Prostate cancer is the second most common cancer among men in the United States. While treatments for prostate cancer exist, none are curative. As a solid tumor, prostate cancer can grow beyond the diffusion limits of oxygen, thereby resulting in a hypoxic environment. While hypoxia can cause death to a variety of cell types, tumor cells can develop resistance to hypoxia and survive under minimal oxygen conditions. Hypoxia in tumor cells has also been associated with poor prognosis, increased metastasis, and decreased efficacy of chemotherapy. BNIP3, a BH-3 only proapoptotic Bcl-2 family member, has been shown to play an important role in cell death under hypoxic conditions in a variety of cell types. In normoxia, BNIP3 shows little to no expression in both cardiomyocytes and many cancer cell types, but is then upregulated under hypoxic conditions. Previous work in our laboratory provides evidence that hypoxia alone, as well as the concomitant increase in BNIP3 expression, cannot cause death of rat neonatal cardiomyocytes. Instead, our studies found that hypoxia with concomitant intracellular acidosis is required. Further studies indicated that BNIP3 is also necessary for hypoxia-acidosis associated cell death in cardiomyocytes. Our results in rat neonatal cardiomyocytes led us to hypothesize that cell death could be induced in hypoxic prostate cancer cells if concomitant acidosis could be induced. Additionally, our intention was to determine if BNIP3 was required for any prostate cancer cell death that may occur under hypoxia-acidosis conditions.

Activity of the hypoxia inducible HIF-alpha transcription factors drive the hypoxic response, resulting in enhancement of angiogenesis, tumour growth, invasion and metastasis. Seeking to uncover a role for Tid1 in control of HIF2-alpha, we used lentiviral shRNA to knock-down Tid1 in 786-0 RCC cells with and without pVHL. In 786-0 cells stably expressing pVHL30, Tid1 knock-down resulted in a dramatic reduction in HIF2-alpha levels relative to controls. Adenoviral-mediated overexpression of Tid1S rescued this decline in HIF2-alpha levels, while overexpression of Tid1L enhanced this decline. A protective role of Tid1S for HIF2-alpha was reproduced in a HEK293 cell model. Immunoprecipitations in HEK293 cells revealed a lack of direct binding between HIF2-alpha and Tid1 in vivo, while adenoviral-mediated overexpression of Tid1 in this model failed to alter in vitro binding between HIF2-alpha and pVHL30. We present a model in which Tid1 regulates HIF2-alpha stability through regulation of pVHL30 nuclear import.

Activity of the hypoxia inducible HIF-alpha transcription factors drive the hypoxic response, resulting in enhancement of angiogenesis, tumour growth, invasion and metastasis. Seeking to uncover a role for Tid1 in control of HIF2-alpha, we used lentiviral shRNA to knock-down Tid1 in 786-0 RCC cells with and without pVHL. In 786-0 cells stably expressing pVHL30, Tid1 knock-down resulted in a dramatic reduction in HIF2-alpha levels relative to controls. Adenoviral-mediated overexpression of Tid1S rescued this decline in HIF2-alpha levels, while overexpression of Tid1L enhanced this decline. A protective role of Tid1S for HIF2-alpha was reproduced in a HEK293 cell model. Immunoprecipitations in HEK293 cells revealed a lack of direct binding between HIF2-alpha and Tid1 in vivo, while adenoviral-mediated overexpression of Tid1 in this model failed to alter in vitro binding between HIF2-alpha and pVHL30. We present a model in which Tid1 regulates HIF2-alpha stability through regulation of pVHL30 nuclear import.

