Thymic epithelial cells (TECs) are indispensable for the development of T cells in the thymus. Two subtypes of TECs exist in the thymus, medullary mTECs and cortical cTECs. Both mTECs and cTECs originate from endodermal thymic epithelial progenitor cells (TEPCs) in the embryo, but how the differentiation of TEPCs is regulated is not well understood. The aims of this thesis were to establish the role of Notch signalling in TEPC differentiation, and how it interacts with known regulators such as FOXN1 and the NFκB pathway. Gene expression data showed that Notch is active in TEPCs and exhibits a correlation with the mTEC lineage. Loss of Notch function led to a significant reduction in the number of mTECs in the thymus, and this can be attributed to aberrant mTEC specification. Furthermore, the duration of Notch activity in determining mTEC number appears limited to the early phase of organogenesis, and precedes RANK/NFκB mediated mTEC proliferation. Gain of Notch function resulted in a considerable shift to a primitive, TEPC-like phenotype, and subsequently a latent increase in mTEC frequency. Finally, transcriptomic and functional analyses pointed to a cross-repressive mechanism between Notch and FOXN1 in TEPCs. Taken together, these results identified Notch as a novel regulator of mTEC specification, likely through maintaining the potency of fetal TEPCs, a prerequisite for mTEC lineage commitment.

Transmissible spongiform encephalopathies (TSEs) are rare, uniformly fatal neurodegenerative disorders that can affect many mammalian species, including humans. A hallmark of these diseases is the conversion of cellular prion protein (PrPC) into an abnormally folded form. This misfolded PrPC is infectious, since it can provide a template for pathogenic conversion of PrPC in a new host. In addition to any toxicity of the misfolded protein, loss of normal PrPC function could be involved in the neurodegenerative processes. However, the physiological role of PrPC is still poorly understood and this project has aimed to address that lack of knowledge. Out of the many putative functions ascribed to PrPC, the most commonly proposed is that it protects cells from stress. In contrast, I have found that stable transfection of the prion protein gene into SH-SY5Y neuroblastoma cells increases cell death in response to serum removal from the culture medium. Following treatment with several chemical toxins, two out of four stably transfected clones did, generally, display greater viability than untransfected cells that do not express detectable levels of PrPC. However, knockdown of PrPC expression by RNA interference had no effect on this stress resistance, indicating that it may not have been mediated directly by PrPC. Given the lack of robust stress protection afforded by PrPC transfection, proteomic analyses of the cells were carried out to identify alternative processes that were perturbed as a result of PrPC expression. The results obtained suggested roles for PrPC in cytoskeletal organisation and cell cycle regulation. Various proteins involved in cytoskeletal organisation were confirmed by western blotting to be differentially expressed in some or all of the stably transfected clones. Additionally, the expression changes to proteins involved in cell cycle regulation resulted in slower proliferation of the clones compared with untransfected cells, a difference that was reduced following RNA interference-mediated knockdown of PrPC. Taken together, these data suggested that specific growth factor-activated pathways were differentially regulated in the stably transfected clones. One candidate pathway was nerve growth factor (NGF) signalling, which promotes neuronal survival and differentiation as well as regulating various processes outside of the nervous system. PrPC-transfection resulted in altered expression of receptors for NGF, suggesting that the stably transfected clones were, indeed, responding differently to NGF stimulation. However, the molecular mechanism responsible for these expression changes remains to be determined, since co-immunoprecipitation experiments did not identify any physical interactions between PrPC and the NGF receptors. Nonetheless, a role for PrPC in modulating NGF signalling has the potential to explain many of the diverse phenotypic observations in PrPC-null mice and might indicate that loss of PrPC function is an important part of TSE pathogenesis.

