L’étude des bases génétiques de ce processus par une analyse de gènes candidats suggère que des gènes PcG et trxG jouent un rôle dans la SD. Par ailleurs, l’asymétrie induite par CycG est aggravée par des mutations des effecteurs de la compétition cellulaire, un mécanisme via lequel des cellules entraînent l’apoptose de cellules voisines de moindre capacité proliférative. Ces résultats soulèvent l’intéressante possibilité d’une implication de la compétition cellulaire dans la SD. / Tissue homeostasis and formation of functional organs during development require a precise balance between proliferation, growth, cell death and differentiation. Drosophila Cyclin G is involved in several of these processes. During my PhD, I showed that Cyclin G is implicated in epigenetic maintenance of cell identities during development. Indeed CycG interacts with genes of the Polycomb (PcG) and trithorax (trxG) groups that encode chromatin factors involved in the maintenance through cell divisions of repressed or active transcriptional state of genes, respectively. These interactions correlate with a change of the homeotic genes Scr and Ubx expression profile, suggesting a role for Cyclin G in the repression of these genes. I also addressed the role of CycG in developmental stability that participates in tissue homeostasis by buffering stochastic variations of developmental processes, also known as developmental noise. CycG overexpression induces a visible asymmetry of wing size, indicating that developmental stability is impaired. Genetic and cellular bases of this process are poorly understood. The results of a gene candidate approach suggest that PcG and trxG genes contribute to developmental stability. Besides, CycG-induced asymmetry is increased by mutating effectors of cell competition, a phenomenon through which cells induce apoptosis of less fit neighbour cells. These observations raise the question of a role for cell competition in developmental stability.

Previous work from our laboratory in the mdx mouse model of Duchenne muscular dystrophy (DMD) demonstrated that a fraction of muscle stem cells (i.e., satellite cells) (MuSCs) progressively lose the expression of myogenic markers during the progression of the disease. In the process of characterizing this aberrant behaviour, we serendipitously discovered that MuSCs might be separated into two distinct subpopulations based on the expression of the GPI-anchored surface protein CD90. Crucially, this separation does not correlate with a divergence from the myogenic lineage; instead, it separates the pool of MuSCs into two subpopulations, both maintaining myogenic properties in healthy muscles. These two newly identified subpopulations do not overlap with any previously reported subpopulation and may be prospectively isolated; present a different response in terms of kinetics of activation and differentiation during the regenerative process induced by acute muscle damage; show a different propensity to enter in GAlert state upon distal injury; display dissimilar pAMPK activity and contain a different amount of mitochondria; are present in different proportions in distinct muscle groups; differentially express ECM encoding genes during quiescence. Moreover, one of the two subpopulations can give rise to the other and therefore appears to be upstream in the lineage hierarchy. Notably, loss of function experiments, in which CD90 was downregulated in MuSCs, diminish the difference in activation displayed by the two subpopulations. This demonstrates that CD90 is a molecular determinant of MuSCs functional diversification. Importantly, although the two subpopulations of MuSCs are numerically similar in healthy limb muscles, one of the two subpopulations is lost with time in dystrophic mdx mice. Based on these data, we are hypothesizing that an imbalance between the two newly identified subpopulations may impair regeneration in the dystrophic muscles. These observations not only increase our knowledge of the molecular and cellular dynamics that are controlling normal and pathological muscle homeostasis but also open the possibility that restoring the proper functional equilibrium between subpopulations of MuSCs may counteract the progression of the dystrophic disease.

The purpose of this thesis is to introduce and demonstrate several biological processes or phenomena using posterior lateral line system of zebrafish as a model. The first part of this work focuses on the highly dynamic tissue remodeling under the control of morphogenetic territories. In particular, eya1 gene is studied to add a susbtantial mechanism during migration of lateral line primordium. I propose that combinatorial Wnt/β-catenin and Fgf signaling control the spatialtemporal profile of Dkk expression to dynamically confine Wnt/β-catenin to the mesenchyme. The second part of this thesis mainly discusses about the existence of tissue compartment whereby Notch signaling involves in governing the regeneration anisotropy. I also discuss the importance of centrifugal movement of hair cell progenitors to propagate and maintain bilateral symmetry in the neuromasts. The last part of this thesis emphasizes on the epigenetic study in order to validate the involvement of macroH2A during development of zebrafish embryos. / La intención de ésta tesis es introducir y demostrar diversos procesos biológicos o fenómenos, usando como modelo el sistema de la línea lateral posterior del pez cebra. La primera parte de éste trabajo se enfoca en el proceso dinámico de remodelación de los tejidos bajo el control de territorios morfogénicos. En particular, se estudia el gen Eya1, como mecanismo sustancial en la migración del primordio de la linea lateral. Propongo que la señalización combinatorial de Wnt/B-catenin y FGF controla el patrón de expresión de Dkk, para confinar dinámicamente Wnt/B-catenin al tejido mesenquimal. La segunda parte de ésta tesis, principalmente discute acerca de la existencia de una compartimentalización de los tejidos, en dónde la señalización via Noch está involucrada en el control de la anisotropía en la regeneración. También se discute la importancia de los movimientos centrífugos de los progenitores de las células ciliadas para propagar y mantener la simetria bilateral en los neuromastos. La última parte de la Tesis emfatiza en el estudio epigenético, para validar la implicación de macroH2A en el desarrolllo de los embriones del pez cebra.

The Tec kinase ITK is activated by TCR stimulation and also required for TCR downstream signaling. Previous studies have reported differential roles of ITK and another Tec family kinase RLK in CD4+ TH differentiation and effector function. However, these findings are confounded by the complex T cell developmental defects in Itk-/- mice. Furthermore, the function of ITK in tissue-resident T cells in the intestine and anti-viral immune response to a persistent infection has not been studied previously. In addition to T cells, recent studies have indicated an expression of ITK in ILC2, but not in other ILC subsets. Yet, the role of ITK in ILC2 has not been characterized. Here, I have examined the role of ITK and RLK in CD4+ TH subsets using a small molecule inhibitor PRN694. I found that PRN694 impaired TH1 differentiation in vitro, and PRN694 administration prevented TH1-mediated colitis progression in vivo. In an MHV68 infection model, Itk-/- mice failed to control viral replication in the intestine, while gut-homing of CD8+ T cells was greatly impaired. Finally, I found that ILC2 number was markedly reduced in the intestine of Itk-/- mice. Gut-specific defect of Itk-/- ILC2 is associated with a low availability of IL-2 in the intestine of Itk-/- mice. Collectively, these data suggest that ITK is important in T cell migration to the intestine and ILC2 homeostasis in the intestine, thereby contributing to the protective response to a latent virus and intestinal tissue homeostasis.