Introducción: El Factor Inducible por Hipoxia 1alpha (HIF-1alpha) es un factor de transcripción cuya actividad permite la adaptación celular a los cambios en las presiones parciales de oxígeno. HIF-1alpha se encuentra sobre-expresado en varios tumores humanos y en algunos de ellos su sobre-expresión se ha asociado con un peor pronóstico, sin embargo, su papel en los carcinomas epidermoides de cabeza y cuello (CECC) es controvertido.Material y Método:Análisis retrospectivo de 116 pacientes con carcinomas epidermoides supraglóticos. Mediante inmunohistoquímica se analizó la expresión de HIF-1alpha, p53, caspasa 3 y CD34. Posteriormente se desarrolló un modelo murino de CECC y en él se analizaron las consecuencias de la sobre-expresión de HIF-1alpha en los tumores inducidos por una línea celular derivada de un carcinoma epidermoide glótico.Resultados:En los carcinomas epidermoides supraglóticos, HIF-1alpha se encuentra frecuentemente sobre-expresado, siendo su expresión independiente del grado de vascularización tumoral. Su expresión de forma aislada no guarda relación con el pronóstico, ni influye en las características anatomopatológicas de los tumores desarrollados por nuestra línea celular.Discusión/Conclusiones:Los carcinomas epidermoides supraglóticos pobremente vascularizados, con sobre-expresión de HIF-1alpha y de p53 y bajos índices apoptóticos, presentan un pronóstico significativamente peor que el resto. En el momento actual, el uso indiscriminado de inhibidores de HIF-1alpha en el tratamiento de los carcinomas epidermoides de cabeza y cuello no esta justificado. El modelo murino de CECC presentado en este trabajo reproduce en múltiples aspectos el comportamiento de estos tumores en humanos, siendo útil tanto para estudiar aspectos básicos de la biología molecular, como para ensayos terapéuticos.

One of today’s most widely spread health problems is urinary incontinence, affecting 60-80% of the US population from age 15 and up. Treatment based on the possibility to implant a scaffold seeded with the patients’ own urine-derived stem cells, hUSC, to regenerate the damaged muscle tissue, would prove effective. A main challenge in regenerating new tissue from cell-seeded scaffolds is the limited cell survival due to insufficient oxygen diffusion to the center of the scaffold. Ways of enhancing cell survival, and thereby, proliferation and differentiation, is by hypoxic preconditioning of the cells or implantation in an oxygen-release material. Hypoxic preconditioning has shown to enhance proliferation as well as the expression of vascular endothelial growth factor, VEGF, in for example human bone marrow derived stem cells, hBMSC. VEGF is involved in the establishment of vasculature structures and an upregulation of its expression may therefore help promote quicker angeogenisis, increasing the oxygen supply and the cell survival. Oxygen-release materials have shown to enhance cell survival and growth both in vitro and in vivo. This study aims to investigate the effect of hypoxia on hUSC, during 9 days of hypoxic culturing (2.0% ± 0.1% O2) with and without oxygen-release material (PLGA 75:25 with 5 w% CPO) in vitro. hBMSC, and human smooth muscle cells, hSMC, have been used as control groups. Cell proliferation, morphology, differentiation, production of VEGF, and expression of hypoxia inducible factor HIF-1α have been studied. According to the results, combining hypoxic preconditioning of hUSC with implantation in oxygen-release material could be an effective way to regenerate muscular tissue. Hypoxic preconditioning enhanced cell proliferation, production of VEGF, and HIF-1α expression. The increase of VEGF and HIF-1α would promote vascularization when implanted. The oxygen-release material showed possible promotion of cell differentiation, which would augment the hUSCs’ myogenic differentiation, while supplying oxygen until the tissue’s vascular structure has been established.

