Dissertação (mestrado)—Universidade de Brasília, Programa de Pós-Graduação em Biologia Molecular, 2014. / Submitted by Marília Freitas (marilia@bce.unb.br) on 2015-10-29T12:33:32Z No. of bitstreams: 1 2014_NestorFabianLeytonCastro.pdf: 3484643 bytes, checksum: 3f92622240d0f7e59822aca3974ed379 (MD5) / Approved for entry into archive by Raquel Viana(raquelviana@bce.unb.br) on 2016-02-12T21:00:47Z (GMT) No. of bitstreams: 1 2014_NestorFabianLeytonCastro.pdf: 3484643 bytes, checksum: 3f92622240d0f7e59822aca3974ed379 (MD5) / Made available in DSpace on 2016-02-12T21:00:47Z (GMT). No. of bitstreams: 1 2014_NestorFabianLeytonCastro.pdf: 3484643 bytes, checksum: 3f92622240d0f7e59822aca3974ed379 (MD5) / A hipóxia ocorre quando as células são expostas a baixas tensões (< 5%) de oxigênio. Isto afeta o metabolismo, a expressão de genes, a secreção e resposta a hormônios e pode garantir a sobrevivência ou algumas vezes iniciar uma cascata apoptótica. O fator 1 induzível por hipóxia (HIF-1) humano regula a transcrição de inúmeros genes envolvidos na resposta à hipóxia e tem importante papel em processos fisiológicos e patológicos. Ele é um heterodímero formado pelas subunidades HIF-1α e HIF-1β, também chamada de transportador nuclear de arilhidrocarbono (ARNT). Em condições de normóxia, o HIF-1 é hidroxilado pelas prolilhidroxilases (PHDs) e marcado para degradação pelo proteasoma. Porém, quando as células se encontram sob hipóxia, as PHDs são inibidas e assim ocorre o acúmulo do HIF-1. Alterações no estado redox podem ter efeito na ativação do HIF-1 e foi verificado que condições de estresse oxidativo podem levar à ativação deste fator. O estresse oxidativo ocorre quando as moléculas antioxidantes e os mecanismos de defesa celular são incapazes de proteger a célula dos efeitos das espécies reativas de oxigênio (EROs) as quais atacam moléculas biológicas vitais, tais como lipídeos, DNA dentre outras. As EROs incluem os radicais livres derivados de oxigênio, entre essas o íon superóxido (O2●-), o radical hidroxila (●OH-), e outras moléculas que embora não possuam elétrons livres são muito reativas devido à sua instabilidade, como o peróxido de hidrogênio (H2O2). Para estudar os efeitos do HIF-1 sob condições de estresse oxidativo, escolhemos a levedura Saccharomyces cerevisiae, também conhecida como levedura de padeiro. A utilização da levedura S. cerevisiae tem provado ser muito útil como um modelo de sistema in vivo para estudar a ação de fármacos, a expressão de proteínas heterólogas e a função básica de fatores de transcrição humanos. Foram usadas três linhagens mutantes, EG103αβ a qual expressa as duas subunidades do fator de transcrição HIF-1, EG103αΔβ que expressa a subunidade beta truncada e a linhagem EG103M2 que possue o vetor vazio. O objetivo deste estudo foi avaliar a resposta ao estresse oxidativo dessas linhagens de leveduras para aprofundar o conhecimento sobre a regulação da atividade de HIF-1. O crescimento das linhagens foi investigado e os resultados mostraram que a linhagem EG103αβ teve um crescimento menor que as outras duas linhagens (EG103αΔβ e EG103M2) em normóxia. Quando tratada com várias concentrações de H2O2 o crescimento da linhagem EG103αβ diferiu do controle não tratado somente nas concentrações de 1 e 2 mM, quando houve uma diminuição do crescimento. Essa resposta foi similar àquela das outras duas linhagens. Então não foi uma resposta específica da linhagem EG103αβ, indicando que a expressão do HIF-1 não alterou a sensibilidade da resposta ao H2O2. Ao tratar as células com alguns agentes oxidantes como Menadiona (MD), hidroperóxido de cumene (CHP) e terc-butilhidroperóxido (t-BHP) os resultados mostraram que a linhagem EG103αβ apresentava uma susceptibilidade menor à MD na concentração 50 μM, em comparação com as outras linhagens. O uso do inibidor da catalase ATZ (10 mM) e H2O2 0,5 mM mostrou que a susceptibilidade da linhagem EG103αβ aumentou em relacão ao controle tratado somente com H2O2. Nas duas outras linhagens o aumento da susceptibilidade ocorreu com uma concentração menor de ATZ (2 mM) e H2O2 0,5 mM. Isto indica que talvez a linhagem EG103αβ tenha maiores concentrações de catalase em relação às outras duas linhagens. O uso do inibidor da síntese de glutationa, butionina-sulfoximina (BSO) não mostrou uma resposta diferenciada da linhagem EG103αΔβ à exposição ao H2O2. O uso do quelante de ferro, deferoxamina (DFO) também mostrou uma ação de proteção contra o dano oxidativo na linhagem EG103αβ, indicando um papel da ausência de ferro na proteção pelo