Maintaining the intrinsic excitability of neurons is crucial for stable brain activity. This can be achieved by the homeostatic regulation of membrane ion channel conductances, although it is not well understood how these processes influence broader aspects of neuron and network function. One of the many mechanisms which contribute towards this task is the modulation of potassium channel conductances by activity-dependent nitric oxide signalling. Here, we first investigate this mechanism in a conductance-based neuron model. By fitting the model to experimental data we find that nitric oxide signalling improves synaptic transmission fidelity at high firing rates, but that there is an increase in the metabolic cost of action potentials associated with this improvement. Although the improvement in function had been observed previously in experiment, the metabolic constraint was unknown. This additional constraint provides a plausible explanation for the selective activation of nitric oxide signalling only at high firing rates. In addition to mediating homeostatic control of intrinsic excitability, nitric oxide can diffuse freely across cell membranes, providing a unique mechanism for neurons to communicate within a network, independent of synaptic connectivity. We next conduct a theoretical investigation of the distinguishing roles of diffusive homeostasis mediated by nitric oxide in comparison with canonical non-diffusive homeostasis in cortical networks. We find that both forms of homeostasis robustly maintain stable activity. However, the resulting networks differ, with diffusive homeostasis maintaining substantial heterogeneity in activity levels of individual neurons, a feature disrupted in networks with non-diffusive homeostasis. This results in networks capable of representing input heterogeneity, and linearly responding over a broader range of inputs than those undergoing non-diffusive homeostasis. We further show that diffusive homeostasis interferes less than non-diffusive homeostasis in the synaptic weight dynamics of networks undergoing Hebbian plasticity. Overall, these results suggest a novel homeostatic mechanism for maintaining stable network activity while simultaneously minimising metabolic cost and conserving network functionality.

Le cancer du poumon est le troisième cancer le plus fréquent chez les femmes et le deuxième chez les hommes, il est la cause principale de décès par cancer dans le monde, avec une mortalité annuelle supérieure à 1 million. Malgré des progrès remarquables dans la thérapie ciblée, la majorité des patients atteints de cancer du poumon sont diagnostiqués dans un stade avancé où ils ne connaissent pas d'amélioration significative de leur survie globale. Les récepteurs à domaine tyrosine kinase, tels que le récepteur du facteur de croissance épidermique (EGFR), traduisent les informations du microenvironnement dans la cellule en activant des voies de signalisation homéostatiques. L'internalisation et la dégradation de l'EGFR suite à la liaison du ligand limitent l'intensité de la signalisation proliférative, ce qui contribue à maintenir l'intégrité cellulaire. Dans les cellules cancéreuses, la dérégulation du trafic de l’EGFR aboutit à des effets divers sur la progression tumorale. Dans cette étude, nous avons identifié la sortiline comme un régulateur clé de l'internalisation de l'EGFR à partir de la membrane plasmique. La perte de l’expression de la sortiline dans les cellules tumorales augmente la prolifération cellulaire en soutenant la signalisation de l’EGFR à la surface de la cellule, ce qui accélère la croissance tumorale. Chez les patients atteints de cancer du poumon, l'expression de la sortiline diminue avec l’augmentation du grade pathologique et l’expression de son gène, SORT1, est fortement corrélée à une meilleure survie globale, en particulier chez les patients présentant une forte expression de l'EGFR. Ainsi, la sortiline représente un nouveau régulateur du trafic intracellulaire de l’EGFR, elle agit en contrôlant l'internalisation de ce récepteur et en limitant la croissance tumorale. / Lung cancer is the third most common cancer in women and the second in men, it is the leading cause of cancer-related death worldwide, with an annual mortality of more than 1 million. Despite remarkable advances in targeted therapy, the majority of patients with lung cancer are diagnosed at an advanced stage where they do not experience a significant improvement in overall survival. Tyrosine kinase receptors such as the epidermal growth factor receptor (EGFR) transduce information from the microenvironment into the cell and activate homeostatic signalling pathways. Internalisation and degradation of EGFR after ligand binding limits the intensity of its proliferative signalling, thereby helping to maintain cell integrity. In cancer cells, deregulation of EGFR trafficking has a variety of effects on tumour progression. Here, we report that sortilin is a key regulator of EGFR internalisation. Loss of sortilin in tumour cells promotes cell proliferation by sustaining EGFR signalling at the cell surface, ultimately accelerating tumour growth. In lung cancer patients, sortilin expression decreases with increased pathologic grade, and the expression of SORT1 (the gene encoding sortilin) is strongly correlated with a better survival, notably in patients with high EGFR expression. Thus, sortilin is a novel regulator of EGFR intracellular trafficking acting by controlling receptor internalisation and limiting tumour growth.