<p>Malignant gliomas are highly lethal cancers characterized by florid angiogenesis. Glioma stem cells (GSCs), enriched through CD133 (Prominin1) selection, are highly tumorigenic and therapy resistance. However, the mechanism through which GSCs promote tumor growth was largely unknown. As we noticed that tumors derived from GSCs contain widespread tumor angiogenesis, necrosis, and hemorrhage, we examined thepotential of GSCs to support tumor angiogenesis. We measured the expression of a panel of angiogenic factors secreted by GSCs. In comparison with matched non-GSC populations, GSCs consistently secreted markedly elevated levels of vascular endothelial growth factor (VEGF), which were further induced by hypoxia. In an in vitro model of angiogenesis, GSC-conditioned medium significantly increased endothelial cell migration and tube formation compared with non-GSC glioma cell-conditioned medium. The proangiogenic effects of GSCs on endothelial cells were specifically abolished by the anti-VEGF neutralizing antibody bevacizumab, which is in clinical use for cancer therapy. Furthermore, bevacizumab displayed potent antiangiogenic efficacy in vivo and suppressed growth of xenografts derived from GSCs but limited efficacy against xenografts derived from a matched non-GSC population. As hypoxia is a key regulator of angiogenesis, I further examined hypoxic responses in GSCs to determine the molecular mechanisms underlying their angiogenic drive. I demonstrated that multiple hypoxia response genes, including the hypoxia-inducible factors (HIFs)-1a and -2a(EPAS-1) were differentially expressed in GSCs in comparison to non-stem glioma cells and normal neural progenitors. GSCs preferentially induced HIF2a; and HIF2a-regulated genes under hypoxia in comparison to non-stem glioma cells. In contrast, neural progenitor/stem cells did not induce HIF2a in response to hypoxia suggesting that the HIF2a hypoxic response is not a general stem cell response. Targeting HIF1a or HIF2a in GSCs using short hairpin RNA (shRNA) inhibited neurosphere formation efficiency, indicating a requirement for HIFs in cancer stem cell self-renewal. HIF1a and HIF2a were also necessary for VEGF expression in GSCs, but HIF2a was not required in matched non-stem glioma cells. In vivo experiments determined that knockdown of HIFs significantly attenuated the tumorigenic capacity of GSCs and increased survival of immunocompromised mice. Together, our work provides the first evidence that that GSCs can be a crucial source of key angiogenic factors in cancers due to their differential hypoxia responses. It also suggests that anti-angiogenic therapies can be designed to target GSC-specific molecular mechanisms of neoangiogenesis, including the expression and/or activity of HIF2a.</p> / Dissertation

Increased vascularization (angiogenesis) is a required adaptation for sustained tumor growth, and the primary mediator of de novo blood vessel formation is vascular endothelial growth factor (VEGF). The central transcriptional activator of VEGF is hypoxia inducible factor-1 (HIF-1), a heterodimeric transcription factor composed of an inducible HIF-1alpha subunit and a constitutively expressed HIF-1beta subunit. In addition to HIF-1, it has recently been reported that signal transducer and activator of transcription 3 (Stat3) is required for VEGF production and angiogenesis. Although it is known that Stat3 is an important mediator of many of the oncogenic signaling pathways that regulate HIF-1alpha, it was not known if Stat3 regulates HIF-1alpha. To answer this important question, the effect of blocking Stat3 signaling on both HIF-1alpha and VEGF expression was examined. Treatment of cells with IL-6, a potent activator of Stat3, resulted in HIF-1alpha and VEGF induction during normoxia. By blocking protein synthesis with cycloheximide, it was determined that IL-6 induction of HIF-1alpha resulted from increased translation. When Stat3 was silenced with siRNA, both basal level expression and IL-6 induction of HIF-1alpha and VEGF were significantly reduced. Furthermore, it is likely that Stat3 is required for HIF-1alpha induction by a variety of growth signals, as both HIF-1alpha and VEGF expression resulting from EGF and heregulin were abolished when Stat3 signaling was blocked. Because we had observed that Stat3 was required for induction of HIF-1alpha by growth signals, we wanted to determine if Stat3 was also required for HIF-1a induction by hypoxia. When Stat3 was silenced and cells exposed to hypoxia, HIF-1a expression was again abolished. Furthermore, the hypoxic induction of VEGF and MMP-2 was also prevented.

Epidemiology studies have established a strong link between chronic arsenic exposure and lung cancer. Currently, contribution of perturbed energy metabolism to carcinogenesis is an intensive area of research. In several human cell culture models (primary, immortal, malignant), we observed that non-cytotoxic exposure to arsenite increased extracellular acidification rate. Lactate accumulation caused by extracellular acidification, could be inhibited by 2-deoxy-D-glucose, a non-metabolized glucose analog. This established that arsenite induces aerobic glycolysis (the Warburg effect), a metabolic shift frequently observed in the acquisition of malignancy. Our studies in BEAS-2B, a non-malignant pulmonary epithelial cell line, found that the metabolic perturbation began early in the course of malignant transformation by arsenite (6 weeks). Correlated with the surge of glycolysis, we found elevated levels of HIF-1A and loss of E-Cadherin during chronic arsenite exposure. Our evidence suggests that this metabolic shift is sustained by HIF-1A (hypoxia-inducible factor 1A). We found that arsenite-exposed BEAS-2B accumulated HIF-1A protein, and underwent transcriptional up-regulation of HIF-1A-target genes. Overexpression of HIF-1A increases glycolysis 15% (vs. control), confirming that HIF-1A can modulate glycolysis in BEAS-2B. Coincident with induction of glycolysis, we observed a decrease in E-cadherin expression, indicating loss of epithelial identity. HIF-1A stable knockdown in BEAS-2B abrogated the arsenite induction of glycolysis, and indicated suppression in colony formation. These findings suggest that the hypoxia-mimetic effect of arsenite plays an important role in arsenite-induced malignant transformation. The significance of this study is that arsenite-induced alteration of energy metabolism represents the type of fundamental perturbation that could extend to many diverse effects caused by arsenic.