HIF-1. Medidas dos níveis de glutationa (GSH-eq) e da enzima superóxido dismutase devem ser repetidas para conclusão definitiva de seu papel na linhagem EG103αβ. Em conclusão, os resultados indicam que a expressão das subunidades que compõem o HIF-1 humano em leveduras reduz seu crescimento, aumenta sua resistência à menadiona e talvez os níveis de catalase e faz com que um quelante de ferro tenha papel protetor nestas células. ______________________________________________________________________________________________ ABSTRACT / Hypoxia occurs when cells are exposed to low oxygen tensions (<5%). It affects the metabolism, gene expression, secretion and response to hormones and can ensure the survival or sometimes initiate an apoptotic cascade. The human hypoxia-inducible factor 1 (HIF-1) regulates the transcription of numerous genes involved in the response to hypoxia and plays an important role in physiological and pathological processes. HIF-1 is a heterodimer formed by the HIF-1α and HIF-1β subunits, the latter also called arylhydrocarbone nuclear transporter (ARNT). In normoxic conditions, HIF-1 is hydroxylated by prolylhidroxilases (PHDs) and marked for degradation by the proteasome. However, when cells are under hypoxia, the PHDs are inhibited and HIF-1 accumulates. Changes in the redox state may have an effect on HIF-1 activation and oxidative stress conditions have been to lead to the activation of this factor. Oxidative stress occurs when the antioxidant molecules and cellular defense mechanisms are unable to protect the cell from the effects of reactive oxygen species (ROS) which attack vital biological molecules such as lipids, DNA and others. ROS include free radicals derived from oxygen, among these the superoxide ion (O2●-), o hydroxyl radical (●OH-), and other molecules that although not having free electrons are very reactive due to its instability, such as hydrogen peroxide (H2O2). To study the effects of HIF-1 under conditions of oxidative stress, we chose the yeast Saccharomyces cerevisiae, also known as Baker's yeast. The use of S. cerevisiae has proven to be very useful as an in vivo model system to study the action of drugs, the expression of heterologous proteins and the basic function of human transcription factors. The three mutant strains, EG103αβ which expresses the two subunits of HIF-1 transcription factor, EG103αΔβ, expressing the truncated beta subunit and EG103M2 strain that possesses an empty vector were used. The objective of this study was to evaluate the oxidative stress response of the yeast strains to increase knowledge about the regulation of HIF-1 activity. The growth of the strains was investigated and the results showed that the strain EG103αβ had a slower growth than the other two strains (EG103αΔβ and EG103M2) under normoxia. When treated with various concentrations of H2O2 the growth of EG103αβ differed from the untreated control only at concentrations of 1 and 2 mM, when there was a decline in growth. This response was similar to that of the other two strains. So it was not a specific response of EG103αβ, indicating that HIF-1 expression did not change the sensitivity response to H2O2. By treating the cells with certain oxidizing agents such as Menadione (MD), cumene hydroperoxide (CHP) and tert-butylhydroperoxide (t-BHP) the results showed that the strain EG103αβ has lower susceptibility to 50 uM MD concentrations, compared to the other strains. The use of the catalase inhibitor ATZ (10 mM) and 0.5 mM H2O2 showed that the susceptibility of EG103αβ increased compared to the control treatment only with H2O2. In the other two strains increased susceptibility was observed with a smaller concentration of ATZ (2 mM) and 0.5 mM H2O2. This indicates that EG103αβ has higher catalase levels in comparison to the other two strains. The use of the inhibitor of glutathione synthesis, buthionine sulfoximine (BSO) showed that EG103αΔβ responded similarly to the other two strains. The use of the iron chelator, deferoxamine (DFO) showed a protective effect against oxidative damage in EG103αβ, indicating a role of iron absence in the protection by HIF-1. Measures of glutathione levels (GSH-eq) and superoxide dismutase should be repeated for definitive conclusion of their role in the EG103αβ lineage. In conclusion, the results indicate that the expression of the subunits that make up the human HIF-1 reduces yeast growth, increases its resistance to menadione and perhaps levels of catalase and makes an iron chelator a protective agent in these cells.

