O adenocarcinoma ductal pancreático (PDAC, pancreatic ductal adenocarcinoma), o tipo mais prevalente de câncer do pâncreas, é uma neoplasia extremamente agressiva e com elevado índice de letalidade. Há uma necessidade premente de identificação de vulnerabilidades no PDAC que possam ser exploradas como alvos terapêuticos, e a utilização de modelos pré-clínicos que recapitulem a complexidade biológica e heterogeneidade clínica da doença é um aspecto central para a realização dessa tarefa. Os xenotransplantes de tecido tumoral derivado de pacientes (PDX, patient-derived tumor tissue xenografts), realizados em camundongos imunodeficientes, replicam com grande similaridade as principais características do tumor original e, assim, constituem uma ferramenta valiosa para o teste de drogas e estudos funcionais. Neste trabalho, 17 amostras cirúrgicas de PDAC humano foram implantadas subcutaneamente em camundongos nude atímicos. Sete tumores (41%) foram enxertados com sucesso e têm sido mantidos em sucessivas gerações de animais receptores. O exame histológico de seis desses xenoenxertos identificou características morfológicas compatíveis com os padrões reconhecidos no PDAC humano, assim como uma consistente similaridade de seu status de diferenciação histológica em relação aos perfis verificados nos tumoresoriginais. O cultivo in vitro de células derivadas de um dos xenotumores resultou em uma nova linhagem de câncer de pâncreas, com morfologia e cinética de crescimento comparáveis às de outras linhagens celulares de câncer pancreático. O potencial tumorigênico dessa nova linhagem foi validado in vivo, com uma consistente formação de tumores após inoculação em camundongos nude. A fim de aproveitar esse recurso para a investigação de potenciais alvos terapêuticos no PDAC, um rastreamento de vulnerabilidades moleculares foi realizado por meio de silenciamento gênico em larga-escala com RNA de interferência (RNAi). Uma biblioteca lentiviral de 4492 shRNAs (short hairpin RNAs), alvejando cerca de 350 genes envolvidos na regulação epigenética, foi empregada para a triagem de genes de suscetibilidade nas células derivadas de PDX, e em outras cinco linhagens tumorais pancreáticas (AsPC-1, BxPC-3, Capan-1, MIA PaCa-2 e PANC-1). Inicialmente, foi realizada uma série de experimentos preliminares, visando à amplificação e controle de qualidade da biblioteca de silenciamento, à produção de vetores lentivirais e à padronização das condições experimentais para a transdução e seleção das células-alvo. Apenas três das linhagens avaliadas (AsPC-1, MIA PaCa-2 e PANC-1) mostraram-se permissíveis à transdução pelos vetores lentivirais, e foram assim utilizadas no screening de alvos epigenéticos. A análise dos dados obtidos nesse ensaio está em curso e os resultados serão utilizados para a definição de potenciais alvos candidatos. Em conclusão, recursos valiosos para apoiar a pesquisa sobre o câncer de pâncreas foram desenvolvidos. A coleção de PDXs estabelecida, bem como a linhagem celular recém-derivada, constituem uma fonte permanente e estável de células de PDAC para análises moleculares e estudos funcionais que busquem elucidar aspectos da doença ainda pouco compreendidos. Adicionalmente, os reagentes gerados e a expertise adquirida com os ensaiosrealizados com a biblioteca de shRNAs contra alvos epigenéticos serão de grande utilidade em futuras investigações para identificar genes com funções importantes na manutenção do fenótipo tumoral, e consequentemente com potencial para serem explorados terapeuticamente. / Pancreatic ductal adenocarcinoma (PDAC), the most prevalent type of pancreatic cancer, is a highly aggressive and lethal neoplasm. There is a pressing need to identify vulnerabilities in PDAC suited to be exploited as therapeutic targets, and the use of preclinical models recapitulating the biological complexity and clinical heterogeneity of the disease is central to this task. Patient-derived tumor tissue xenografts (PDX), established in immunodeficient mice, replicate with great similarity the main characteristics of the original tumor and thus constitute a valuable tool for drug testing and functional studies. In this work, 17 surgical samples of human PDAC were implanted subcutaneously in athymic nude mice. Seven tumors (41%) were successfully grafted and have been maintained through successive generations of recipient animals. Histological examination of six of these xenografts identified morphological characteristics compatible with the recognized patterns of human PDAC, as well as a consistent similarity of their histological differentiation status in relation to the profiles verified in the original tumors. In vitro culture of cells derived from one of these xenografts resulted in a new pancreatic cancer cell line, with morphology and growth kinetics comparable to those of other pancreatic tumor cells. The tumorigenic potential of this freshly derived cell line was validated in vivo, with a consistent tumor formation following inoculation into nude mice. To take advantage ofthis resource to investigate potential therapeutic targets in PDAC, a screening of molecular vulnerabilities was performed through large-scale gene silencing with RNA interference (RNAi). A lentiviral library containing 4492 short hairpin RNAs (shRNAs), targeting about 350 genes involved in epigenetic regulation, was employed for the search of susceptibility genes in the PDX-derived cells and in other five pancreatic tumor cell lines (AsPC-1, BxPC -3, Capan-1, MIA PaCa-2 and PANC-1). Initially, a series of preliminary experiments were carried out aiming at the amplification and quality control of the silencing library, production of lentiviral vectors and adjustment of the experimental conditions for transduction and selection of the target cells. Only three of the cell lines evaluated (AsPC-1, MIA PaCa-2 and PANC-1) were permissible for transduction by the lentiviral vectors, and were accordingly used in the screening of epigenetic targets. The analysis of data obtained in this trial is ongoing and the results will be used for definition of potential candidate targets. In conclusion, valuable resources to support research on pancreatic cancer have been developed. The established collection of PDXs as well as the newly derived cell line constitutes a permanent and stable source of PDAC cells for molecular analyzes and functional studies seeking to elucidate aspects of this disease that are still poorly understood. Additionally, both the reagents generated and the expertise gained from the RNAi assay against epigenetic targets will have inordinate usefulness in future investigations to identify genes with major functions in maintaining the malignant phenotype, and consequently with the potential to be exploited therapeutically.