Research Doctorate - Doctor of Philosophy in Clinical Psychology / Since 1973, mental health professionals have rejected the historical view of homosexuality as being inherently pathological (American Psychiatric Association, 1973; Le Vay, 1996). However, research shows that some, but not all, gay men are at increased risk of a range of difficulties, including substance use, depression, anxiety, and suicide (e.g., Ashman, 2004; Fergusson, Horwood, & Beautrais, 1999; Gonsiorek, 1988; Kulkin, Chauvin, Percle, 2000; Meyer, 2003). The current research aimed to investigate (a) whether psychosocial well-being varied according to stage of gay identity development based on Cass’ (1979) model of homosexual identity formation (HIF); and (b) why such stage-based variations in well-being occur. Participants were self-identified gay men who completed internet-based questionnaires. Studies 2, 3 and 4 included experimental manipulations. Study 1 revealed that the relationship between HIF stage and psychosocial well-being represented a U-shaped function. The early confusion and comparison stages and late pride and synthesis stages of HIF were associated with good psychosocial well-being. In contrast, the middle tolerance and acceptance stages of HIF were associated with poor well-being. Study 2 revealed that acceptance stage participants demonstrated more closeting, lower in-group identification, lower membership collective self-esteem, and lower private collective self-esteem than did synthesis stage participants. However, none of these variables mediated the effects of HIF stage on well-being. In Study 3, I used improved measures of in-group identification and closeting and found that, compared to synthesis participants, acceptance participants reported greater identity salience and less global identification and used acting straight and closeting strategies to a greater extent. Importantly, in Study 4, I found that global identification, identity salience and the acting straight strategy independently mediated the effects of HIF stage on psychosocial well-being. These latter findings suggest that acceptance stage people have poorer well-being because (a) they identify less with the gay in-group, (b) they are more preoccupied with their gay identity, and (c) they make greater use of an acting straight strategy to manage their identity. Taken together, these findings lend empirical support to Cass’ (1979) model of HIF and contradict the notion that homosexuality is inherently pathological. Rather, individuals’ responses to membership of a negatively valued social group hold significant implications for their well-being. Two key implications follow from this research. First, at the individual level, the nature and timing of clinical interventions to assist gay men must be appropriate to their stage of HIF. Second, at the society level, broad social change is required to reduce stigma associated with gay identity.

