Kisspeptin (encoded by Kiss1) is a hypothalamic neuropeptide that is directly regulated by sex steroids and directly stimulates gonadotropin-releasing hormone (GnRH) neurons. Kisspeptin cell models were established in order to facilitate future molecular analysis of kisspeptin. mHypoA-51 and mHypoA-63 cell lines were found to express kisspeptin, estrogen receptor α and β, substance P, but not tyrosine hydroxyase. Furthermore, estrogen decreased Kiss1 expression in both cell lines. Based on these results, it was concluded that mHypoA-51 and mHypoA-63 are representative of arcuate kisspeptin neurons. Accumulating evidence also indicates that kisspeptin indirectly stimulates GnRH neurons through afferent neurons. Kisspeptin receptor expression was detected in native neuropeptide Y (NPY) neurons. Using the mHypoE-38 cell line, kisspeptin was found to directly regulate NPY mRNA expression and secretion via the ERK1/2 and p38 MAPK pathways. This is the first evidence that kisspeptin directly stimulates NPY neurons to potentially exert indirect effects on GnRH neurons.

Kisspeptin (encoded by Kiss1) is a hypothalamic neuropeptide that is directly regulated by sex steroids and directly stimulates gonadotropin-releasing hormone (GnRH) neurons. Kisspeptin cell models were established in order to facilitate future molecular analysis of kisspeptin. mHypoA-51 and mHypoA-63 cell lines were found to express kisspeptin, estrogen receptor α and β, substance P, but not tyrosine hydroxyase. Furthermore, estrogen decreased Kiss1 expression in both cell lines. Based on these results, it was concluded that mHypoA-51 and mHypoA-63 are representative of arcuate kisspeptin neurons. Accumulating evidence also indicates that kisspeptin indirectly stimulates GnRH neurons through afferent neurons. Kisspeptin receptor expression was detected in native neuropeptide Y (NPY) neurons. Using the mHypoE-38 cell line, kisspeptin was found to directly regulate NPY mRNA expression and secretion via the ERK1/2 and p38 MAPK pathways. This is the first evidence that kisspeptin directly stimulates NPY neurons to potentially exert indirect effects on GnRH neurons.

This dissertation evaluated the feasibility of using commercially available immortalized cell lines in building a tissue engineered in vitro blood-brain barrier (BBB) co-culture model for preliminary drug development studies. Mouse endothelial cell line and rat astrocyte cell lines purchased from American Type Culture Collections (ATCC) were the building blocks of the co-culture model. An astrocyte derived acellular extracellular matrix (aECM) was introduced in the co-culture model to provide a novel in vitro biomimetic basement membrane for the endothelial cells to form endothelial tight junctions. Trans-endothelial electrical resistance (TEER) and solute mass transport studies were engaged to quantitatively evaluate the tight junction formation on the in-vitro BBB models. Immuno-fluorescence microscopy and Western Blot analysis were used to qualitatively verify the in vitro expression of occludin, one of the earliest discovered tight junction proteins. Experimental data from a total of 12 experiments conclusively showed that the novel BBB in vitro co-culture model with the astrocyte derived aECM (CO+aECM) was promising in terms of establishing tight junction formation represented by TEER values, transport profiles and tight junction protein expression when compared with traditional co-culture (CO) model setups and endothelial cells cultured alone. Experimental data were also found to be comparable with several existing in vitro BBB models built from various methods. In vitro colorimetric sulforhodamine B (SRB) assay revealed that the co-cultured samples with aECM resulted in less cell loss on the basal sides of the insert membranes than that from traditional co-culture samples. The novel tissue engineering approach using immortalized cell lines with the addition of aECM was proven to be a relevant alternative to the traditional BBB in vitro modeling.