The largest U.S. cancer health disparity exists in prostate cancer, with African American men having the highest incidence and mortality rates. The present study evaluated the effects of ZIC2 and the underlying mechanisms in the E006 parental African-American cell line that produces tumors at accelerated growth rates because of the increase of ZIC2 genes in African-American males. We analyzed the experimental research that the overexpression of ZIC2 contributes to progression of prostate cancer. E006AA cells with overexpressed or suppressed ZIC2 were analyzed to determine phenotypic differences, PCR, cell proliferation and immunoblot assays. The expression levels of ZIC2 were analyzed by CRISPR-Cas9, Western blot and proliferation growth curves. We discovered using these experimental techniques to knockout ZIC2, reduced cell proliferation occurred. This research investigated the role of ZIC2 in prostate cancer progression and the effects of the loss or gain of function of ZIC2 by using CRISPR-Cas 9 genome editing technology.

Cultura de células de carrapatos, é uma ferramenta importante, para isolar e estudar os agentes de doenças transmissíveis. Amblyomma sculptum é o principal vetor de Rickettsia rickettsii, o agente da febre maculosa. O objetivo deste estudo foi estabelecer e caracterizar as células deste carrapato, além de testar seu potencial uso, como substrato para o crescimento e isolamento de patógenos. A partir de massas de ovos de A. sculptum, culturas primárias foram, preparadas em meio L-15B, subcultivadas ao atingirem a confluência necessária e criopreservadas em diversas passagens. A boa recuperação celular estabeleceu a linhagem IBU/ASE-16, a qual foi testada para, Rickettsia spp, Trypanosoma theileri e Leishmania infantum chagasi, obtendo-se bons resultados. A citometria de fluxo analisou a expressão de marcadores de células-tronco, proliferação, diferenciação e regulação do ciclo celular, nas IBU/ASE-16. O Δψm mostrou atividade das mitocôndrias e ausência de células inativas. A microscopia confocal, localizou estruturas celulares marcadas com fluorocromos, enquanto que a MEV,mostrou, junções intercelulares, matriz extracelular e redes neuronais. O teste de tumorigênese em camundongos Balb/c nu/nu, não resultou em crescimento de tumores. / Cell cultures of ticks are important tools to isolate and study agents of transmissible diseases. Amblyomma sculptum is the main vector of Rickettsia rickettsii, the agent for spotted fever. The aim of this study was to establish and characterize the cells of this tick, and test their potential use as a substrate for the growth and isolation of pathogens. From a egg masses of A. sculptum, primary cultures were prepared in L-15B medium, subcultured as they reached the necessary convergence and cryopreserved in several passages. The good cell recovery established IBU/ASE-16 lineage, which was tested for Rickettsia spp, Trypanosoma theileri and Leishmania infantum chagasi, yielding good results. Flow cytometry analyzed the expression of stem cell markers, proliferation, differentiation and regulation of the cell cycle, in the IBU/ASE-16 lineage. The Δψm showed activity of mitochondria and absence of inactive cells. Confocal microscopy located cell structures marked with fluorochromes, while MEV showed intercellular junctions, extracellular matrix and neural networks. Tumorigenesis tests in Balb/c nu/nu mice resulted in no tumor growth.