The tumor-selective viral replication capacity and pro-apoptotic effects of oncolytic reovirus have been reported to be dependent on the presence of an activated RAS pathway in several solid tumor types. However, the mechanisms of selective anticancer efficacy of the reovirus-based formulation for cancer therapy (Reolysin, pelareorep) have not been rigorously studied in soft tissue sarcomas (STS). Here we report that Reolysin triggered a striking induction of the anti-angiogenic chemokine interferon-gamma-inducible protein 10 (IP-10)/CXCL10 (CXC chemokine ligand 10) in both wild type and RAS mutant STS cells. Further analysis determined that Reolysin treatment possessed significant anti-angiogenic activity irrespective of RAS status. In addition to CXCL10 induction, Reolysin dramatically downregulated the expression of hypoxia inducible factor (HIF)-1 alpha, HIF-2 alpha and inhibited vascular endothelial growth factor (VEGF) secretion. CXCL10 antagonism significantly diminished the anti-angiogenic effects of Reolysin indicating that it is a key driver of this phenomenon. Xenograft studies demonstrated that Reolysin significantly improved the anticancer activity of the anti-angiogenic agents sunitinib, temsirolimus, and bevacizumab in a manner that was associated with increased CXCL10 levels. This effect was most pronounced following treatment with Reolysin in combination with temsirolimus. Further analysis in additional sarcoma xenograft models confirmed the significant increase in CXCL10 and increased anticancer activity of this combination. Our collective results demonstrate that Reolysin possesses CXCL10-driven anti-angiogenic activity in sarcoma models, which can be harnessed to enhance the anticancer activity of temsirolimus and other agents that target the tumor vasculature.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20974号 / 医博第4320号 / 新制||医||1026(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 増永 慎一郎, 教授 高田 穣, 教授 武田 俊一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

A tuberculose (Tb) é uma doença infecciosa crônica ocasionada pelo bacilo Mycobacterium tuberculosis (Mtb). No espaço alveolar, o bacilo entra em contato com células do sistema imune inato como as células dendríticas (DCs), assim como células epiteliais (AEC). Na Tb, o fator de transcrição HIF-1? (Fator Induzido por Hipóxia 1 alfa) se encontra elevado em macrófagos infectados e células epiteliais alveolares adjacentes. Por isso, o objetivo deste trabalho foi investigar o papel de HIF-1? na resposta pró- inflamatória de AEC do tipo II (AEC-II) e na modulação da função de DCs em contato com AEC-II durante a infecção por Mtb. Células MLE-15 foram infectadas com Mtb H37Rv (MOI10) para avaliação da permissividade à infecção (microscopia eletrônica), crescimento dos bacilos (CFU) e ativação através da análise de citocinas (ELISA), nitrito (ensaio de Griess), TLR2, TLR9 e HIF-1? (qPCR e/ou Western blotting). A modulação DCs derivadas da medula óssea (BMDCs) por AEC-II, foi analisada de forma direta (contato) ou indireta (meio condicionado - CM) de MLE-15 não infectadas (CM - NIC) ou infectadas (CM - IC). Foram determinadas citocinas (ELISA), nitrito (Ensaio de Griess), expressão de HIF-1?, enzimas glicolíticas, moléculas co-estimuladoras e CCR7 (citometria de fluxo). Ensaio de migração foi realizando em câmera de boyden. A proliferação de células T CD4+ naive em co-cultura com BMDCs e a manutenção da Th17 foram avaliadas por citometria de fluxo. Eventualmente, BMDCs foram previamente infectadas com Mtb (MOI2) e, em seguida, estimuladas com CM-NIC ou CM-IC. Células MLE-15 mostraram-se permissivas à infecção, não conseguindo controlar o crescimento dos bacilos. A infecção induziu aumento da produção de IL-6, NO2-,CCL5, S100A9 e IFN- ?. Apesar do acúmulo inicial de HIF-1?, a expressão gênica caiu com o passar do tempo. A expressão gênica de TLR2 e TLR9 também estava aumentada. A regulação positiva10 de HIF-1? em