The Hedgehog signalling pathway is an essential developmental pathway present in all bilaterians that is involved in embryogenesis, body patterning and stem cell homeostasis. Dysregulation of the Hh pathway leads to various kinds of cancer, such as basal cell carcinoma and medulloblastoma. Smoothened (SMO), a Frizzled-type G-protein coupled receptor (GPCR), is the essential transmembrane signal transducer within the Hh pathway, conveying the signal from the upstream transmembrane protein, Patched1 (Ptc1), to the downstream intracellular proteins. The mechanisms by which SMO transmits the Hh signal from the extracellular environment, through the plasma membrane and to the intracellular proteins are not known. In this thesis, I present my work into the structural and functional characterisation of the extracellular and transmembrane domains (TMD) of human SMO in order to better understand the molecular mechanisms of its signal transduction. The extracellular region of SMO contains a highly conserved cysteine-rich domain (CRD) and a linker domain (LD). I present the first crystal structure of the CRD, LD and TMD of SMO, which is also the first crystal structure of a GPCR with a large functional extracellular domain. This structure revealed a domain architecture for SMO that enables regulation of its transmembrane domain by its extracellular domains. It also revealed a cholesterol molecule bound to the CRD, which we subsequently determined to be a new endogenous small-molecule agonist for SMO. I present five further structures of SMO bound to different small molecule agonists and antagonists. Together, these structures demonstrate that the position of the CRD relative to the TMD reflects the activation state of SMO. We also generated nanobodies against the extracellular region of SMO in order to stabilise its conformation. These studies not only improve our understanding of the workings of a key transmembrane protein within a fundamental signalling pathway but will also aid efforts to develop better therapeutics for an important cancer target.

The mammalian testis is divided into two distinct compartments which carry out its principal functions. Spermatogenesis occurs within the seminiferous tubules and androgen biosynthesis primarily occurs in the interstitial space. Both these processes are entirely dependent upon the two major testicular somatic cell populations - the Sertoli and Leydig cells respectively. In human males, testicular spermatogenic and endocrine function declines during the ageing process. Of particular significance is the reported age-related decrease in Leydig cell androgen production as androgens have been suggested to play a crucial role in supporting lifelong general health in men, with low circulating testosterone linked to an increased risk of developing chronic age-related cardiometabolic diseases. However, the relationship between ageing, testicular function and disease is not fully understood, impeding the development of novel therapeutic strategies to treat age-related testicular dysfunction. In one set of studies undertaken herein, a series of novel mouse models of premature ageing were utilised to begin to dissect the process of age-related testicular degeneration. Firstly, a novel knockout-first conditional allele of a previously reported premature-ageing model driven by Cisd2 (CDGSH Iron Sulphur Domain 2) deficiency was validated and the testicular phenotype characterised and compared to that of naturally aged mice at 18-months of age. Histological analyses revealed premature testicular atrophy at 6-months of age in CISD2 deficient mice, consistent with observations of the naturally aged testis. Circulating testosterone was significantly lower in CISD2-deficient mice compared to wild-type controls at 6-months of age and the luteinising hormone/testosterone ratio was significantly elevated, indicative of compensated Leydig cell failure. mRNA expression of key genes involved in androgen production were also significantly reduced in the CISD2-deficient testis, pointing to Leydig cell dysfunction in this model of premature aging. Next, Cre/LoxP technology was used to delete Cisd2 from specific testicular cell populations to determine which cell types control/support Leydig cell function during the ageing process. Testosterone production was unaffected when Cisd2 was disrupted in either the