Prostate cancer (PCa) accounts for an estimated 20% of new cancer cases and 10% of deaths in just US males in 2020. Despite this prevalence, the molecular basis of its development and initiation remains unclear. To help identify the molecular basis of PCa progression, it is important to generate a collection of human prostate epithelial cells (hPrEC) that remain karyotypically normal and represent the epithelial cell types present in the human prostate. hPrEC can only go through a limited number of passages before they become senescent. Immortalization prevents senescence and enables continuous cell division. Our lab previously immortalized hPrEC cells by the expression of human telomerase (hTERT) with concomitant CRISPR inactivation of the CDKN2A locus, which directs the expression of both p16INK4A and p14ARF genes.Characterization of the two clonal cell lines that were generated showed that they maintained normal cell growth characteristics with intact p53 and pRb pathways, near normal karyotypes and have characteristics of basal cell origin. Subsequently, our lab sought to determine if expression of hTERT with knockout of just p16INK4A alone was also sufficient for immortalization, using CRISPR technology to inactivate exon 1α of the CDKN2A locus along with ectopic expression of the hTERT transgene. Knockout of p16INK4A but not p14ARF along with exogenous expression of hTERT resulted in the generation of a new immortal clone. Using these immortalized clones, along with primary hPrEC from ATCC our goal is to further characterize these cells to aid in future attempts aimed at immortalizing normal PrEC from multiple individuals and for the efficient establishment of a primary prostate cancer cell line. Our first approach included immunophenotyping our generated immortal hPrEC clones and ATCC hPrEC’s to identify the cell populations defining each of our clones and the different cell populations present in the primary hPrEC. We also characterized the expression of cells using 3D cell culture to determine their morphology and the expression of relevant markers. Finally, we identified the differentially expressed genes by RNA-seq in our immortalized hPrEC clones and ATCC hPrEC to determine their closest lineage identity as well as find suitable markers to use for future studies. These cell lines will also serve as a model to study transformation of PrEC in culture and xenograft tumorigenesis in mice. / Biomedical Sciences

No description available.

<p>Angiogenesis is the formation of new blood vessels from the pre-existing blood vessels. Blood vessels are composed of endothelial cells and supporting musculature. Angiogenesis is regulated by numerous soluble ligands and by cell-matrix interactions. We have studied the molecular mechanisms in fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor-A (VEGF-A)-induced angiogenesis using immortalized endothelial cell lines in different angiogenesis assays.</p><p>The role of the signaling protein H-Ras in FGF-2-induced <i>in vitro</i> angiogenesis was studied by expressing mutated versions of H-Ras in immortalized mouse brain endothelial cells using a tetracycline-regulated expression system. <i>In vitro</i> angiogenesis was analyzed as the ability of cells to invade a fibrin matrix and form branching structures in response to a combination of FGF-2 and tumor necrosis factor-α (TNF-α). Inhibition of H-Ras through the expression of dominant negative (S17N) H-Ras or pharmacological inactivation of H-Ras with a farnesyl transferase inhibitor, did not inhibit growth factor-induced invasion. In contrast, expression of constitutively active (G12V) H-Ras caused cells to adopt a transformed phenotype which inhibited invasive growth and cells formed solid tumors when injected in nude mice. These studies suggest that H-Ras activity is not required for differentiation but its activity must be tightly regulated as aberrant activity impairs endothelial cell differentiation.</p><p>In order to screen for both known and novel genes that regulate angiogenesis we used large scale microarray analysis. In VEGF-A-stimulated telomerase immortalized human microvascular endothelial cells undergoing invasive growth in fibrin gels, or forming cord-like structures on collagen, we identified several genes that were differentially expressed. Some of these are known to be important for endothelial cell functions and angiogenesis while others have no previous connection with endothelial cell function or were transcripts with no assigned function. Further analysis of these proteins will aid in elucidating the molecular mechanisms underlying endothelial cell differentiation. </p>