células epiteliais induziu uma redução de IL-6 e NO, sem, no entanto, interferir significativamente no número de CFU. BMDCs estimuladas com CM - IC mostraram maior produção de IL-1?, IL-12, IL-6 e IL-10, maior expressão gênica de GLUT1 e HK2, além do acúmulo inicial de HIF-1?, que foi degradado em 24 horas, acompanhado de baixa expressão de iNOS. A expressão MHC-II, CD80, CD86 e CCR7 estava aumentada em BMDCs submetidas ao CM - IC, enquanto a indução do acúmulo de HIF-1? através do seu estabilizador, DMOG, foi capaz de reverter negativamente essa resposta. A maior maturação de BMDCs ocasionou maior proliferação de células TCD4+ naive, desfavorecendo a indução de células T CD4+ IFN?+. Entretanto, as citocinas produzidas favorecerem a manutenção de células TCD4+ produtoras de IL-17. No entanto, o fenótipo de maior maturação foi perdido em BMDCs infectadas estimuladas com CM - IC, aliado à baixa produção de TNF e alta produção de IL-10. Em conclusão, HIF-1? mostrou uma função anti-inflamatória, reduzindo a produção de moléculas pró- inflamatórias por AEC-II e regulando negativamente a maturação e a migração de DCs. Além disso, apesar de AEC-II infectadas por Mtb favorecerem a maturação e migração de DCs, o Mtb é capaz de subverter essa resposta / Tuberculosis (Tb) is a chronic infectious disease caused by the bacillus Mycobacterium tuberculosis (Mtb). In the alveolar space, the bacillus contacts cells from the immune system, such as dendritic cells (DCs), as well as alveolar epithelial cells (AEC). In Tb, the transcription factor HIF-1? (Hypoxia-inducible factor 1- alpha) is accumulated in infected macrophages and adjacent alveolar epithelial cells. Therefore, the aim of this work was to investigate the role of HIF-1? in the proinflammatory response of type II AEC (AEC-II) and modulation of DCs function upon contact with AEC-II during Mtb infection. MLE-15 cells were infected with Mtb H37Rv (MOI10) to evaluate the permissiveness to infection (electron microscopy), killing ability (CFU) and cell activation through cytokine analysis (ELISA), nitrite (Griess assay), TLR2, TLR9 and HIF-1? (qPCR and / or Western blotting). Bone marrow derived DCs (BMDCs) modulation by AEC-II, were analyzed directly (contact) or indirectly (conditioned medium - CM) of uninfected (CM-NIC) or infected (CMIC) MLE-15. We determined cytokines (ELISA), nitrite (Griess Assay), HIF-1? expression, glycolytic enzymes, co-stimulatory molecules and CCR7 (flow cytometry). Chemotaxie assay was performed on Boyden camera. Proliferation of naive CD4 + T cells in co-culture with BMDCs and maintenance of Th17 were assessed by flow cytometry. Eventually, BMDCs were previously infected with Mtb (MOI2) and then stimulated with CM-NIC or CM-IC. MLE-15 cells were permissive to infection, failing to control the bacilli growth. Infection induced increased production of IL-6, NO2-, CCL5, S100A9 and IFN-?. Despite the initial accumulation of HIF-1?, gene expression dropped over time. The gene expression of TLR2 and TLR9 was also increased. Positive regulation of HIF-1? in epithelial cells induced a reduction of IL-6 and NO, but did not significantly interfere with the number of CFU. BMDCs stimulated with CM-IC showed higher production of IL-1?, IL-12, IL-6, and IL-10, greater GLUT1 and HK2 gene expression, in addition to the initial12 accumulation of HIF-1?, which was degraded within 24 hours, accompanied by low iNOS expression. The expression of MHC-II, CD80, CD86 and CCR7 was increased in BMDCs undergoing CM-IC, while the induction of HIF-1? accumulation through its stabilizer, DMOG, was able to negatively revert this response. The higher maturation of BMDCs resulted in a greater proliferation of naive CD4 + T cells, but hampered induction of CD4 + IFN? + T cells. However, cytokines produced favor the maintenance of IL-17 producing CD4 + cells. The phenotype of higher maturation was lost in Mtb-infected BMDC, accompanied by low TNF production and high IL-10 production. In conclusion, HIF-1? showed an anti-inflammatory function, reducing the production of proinflammatory molecules by AEC-II and negatively regulating the maturation and migration of DCs. In addition, although Mtb-infected AEC-II favor maturation and migration of DCs, Mtbinfection of DCs is capable of subverting this response