Leydig cell population or Sertoli cell population. These observations suggest that disruption to the testicular microenvironment in which Leydig cells reside, rather than intrinsic Leydig cell ageing, may play a significant role in age-associated Leydig cell dysfunction. A second set of studies were carried out to investigate the role of leukemia inhibitory factor (LIF) signalling in the maintenance of testicular function. LIF is a pleiotropic cytokine belonging to the interleukin-6 family. In the rodent testis, LIF is expressed in fetal life and adulthood; the peritubular myoid cells thought to be the main site of production. Given their anatomical location within the testis, LIF produced by peritubular myoid cells may act on both intratubular and interstitial cells to influence spermatogenesis and steroidogenesis respectively. Indeed, LIFR is expressed in germ cells, Sertoli cells, Leydig cells as well as testicular macrophages suggesting that LIF may be a key paracrine regulator of testicular function. However, the precise role of LIF/LIFR signalling in the testis is largely unknown. As such, models of testicular cell-specific Lifr deletion were generated using Cre/LoxP technology. Analysis of these novel models of conditional LIFR ablation revealed that LIFR is dispensable in germ cells for normal spermatogenesis. However, LIFR ablation from Sertoli cells resulted in a progressive degenerative phenotype, characterised by abnormal germ cell loss, sperm stasis, seminiferous tubule distention and subsequent atrophy of the seminiferous tubules. In a final set of studies, a rat model of Leydig cell ablation-regeneration was used to determine the regenerative capacity of human adipose-derived perivascular stem cells (hAd-PSC) as a potential therapy for testicular dysfunction. Following ethane dimethanesulphonate (EDS) mediated Leydig cell ablation, primary hAd-PSCs, cultured with or without LH, IGF-1, PDGFBB, T3 and ITS supplement, were transplanted into the rat testis and Leydig cell regeneration was monitored via serial measurements of circulating luteinising hormone (LH) and testosterone. Overall, hAd- PSCs had no impact on the recovery of circulating testosterone levels. However, when pre-cultured with the cocktail of hormone/growth factor supplements, the LH spike induced by the removal of testosterone negative feedback was dampened, suggesting the transplanted cells may promote Leydig cell regeneration. Whether these cells differentiate into Leydig cells, or simply provide paracrine support to the regenerating Leydig cells remains to be determined. Although Ad-PSCs may enhance regeneration kinetics, the transplanted cells were undetectable in the testis 5 weeks post transplantation suggesting they may not survive in the context of long term xenogeneic transplantation.

Orientadores: Michel Georges Albert Vincentz, Juliana de Maria Felix / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-11T18:43:41Z (GMT). No. of bitstreams: 1 Branco_DianaSantos_M.pdf: 18385613 bytes, checksum: 43238e785391538d38599321de0ac5ca (MD5) Previous issue date: 2008 / Resumo:Além de fonte primária de carbono e energia para os principais tipos celulares, os açúcares produzidos pela fotossíntese adquiriram importantes funções ao longo da evolução das plantas, no controle do crescimento e desenvolvimento, do metabolismo e na resistência a estresses abióticos (osmótico, energético) e bióticos (potógenos). Os açúcares atuam como sinalizadores ativando cascatas de transdução e, desta forma, promovendo mudanças na programação da expressão gênica. Com o objetivo de entendermos como a sinalização por açúcares diversificou-se em angiospermas, iniciamos uma análise comparativa dos perfis de expressão gênica em resposta aos açúcares sacarose e glicose em plântulas da monocotiledônea Saccharum spp e da eudicotiledônea Arabidopsis thaliana. Para tanto, duas abordagens foram utilizadas. O primeiro aspecto do trabalho estabeleceu relações entre elementos de resposta rápida (resposta primária) a açúcar e acúmulo de sacarose em genótipos de cana contrastantes para teor de sacarose. Outra abordagem, mais abrangente, procurou identificar genes diferencialmente expressos em resposta à sacarose. Na primeira parte do trabalho, a análises por qRT-PCR revelaram uma clara relação entre genes envolvidos em acúmulo de sacarose em cana-de-açúcar e sinalização