Diversas linhagens de células de carrapatos já foram estabelecidas em outras regiões do mundo para isolamento de patógenos. O objetivo deste estudo é obter cultivos de células de Rhipicephalus sanguineus para testar o crescimento de alguns bioagentes. O primeiro capítulo trata do estabelecimento da linhagem celular e o segundo, do uso da linhagem como substrato para infecção de patógenos. Ovos de R. sanguineus foram preparados em meio L-15B e mantidos à 30 °C. Quando as células se tornaram confluentes, as culturas foram propagadas e criopreservadas. O descongelamento foi bem sucedido a partir da terceira passagem e a identidade celular foi confirmada por sequenciamento utilizando o fragmento 16S rDNA mitocondrial. As células foram infectadas com Erhlichia canis, Leishmania infantum chagasi e Trypanosoma cruzi, sendo eficientes somente para E. canis. / Several lines of embryonic cells of ticks already been established in other regions of the world, and are used to isolate and propagate pathogens. The aim of this study, is to obtain cell cultures from Rhipicephalus sanguineus to test the growth of some bioagents.The first addressing consists of the establishment of the cells and the second, using the cell lineage as a substrate for the pathogens infection. The egg masses of R. sanguineus were prepared in L-15B medium and cultures were maintained at 30 °C. When a confluent cellular monolayer was obtained, the cultures were passaged and frozen. Defrosting of cryopreserved cultures from the third passage was successful. Cell identity was confirmed by sequencing using 16S rDNA gene fragment. Cells were infected with Erhlichia canis, Leishmania infantum chagasi and Trypanosoma cruzi. Only for E. canis the cells of R. sanguineus were effective as a substrate for growth of this pathogen.

Respiratory distress syndrome and pulmonary oedema often result in poor health and in the worst case scenario, death. Several studies have proposed that the eventual resolution of these dangerous conditions is due to active sodium reabsorption through the epithelial sodium channel (ENaC), which is crucial for lung fluid clearance. Although clinical prognosis can be improved by using glucocorticoid hormones to augment the ENaC-dependent removal of liquid from the lungs, we still require a better understanding of the underlying mechanism in order to improve treatments in the future. This thesis, therefore explores the role of serum / glucocorticoid-inducible protein kinase 1 (SGK1) and protein kinase A (PKA) in the responses of hormone-stimulated H441 human airway cells. Dexamethasone, a synthetic glucocorticoid hormone, is thought to evoke expression of the gene encoding SGK1 and, to become catalytically active, this gene product must then be phosphorylated via TORC2 and PDK1, protein kinases activated via the P13-kinase pathway. Once activated, SGK1 appears to exert control over the surface abundance of ENaC subunits by phosphorylation, and thus inactivating, a ubiquitin ligase (Nedd4-2), that normally mediate the withdrawal of ENaC subunits from the plasma membrane. Protein kinase A (PKA) may contribute to this control mechanism by also phosphorylating Nedd4-2. In order to clarify the way in which these pathways contribute to glucocorticoid-induced lung liquid clearance, the present thesis has explored the effects of dexamethasone and / or PKA activation upon the overall / surface expression of ENaC subunits, the activities of SGK1 and PKA and the phosphorylation status of physiologically-important residues within Nedd4-2 itself.