A temperatura é um fator abiótico fundamental que influencia diretamente o comportamento e a sobrevivência dos organismos, incluindo peixes. Por serem ectotérmicos, peixes respondem diretamente à variação de temperatura. Eles possuem mecanismos celulares de termoestabilidade, com diferentes expressões, ajustados evolutivamente às condições do meio. O presente trabalho tem como objetivo estudar os efeitos do aumento gradual da temperatura (2°C/h) a partir da temperatura ambiente de inverno (22°C) até 26, 30, 32, 34 e 36°C e temperatura crítica de sobrevivência (TCS) em peixes pampo, Trachinotus carolinus. Analisou-se o comportamento a partir de vídeos; a expressão das proteínas relacionadas à hipóxia tecidual HIF-1~a e VEGF em músculo e fígado através da técnica de imuno-histoquímica; e análises celulares através da quantificação de mitocôndrias e mensuração de sua área. Os resultados mostraram que o comportamento dos pampos é alterado com o aumento da temperatura, apresentando um padrão definido. As maiores expressões proteicas foram em temperaturas elevadas, a 34°C no caso do VEGF e a 36°C no caso do HIF-1 ~a. Houve aumento no número de mitocôndrias e queda em sua área conforme o aumento da temperatura. Portanto, a temperatura tem influência em todos os níveis de organização estudados, ou seja, celular, tecidual e do organismo / Temperature is an important abiotic factor that influences directly the behavior and survival of organisms, including fish. Fishes are ectothermic, and respond directly to temperature variation. They have cellular mechanisms of thermostability that were adjusted during evolution to environmental conditions. The present work aims to study the effects of the gradual increase in temperature (2°C / h) from the winter temperature (22°C) to 26, 30, 32, 34 and 36°C and critical survival temperature (TCS) in fish pompano, Trachinotus carolinus. We analyzed the behavior out of vídeos; the expression of proteins related to tissue hypoxia HIF-1 ~a and VEGF in muscle and liver through the technique of immunohistochemistry; and determined the number and the área of cellular mitochondria. The results showed that the behavior of pampano changes with increasing temperature, in a gradual scale pattern. The protein expressions were higher at elevated temperatures, at 34°C in the case of VEGF and at 36°C in the case of HIF-1 ~a. An increase in the number of mitochondria, and a decrease in their area were observed with increasing temperature. Therefore, temperature had measurable effects at all levels of organization studied, i.e., cellular, tissular and organismic

A temperatura é um fator abiótico fundamental que influencia diretamente o comportamento e a sobrevivência dos organismos, incluindo peixes. Por serem ectotérmicos, peixes respondem diretamente à variação de temperatura. Eles possuem mecanismos celulares de termoestabilidade, com diferentes expressões, ajustados evolutivamente às condições do meio. O presente trabalho tem como objetivo estudar os efeitos do aumento gradual da temperatura (2°C/h) a partir da temperatura ambiente de inverno (22°C) até 26, 30, 32, 34 e 36°C e temperatura crítica de sobrevivência (TCS) em peixes pampo, Trachinotus carolinus. Analisou-se o comportamento a partir de vídeos; a expressão das proteínas relacionadas à hipóxia tecidual HIF-1~a e VEGF em músculo e fígado através da técnica de imuno-histoquímica; e análises celulares através da quantificação de mitocôndrias e mensuração de sua área. Os resultados mostraram que o comportamento dos pampos é alterado com o aumento da temperatura, apresentando