Angiogenesis is the formation of new blood vessels from the pre-existing blood vessels. Blood vessels are composed of endothelial cells and supporting musculature. Angiogenesis is regulated by numerous soluble ligands and by cell-matrix interactions. We have studied the molecular mechanisms in fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor-A (VEGF-A)-induced angiogenesis using immortalized endothelial cell lines in different angiogenesis assays. The role of the signaling protein H-Ras in FGF-2-induced in vitro angiogenesis was studied by expressing mutated versions of H-Ras in immortalized mouse brain endothelial cells using a tetracycline-regulated expression system. In vitro angiogenesis was analyzed as the ability of cells to invade a fibrin matrix and form branching structures in response to a combination of FGF-2 and tumor necrosis factor-α (TNF-α). Inhibition of H-Ras through the expression of dominant negative (S17N) H-Ras or pharmacological inactivation of H-Ras with a farnesyl transferase inhibitor, did not inhibit growth factor-induced invasion. In contrast, expression of constitutively active (G12V) H-Ras caused cells to adopt a transformed phenotype which inhibited invasive growth and cells formed solid tumors when injected in nude mice. These studies suggest that H-Ras activity is not required for differentiation but its activity must be tightly regulated as aberrant activity impairs endothelial cell differentiation. In order to screen for both known and novel genes that regulate angiogenesis we used large scale microarray analysis. In VEGF-A-stimulated telomerase immortalized human microvascular endothelial cells undergoing invasive growth in fibrin gels, or forming cord-like structures on collagen, we identified several genes that were differentially expressed. Some of these are known to be important for endothelial cell functions and angiogenesis while others have no previous connection with endothelial cell function or were transcripts with no assigned function. Further analysis of these proteins will aid in elucidating the molecular mechanisms underlying endothelial cell differentiation.