primária por carboidratos. A partir de 34 SAS (Sugarcane Assembled Sequence) testados envolvidos em acúmulo de sacarose em cana, 24 deles também foram responsivos à glicose e/ou sacarose, sendo que 9 deles responderam em um mesmo sentido em genótipos de cana-de-açúcar que acumulam maior quantidade de sacarose (alto Brix). Dos 24 SAS responsivos à sacarose e/ou glicose, apenas 6 deles apresentaram genes ortólogos em Arabidopsis thaliana cuja regulação por estes açúcares ocorreu de maneira similar. Dentre eles, temos o fator de transcrição IAA16, que se mostrou reprimido por sacarose e glicose, constituindo um possível gene de interação entre sinalização por açúcares e auxina. Duas SNFs quinases parálogas de cana-de-açúcar tem como ortólogo um único gene de Arabidopsis thaliana. Os três genes foram reprimidos por sacarose e glicose, sendo outra parte conservada, na via de sinalização a açúcares entre as duas espécies. Outro gene de particular interesse corresponde a uma deidrina, reprimida por sacarose e glicose em cana, assim como seu ortólogo em Arabidopsis e genótipos alto Brix, sugerindo importante papel deste gene em processos relacionados a sinalização/acúmulo de sacarose. Na segunda parte do trabalho, utilizando-se a técnica de microarranjos de cDNA a partir do chip SUCAST, encontramos 55 genes diferencialmente expressos em resposta à sacarose. Destes, apenas 3 apresentaram genes ortólogos de Arabidopsis regulados por açúcar num mesmo sentido que em cana, correspondentes a duas proteínas quinases e a um gene pseudo-response-regulator. Este estudo preliminar identificou genes conservados da sinalização por açúcares em angiospermas que representam possíveis nós importantes das redes de controle relacionadas a carboidratos. O estabelecimento de um possível envolvimento de alguns destes genes no controle da capacidade de acumular mais sacarose no colmo da cana, abriu novas perspectivas na análise molecular desta importante característica. Estudos mais abrangentes são necessários para melhorar os conhecimentos sobre o grau de diversificação da sinalização por açúcares em angiospermas e os valores adaptativos associados. / Abstract: Besides act as carbon primary source in the major types of cells, sugars produced by photosynthesis acquired important functions in the course of plant's evolution like controlling growth, development, and metabolism and acting in resistance to abiotic and biotic stresses like osmotic, energetic and response to pathogens. Sugars can be signals that active signal transduction pathways to change genes expression programs. In order to access the diversification of sugar pathway signaling in angiosperms we conduct comparative analysis of the gene expression in response to sucrose and glucose in seedlings of the monocot Saccharum sp. and the eudicot Arabidopsis thaliana. We also aimed to access the possible correlation between genes related to sucrose storage in sugar-cane and genes related to primary sugar responses. Another aim was to identify deferentially expressed genes in sucrose response. A clearly relation between genes related to sucrose storage in sugar-cane and quickly primary response to sugars was obtained by qRT-PCR analysis. We tested 34 SAS (Sugar Assembled Sequence) related to sucrose storage in sugar-cane and we found that 24 of them were responsive to glucose and/or sucrose. Nine genes showed the same expression pattern (induction or repression) in response to sugar as seen in high Brix genotypes. Six, of this 24 genes, have Arabidopsis orthologues regulated in the same direction (induced or repressed). One is an IAA16 transcription factor that is repressed by both, glucose and sucrose, and may play a role in an integrative pathway of sugar and auxin responses. We also find two SNFs kinases (paralogues) related to a single Arabidopsis ortholog showing the repression response. Another interesting gene is a dehydrin that was repressed in response to sucrose and glucose in sugar-cane and Arabidopsis (its ortholog) and in the high Brix sugar-cane genotypes. It suggests an important role for this dehydrin in processes related to sucrose signaling and storage. In the second part of this work, the sugar-cane cDNA microarray chip, called SUCAST, allow us to identify 55 deferentially expressed in response to sucrose. Only three of these genes have orthologues regulated in same way in sugar-cane