um padrão definido. As maiores expressões proteicas foram em temperaturas elevadas, a 34°C no caso do VEGF e a 36°C no caso do HIF-1 ~a. Houve aumento no número de mitocôndrias e queda em sua área conforme o aumento da temperatura. Portanto, a temperatura tem influência em todos os níveis de organização estudados, ou seja, celular, tecidual e do organismo / Temperature is an important abiotic factor that influences directly the behavior and survival of organisms, including fish. Fishes are ectothermic, and respond directly to temperature variation. They have cellular mechanisms of thermostability that were adjusted during evolution to environmental conditions. The present work aims to study the effects of the gradual increase in temperature (2°C / h) from the winter temperature (22°C) to 26, 30, 32, 34 and 36°C and critical survival temperature (TCS) in fish pompano, Trachinotus carolinus. We analyzed the behavior out of vídeos; the expression of proteins related to tissue hypoxia HIF-1 ~a and VEGF in muscle and liver through the technique of immunohistochemistry; and determined the number and the área of cellular mitochondria. The results showed that the behavior of pampano changes with increasing temperature, in a gradual scale pattern. The protein expressions were higher at elevated temperatures, at 34°C in the case of VEGF and at 36°C in the case of HIF-1 ~a. An increase in the number of mitochondria, and a decrease in their area were observed with increasing temperature. Therefore, temperature had measurable effects at all levels of organization studied, i.e., cellular, tissular and organismic

PARP-1 has been identified as a major player in apoptotic pathways. Its excessive activation causes mitochondrial dysfunction, permeability, and AIF mitochondrion-to-nucleus translocation. It has been suggested that PARP-1 interacts indirectly with Bnip3, a mitochondrial pro-apoptotic factor. However, the mechanistic linkage is still not well understood. Our lab has shown that cytosolic/nuclear NAD+ depletion is a hallmark for PARP-1 over activation and inhibition of sirtuin activity. Specifically in my project, we think that PARP-1 induced- NAD+ depletion and sirtuin inhibition causes hyperacetylation of the α subunit of the transcription factor HIF-1 allowing increased HIF-1 binding to Bnip3 upstream promoter, and increased Bnip3 expression. Indeed, our PARP-1 Knock out neurons, MNNG and PJ34 treatment, chromatin immunoprecipitation, and HIF-1α loss of function studies strongly confirmed the necessity of HIF-1 to increase Bnip3 expression in hypoxia. Overall, our research suggests a role for HIF-1 in increasing PARP-1 dependent Bnip3 expression in hypoxic models. / May 2016

KCN-1 is known to reduce tumor growth 6-fold in mice implanted with LN229 glioma cells. Although this inhibitor is effective, the mechanism of action for KCN-1 is not well understood. Based on preliminary studies, KCN-1 reduces tumor growth by disrupting the HIF 1 (hypoxia-induced factor-1) pathway. The binding target of KCN-1 needs to be investigated in order to develop KCN-1 or its analogs for therapeutic applications. In this research, a molecule was designed and synthesized for the identification of the binding target of KCN-1. Specifically, this molecule contains the inhibitor (KCN-1), a photocleavable linker, beads, and the affinity label (L DOPA). When UV light shines on the linker, the trans-alkene isomerizes to cis-alkene and undergoes intramolecular ring-closing reaction, which helps cleave the immobilized bead from the linker. The immobilized bead is used to separate the binding fragment attached to the photocleavable linker from the solution after enzyme digestion. The affinity label (L-DOPA) reacts with a nucleophile from the binding target and creates a covalent bond. If the design is successful, this method is able to analyze the mass of the peptide sequence and determine the binding target of KCN-1.