Sabe-se há décadas que mutações nos genes ras estão presentes em cerca de 20% dos cânceres humanos, mas o desenvolvimento de terapias eficazes para o tratamento de câncer dependente dos oncogenes ras permanece um desafio científico importante. Nesse contexto, o nosso grupo publicou recentemente resultados interessantes mostrando que FGF2 exógeno ou PMA, contrariamente à expectativa geral, inibem a proliferação de células de camundongo malignas dependentes dos oncogenes H- ou K-Ras. Para dar continuidade a estes estudos o projeto desta tese foi planejado para investigar os mecanismos subjacentes a possíveis efeitos citotóxicos de FGF2 e PMA em células humanas transformadas por ras. Para esse fim, a linhagem humana imortalizada HEK 293 foi condicionalmente transformada pela expressão ectópica da construção quimérica de DNA ER:H-rasV12, que codifica a oncoproteína de fusão ER:H-RasV12, cuja atividade é induzível por 4-hidroxi-tamoxifen (4OHT). Essa abordagem nos permitiu verificar os efeitos de FGF2 e PMA em sublinhagens HEK/ER:HrasV12 fenotipicamente \"normais\" ou transformadas por níveis crescentes da oncoproteína H-RasV12. Os principais resultados mostraram que tanto FGF2 como PMA tem efeito dual promovendo ou inibindo a proliferação das células transformadas em função da concentração intracelular crescente de H-RasV12. Ensaios de crescimento de colônias em suspensão de agarose mostraram que: a) as células parentais HEK293 não desenvolveram colônias mesmo quando tratadas com FGF2 ou PMA, resultados que estão de acordo com seu fenótipo não tumoral; b) mas, as sublinhagens HEK/ER:HrasV12 deram origem a colônias mesmo quando tratadas com concentrações pequenas de 4OHT, que condicionaram níveis intracelulares baixos de ER:HRasV12; nestas condições experimentais, FGF2 foi um forte promotor do crescimento de colônias, condizente com sua reconhecida atividade promotora do crescimento de células tumorais em suspensão; ainda nestas condições, PMA não teve efeito significante sobre o crescimento de colônias; c) coerentemente, concentrações elevadas de 4-OHT levaram aos níveis intracelulares mais altos de ER:HRasV12 e, por conseguinte, a desenvolvimento máximo de colônias de células HEK/ER:HrasV12, no entanto, nestas condições, ambos FGF2 e PMA inibiram completamente o crescimento de colônias. Por outro lado, transformação de HEK293 com um vetor de expressão constitutiva de HrasV12 levou à seleção e isolamento das sublinhagens tumorais HEK/HrasV12, cujo fenótipo se caracterizou por: a) nenhum efeito de FGF2 sobre a sua proliferação e b) forte inibição de sua proliferação por PMA. A ação citotóxica de PMA exclusivamente observada em células HEK 293 transformadas por H-rasV12 se caracterizou por: a) total dependência de PKC, provavelmente mediada pela ativação proteolítica específica de PKC &#948;; b) envolvimento de níveis elevados e sustentados de ROS com disparo tardio de apoptose. / It is known for nearly 20 years that mutated ras oncogenes are found in 20% of human malignancies, however efficacious therapies are not yet available for Ras-driven cancer. Along of these lines, our group recently published provocative results showing, against common belief, that FGF2 and PMA inhibited proliferation of Ras-dependent malignant mouse cells. Aiming to gain insight into this intriguing phenomenon, the present thesis project was planned to investigate the possible cytotoxicity of FGF2 and PMA in human Ras-driven malignant cells. To this end an immortalized non-tumorigenic human cell line (HEK293) was stably transformed with the DNA construction ER:H-rasV12, which encodes the fusion protein ER:H-RasV12, whose activity requires activation by 4-hidroxitamoxifen (4-OHT). This approach allowed us to evaluate FGF2 and PMA effects on HEK/ER:HrasV12 sublines under switching from \"normal\" to transformed phenotypes upon 4-OHT induction. Our main results have shown that both FGF2 and PMA displayed dual effects promoting or inhibiting proliferation of HEK/ER:HrasV12 cells in function of ER:HRasV12 intracellular levels. Clonogenic assays in agarose suspension have shown: a) parental HEK293 line did not develop colonies under FGF2 and PMA treatment or not, in agreement with its non-tumorigenic nature; b) however, HEK/ER:HrasV12 sublines developed colonies even under low 4-OHT concentrations, which led to low ER:HRasV12 intracellular levels; under these conditions FGF2 strongly promoted colony growth and PMA had no effect; c) furthermore, in HEK/ER:HrasV12 sublines, elevated 4-OHT concentrations led to high ER:HRasV12 intracellular levels and maximal colony growth; but, under these experimental conditions both FGF2 and PMA abolished colony growth. On the other hand, HEK293 transformation with a vector that constitutively express HrasV12 yielded HEK/ER:HrasV12 sublines displaying the following phenotypic traits: a) non FGF2 effects on proliferation and b) severe proliferation inhibition by PMA. PMA toxicity, exclusively observed in HrasV12 -transformed HEK293 cells, was characterized by: a) total dependency on PKC, likely mediated by specific proteolytic activation of PKC&#948;; b) involvement of high and sustained ROS levels correlated with late apoptosis triggering.

The chimeric Alb-uPA SCID mouse that has been transplanted with human hepatocytes is a model to facilitate in vivo study of HCV. We explored further development of the model by using repopulation with immortalized human hepatocytes (IHH) in place of primary human hepatocyte (PHH) transplantation to support HCV infection. In vitro HCV studies typically utilize a human hepatoma cell line (Huh7) and rely on transfection with transcribed genomic RNA derived from a unique HCV strain (JFH1). Unfortunately, this system has not been successful in support of infection with serum-derived HCV (HCVser). IHH may offer an alternative since their differentiation status remains close to that of PHH. IHH transfected with HCV RNA (H77 or JFH1) or infected with HCVser showed stable intracellular and supernatant HCV RNA by real-time RT-PCR. IHH showed intracellular HCV NS3 proteins. HCV transfected or infected IHH secrete infectious HCVcc for in vivo and vitro. / Experimental Surgery

No description available.