and Arabidopsis. These genes correspond to two protein-kinase and a pseudo-response regulator. This preliminary approach leads us to identify conserved genes in sugar signaling among angiosperms that possibly represents important nodes in the regulatory networks in response to sugars. Establishing the involvement of some of these genes in the ability of sucrose storage in sugar-cane's culm will lead us to new perspectives in the molecular basis of this characteristic. More specific works are also needed to improve the knowledge about the real degree of evolutive diversification in sugar signaling among angiosperms and associated genetic fitness. / Mestrado / Genetica Vegetal e Melhoramento / Mestre em Genética e Biologia Molecular

The objective with this thesis is to describe security mechanisms that are inte-grated or are proposed to be integrated with the Session Initiation Protocol (SIP). SIP is used for establishing, modifying, and terminating multimedia ses-sions over the IP network. This thesis is divided into two main parts, where the first part describes the implemented security mechanisms in SIP and the second part describes a number of proposed security mechanisms that may be implemented in SIP. At the end of the report there is a section that presents the scripts and results from different security tests that were performed on two implementations of SIP. Apart from describing different security mechanisms in the first part of this thesis, this section also contains an analysis on how possible security threats against SIP may be used to launch different attacks. The analysis also describes how these attacks may be prevented, if possible, by using the secu-rity mechanisms provided by SIP. The second part also contains an analysis section, which is focusing on finding the advantages and disadvantages of using a specific security mechanism compared to a similar security mechanism that is currently used or has been used in SIP. In the last section of this thesis I present my conclusions and a summary of the results.

Abstract Secreted signalling molecules are important for the regulation of developmental cell responses. In the developing kidney, signalling occurs between epithelial ureteric bud and metanephric mesenchyme and in between their derivatives. Wnt6 gene activity was localized to the ureteric bud and newly formed branches of the ureteric tree during early stages of kidney development. In a classic organ culture system, Wnt6 signalling induced the activation of marker genes for early nephrogenesis. The metanephric mesenchymes isolated from the Wnt4 deficient embryos were also induced, and the Wnt4 gene became activated in the presence of a Wnt6 signalling source. We propose that Wnt-6 is involved as a metanephric inducer and controls nephrogenesis. Wnt4 is essential for nephrogenesis in mouse and we indicate an additional role for Wnt4 in the control of periureteric stromal differentiation. A failure in vascular development was also found. Bmp4 expression in the medullar stroma of the Wnt4-deficient kidneys was absent concomitantly with a loss of expression of the smooth muscle marker, α-SMA. In vitro Wnt4 signalling induced Bmp4 expression and local α-SMA production. Hence, we conclude that lack of Wnt4 signalling leads to a loss of the periureteric smooth muscle cells, and Wnt4 may locally regulate this cell population in normal kidneys via regulation of Bmp4 signalling. The pluripotent neural crest cells are proposed to play regulatory roles in the early metanephros. Here, the use of transgenic animals allowed visualisation of the lumbo-sacral neural crest (NC) cells in close proximity to the early metanephros. The NC cells, however, disappeared in most part of the kidney by E12.5. The Splotch embryos lack the NCs from the early urogenital region. A developmental defect in the kidneys of Splotch embryos was not observed in vivo or in vitro. The results suggest that the neural crest is not essential for early embryonic kidney development. In sum, the work presented indicates an important role for Wnt6 in the induction of kidney tubules in vitro, for Wnt4 in the specification of kidney smooth muscle cells and for endothelial development in kidney. The neural crest cells apparently have no active morphogenetic role in early kidney development.

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