A síndrome de Mayer-Rokitansky-Kuster-Hauser (MRKH) caracteriza-se pela aplasia congênita dos ductos Mullerianos. Devido a algumas características peculiares, as células-tronco mesenquimais estão sendo vistas como uma nova alternativa de tratamento em pacientes acometidos pela síndrome de MRKH. Considerando que alguns biomateriais servem como suporte estrutural e interferem positivamente na regeneração tecidual, a associação da linhagem celular de mucosa vaginal HMV-II e das MSC derivadas de tecido adiposo humano com biomateriais apresenta uma nova possibilidade na criação de neovagina. Nesta perspectiva, foram cultivadas células HMV-II com diferentes biomateriais (Membracel, Biofilme, Cellprene, PLGA PI quimicamente modificado) a fim de selecionar o melhor material alternativo. Ambas as células, associadas ao biomaterial selecionado, foram submetidas à análise morfológica, coloração ácido periódico-Schiff (PAS), expressão de marcadores epiteliais específicos por imunofluorescência e microcopia eletrônica de varredura (MEV). As células que interagiram com o biomaterial apresentaram marcadores epiteliais específicos e características morfológicas epiteliais. Estes resultados indicam que a interação do biomaterial com ambas as células testadas tem potencial capacidade para uma epitelização eficiente da neovagina. O crescimento das MSC com o biomaterial selecionado para implantação subsequente em pacientes com síndrome de MRKH pode representar uma alternativa válida e promissora para a reconstrução vaginal. / The Mayer-Rokitansky-Kuster-Hauser (MRKH) syndrome is characterized by congenital aplasia of the Mullerian ducts. Because mesenchymal stem cells (MSCs) secrete paracrine factors, they have been seen as a new treatment option for several diseases. Considering that some biomaterials can be used as scaffolds and interfere positively in tissue regeneration, the association of human vaginal mucosa (HMV-II) cell line and MSCs with biomaterials appears as a new option for the creation of neovagina. In this study we cultured HMV-II cells with different biomaterials (Membracel, Biofilm, Cellprene, chemically modified PLGA PI) to select the best alternative material. For that both cells were cultured with the selected biomaterial and evaluated by morphological analysis, periodic acid-Schiff (PAS) staining, expression of epithelial markers by immunofluorescence and scanning electron microscopy (SEM). The analysis of the in vitro cell-biomaterial interactions showed specific epithelial markers and epithelial morphological features for both cells. These results indicate that the interaction of the biomaterial with the two tested cells has the potential capacity for an efficient epithelialization of the neovagina. Therefore, growth of MSCs with the selected biomaterial for subsequent implantation in patients with MRKH syndrome may represent a valid and promising alternative for vaginal reconstruction.

This thesis contends that technologically-derived resurrections of human bodies and bodily fragments can be viewed as indicative of a 'post-biological' ontology. Drawing from examples in which human bodies are resurrected, both figuratively and actually, this thesis puts forward the term 'post-biological subject' as an ideological framework for conceptualising the reconfiguration of human ontology that results from various medical technologies that 'resurrect' the human body. In this instance, the term 'postbiological', borrowed from Hans Moravec who uses it denote a future in which human being is radically disembodied and resurrected within a digital realm, is used somewhat ironically: where Moravec imagines an afterlife in which the body is discarded as so much 'meat', the post-biological afterlife of the body in this thesis centres around a form of corporeal resurrection. Corpses, living organs and excreta may all be resurrected, some of them in digital format, yet this kind of resurrection departs radically from the disembodied spiritual bliss imagined in many conceptualisations of resurrection. The post-biological subject resists ontological delineation and problematises boundaries defining self and other, living and dead, and human and nonhuman and is fraught with a number of cultural anxieties about its unique ontological status. These concerns are analysed in the context of a number of phenomena, including melancholy, horror, monstrosity and the uncanny, all of which similarly indicate an anxious fixation with human ontology. The purpose of discussing post-biological bodies in relation to phenomena such as melancholy or the uncanny is not to reinstate as ideological frameworks the psychoanalytic models from which these concepts are derived, but rather to use them as starting points for more complex analyses of postbiological ontology. The first and second chapters of this thesis discuss instances in which the human body is posthumously modified, drawing on Gunther von Hagens's Body Worlds exhibition and the Visible Human Project. The Body Worlds plastinates are situated in a liminal and ambiguous ontological space between life and death, and it is argued that their extraordinary ontological status evokes a form of imagined melancholy, wherein the longed-for and lost melancholic object is a complete process of death. In the case of the Visible Human Project, it is argued that the gruesome and highly technologised process of creating the Visible Male, wherein the corpse is effectively dehumanised and iv rendered geometric, evokes the trope of horror, while at the same time being fraught with a nostalgic longing for a pre-technological, anatomically 'authentic' body. The third and fourth chapters of this thesis discuss instances in which the living human body is reconfigured, focusing on immortal cell lines and organ transplantation, and on medical imaging technologies such as computed tomography and magnetic resonance imaging. In the third chapter it is argued that organ transplantation and the creation of immortal cell lines give rise to profound anxieties about ontological contamination through their capacity to render permeable the imagined boundaries defining self, and in this way invoke the monstrous. The fourth chapter interrogates the representation of medical imaging in Don DeLillo?s novel White Noise, arguing that the medical representation of the body functions as a form of double, a digital doppelganger that elicits an uncanny anxiety through its capacity to presage death.