Made available in DSpace on 2016-06-02T20:19:13Z (GMT). No. of bitstreams: 1 2912.pdf: 5230025 bytes, checksum: e6829f4e9fed0f66d6055d44a7e1c4f0 (MD5) Previous issue date: 2010-03-26 / Universidade Federal de Sao Carlos / Skin flaps are widely used in plastic surgery, mainly in reconstruction surgeries (transference of skin graft, pre-made tissues). After the surgery one of the major complication is ischemia, which may cause necrosis of the flap. Several features have been studied with the aim of increasing the viability flaps. Among these features, the low laser therapy is an alternative treatment, since it can promote an increase in microcirculation and vascular neoformation. However, there are discrepancies in the literature of the parameters employed in the use of laser, especially the fluence used in treatment . The aim of this study was to investigate the effects of 2 different laser wavelengths (660nm e 780nm) at 30 and 40J/cm2, on the viability of skin flap in rats evaluated by the paper template, vessels blood counting, activity of matrix metalloproteinase-2 (MMP-2), evaluation of plasma levels of NO, and expression hypoxiainducible Factor 1_. Sixty male animals Wistar were used in this study and they were distributed into the following groups (n=12 each group): control group, group irradiated with 660nm, at 30J/cm2; group irradiated with 660nm, at 40J/cm2 group irradiated with 780nm, at 30J/cm2, and group irradiated with 780nm, at 40J/cm2. The skin flap was performed on the back of all animals studied, with a plastic sheet interposed between the flap and the donor site. The animals received laser irradiation immediately after surgery and within 4 days, using the technical point of contact, on 24 points on the skin surface and around it. On the seventh postoperative day was evaluated the percentage of necrotic area and collected samples of tissue for histological analysis, zymography and protein expression by Western blotting, animals were euthanized by exsanguination, and blood were evaluated plasma levels of NO. The data obtained from the evaluation by the method of the paper template showed no increase in the viability of skin flaps after laser treatment. For biochemical analysis we found that the laser modulates the activity of MMP-2 and expression of HIF-1_ and induced an increase in the number of vessels especially in the groups irradiated with 40J/cm2 Measurement plasma level of NO did not differ between groups. Molecular analysis showed that the application of laser parameters used here, although it stimulated angiogenesis by modulating HIF-1_ and activity of MMP-2 was not able to improve the viability of skin flaps. Thus, we conclude that to find the beneficial effects of laser therapy we need to understand their mechanisms of action and know the best parameters to use. / Os retalhos cutâneos são amplamente utilizados na cirurgia plástica, principalmente na reconstrutiva. Após o procedimento operatório, uma das principais complicações é a isquemia, podendo ocasionar a necrose do retalho. Vários recursos têm sido estudados com o intuito de aumentar a viabilidade desses retalhos. Dentre esses recursos, o laser de baixa intensidade é uma alternativa de tratamento, uma vez que pode promover um aumento da microcirculação e da neoformação vascular. Entretanto, existem discrepâncias na literatura em relação aos parâmetros empregados no uso do laser de baixa intensidade, principalmente das fluências utilizadas nos tratamentos. Este estudo teve como objetivo verificar o efeito de 2 comprimentos de onda diferentes (660nm e 780nm), com fluências de 30 e 40J/cm2, na viabilidade do retalho cutâneo randômico em ratos, avaliados pelo método do gabarito de papel, contagem dos vasos sanguíneos, atividade da metaloproteinase de matriz -2 (MMP-2), avaliação dos níveis plasmáticos de NO, e expressão do fator induzível por hipóxia (HIF-1_). Sessenta ratos da linhagem Wistar foram usados nesse estudo, sendo distribuídos em 5 grupos (n=12): grupo controle, grupo irradiado com 660nm a 30J/cm2; grupo irradiado com 660nm a 40J/cm2, grupo irradiado com 780nm a 30J/cm2, e grupo irradiado com 780nm a 40J/cm2. O retalho cutâneo foi realizado no dorso dos animais com dimensões de 10 X 4cm e uma barreira plástica foi interposta entre o retalho e o leito doador. Os animais receberam irradiação laser imediatamente após a cirurgia e nos 4 dias seguintes, utilizando-se a técnica pontual em contato em 24 pontos distribuídos sobre e ao redor do retalho. No sétimo dia pósoperatório foi avaliado a porcentagem de área de necrose e coletado amostras de tecido para análise histológica, zimografica e de expressão protéica por western blotting, os animais foram eutanaziados por exsanguinação, e no sangue foram avaliados os níveis plasmáticos de NO. Os dados obtidos a partir da avaliação pelo método de gabarito de papel mostraram que não houve aumento na viabilidade dos retalhos cutâneos após tratamento com laser. Pelas análises bioquímicas observamos que o laser modulou a atividade de MMP-2 e a expressão de HIF-1&#61537;, bem como induziu o aumento no número de vasos especialmente nos grupos irradiados com 40J/cm2 independente do comprimento de onda utilizado. A medida do NO circulante não apresentou diferença entre os grupos. A análise molecular mostrou que a aplicação do laser com os parâmetros aqui utilizados, embora tenha estimulado a angiogênese por modular HIF-1&#61537;&#61472;e a atividade de MMP-2 não foi capaz de melhorar a viabilidade dos retalhos cutâneos. Assim, concluímos que para encontrarmos os efeitos benéficos e seguros da laserterapia precisamos compreender seus mecanismos de ação e conhecer os melhores parâmetros de utilização.