Dissertation (Ph.D.)--University of South Florida, 2008. / Includes vita. Includes bibliographical references. Also available online.

The fundamental goal of proteomics is to gain an understanding of the expression and function of the proteome on the level of individual proteins, on the level of defined cell types and on the level of the entire organism. In this thesis, the human proteome is explored using membrane protein topology prediction methods to define the human membrane proteome and by global protein expression profiling, which relies on a complex study of the location and expression levels of proteins in tissues and cells. A whole-proteome analysis was performed based on the predicted protein-coding genes of humans using a selection of membrane protein topology prediction methods. The study used a majority decision-based method, which estimated that approximately 26% of the human genes encode for a membrane protein. The prediction results are displayed in a visualization tool to facilitate the selection of antigens to be used for antibody generation. Global protein expression profiles in a large number of cells and tissues in the human body were analyzed for more than 4000 protein targets, based on data from the antibody-based immunohistochemistry and immunofluorescence methods within the framework of the Human Protein Atlas project. The results revealed few cell-type specific proteins and a high fraction of human proteins expressed in most cells, suggesting that cell and tissue specificity is attained by a fine-tuned regulation of protein levels. The expression profiles were also used to analyze the relationship between 45 cell lines by hierarchical clustering and principal component analysis. The global protein expression patterns overall reflected the tumor origin of the cells, and also allowed for identification of proteins of importance for distinguishing different categories of cell lines, as defined by phenotype of progenitor cell. In addition, the protein distribution in 16 subcellular compartments in three of the human cell lines was mapped. A large fraction of proteins were localized in two or more compartments and, in line with previous results, a majority of proteins were detected in all three cell lines. Finally, mass spectrometry-based protein expression levels were compared to RNA-seq-based transcript expression levels in three cell lines. Highly ubiquitous mRNA expression was found and the changes of expression levels between the cell lines showed high correlations between proteins and transcripts. Large general differences in abundance of proteins from various functional classes were observed. A comparison between categories based on expression levels revealed that, in general, genes with varying expression levels between the cell lines or only expressed in one cell line were highly enriched for cell-surface proteins. These studies show a path for a systematic analysis to characterize the proteome in human cells, tissues and organs. / QC 20110317 / The Human Protein Atlas project

Fertility is dependent on the precisely orchestrated communication of an array of effectors within the reproductive axis, all of which impinge on gonadotropin-releasing hormone (GnRH) neurons. A novel reproductive inhibitor was identified in avian species and growing evidence suggests that the functional mammalian homologue, RFamide-related peptide-3 (RFRP-3 or GnIH) can inhibit GnRH neuronal activity and gonadotropin release. To date, the regulation and effects of RFRP-3 at the hypothalamic level are poorly understood. We established an Rfrp-expressing neuronal cell model to investigate the mechanisms of transcriptional regulation of the genes for RFRP and the RFRP receptor, GPR147 by dexamethasone and estradiol. We show that the RFRP system is a direct target for stress-associated transcriptional regulation. Further, employing a novel GnRH-secreting cell line, we report that GnRH neurons express Gpr147 and RFRP-3 represses the transcription of GnRH. These data further our understanding of the level and regulatory effects at which RFRP-3 modulates reproduction.