Žmogaus organizme širdies ir kraujagyslių sistema, kraujodaros organai bei kvėpavimo organų sistema leidžia organizmui palaikyti deguonies pusiausvyrą. Širdies ligos, vėžys, smegenų kraujagyslių ligos ir chroniška obstrukcinė plaučių liga- tai susirgimai, nuo kurių dažniausiai miršta žmonės visame pasaulyje. Visų šių ligų viena iš priežasčių yra deguonies pusiausvyros sutrikimas (Semenza, 2001). Ląstelės atsakas į stresines situacijas, tokias kaip deguonies trūkumas ląstelėje, seniai domino mokslininkus. Specifinis atsakas į deguonies koncentraciją ląstelėje buvo nustatytas bakterijose ir mielėse, bet ilgai buvo neaptinkamas aukštesniuose organizmuose. Atlikus visą eilę tyrimų, buvo aptikta sąsaja tarp deguonies kiekio ląstelėje kitimo ir transkripcijos įvykių kaskados, reguliuojančios aukštesniųjų organizmų ląstelių atsaką į hipoksiją. Vieno iš ryškiausių homeostazės atsako į hipoksiją- hematopoetinio augimo faktoriaus eritropoetino molekulinė analizė leido aptikti hipoksijos indukuojamą transkripcijos faktorių (HIF). Dabar jau nustatyta, kad ši sistema funkcionuoja visose žinduolių ląstelėse. (Schofield and Ratcliffe, 2004). HIF faktoriai- tai visa šeima baltymų, vaidinančių nemažą vaidmenį vėžinių auglių vystymesi žmogaus organizme. Tai daro šių faktorių raiškos reguliacijos tyrimus labai svarbiais, nes gauti rezultatai tiesiogiai turi ir praktinės reiksmės vėžinių auglių diagnostikoje bei gydyme. / Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric protein that consist of two proteins- HIF-1&#945; and HIF-1&#946;. HIF-1 activates the transcription of many genes that code for proteins that are involved in angiogenesis, glucose metabolism, cell proliferation/survival and invasion/metastasis. HIF-1&#945; protein degradation is regulated by O2-dependent prolyl hydroxylation, whitch targets the protein for ubiquitylation by E3 ubiquitin-protein ligases. These ligases contain the von Hippel-Lindau tumour-suppressor protein (VHL), whitch binds specifically to hydroxylated HIF-1&#945; and ubiqutinates it. Ubiquitylated HIF-1&#945; is rapidly degraded by the proteosome. HIF-1&#945; interacts with the molecular chaperone Hsp90. Hsp90 plays a pivotal role in mediating the proper folding and subsequent activation of its numerous “client” proteins. Hsp90 also cooperates with the proteosomal pathway to eliminate misfolded cellular proteins. The antibiotic geldanamycin (GA) binds to Hsp90 and modulates its chaperone function, thereby accelerating the degradative activity associated with Hsp90. However, whether Hsp90 modulates HIF-1&#945; activity by stabilization of the protein or by another mechanism is not clear. We tested the hypothesis weather and how inactivation (i.e. with geldanamycin) of Hsp90 protein function influences HIF-1&#945; protein expression levels. Our obtained results shows that Hsp90 is required to HIF-1&#945; protein under normoxia as well as hypoxia and is... [to full text]

Increased glucose metabolism may occur in malignant tumours due to altered gene expression or a response to hypoxia. It has been shown that tumours with high levels of glycolysis, indicated by elevated lactate, are less responsive to radiotherapy. It is not clear whether this effect is caused by lactate itself or rather that high lactate is a surrogate for a radioresistant property such as hypoxia. Furthermore, we are not aware of studies that examine the manipulation of lactate production in tumours to alter radiation response. We propose a novel approach of metabolic targeting of HIF-1 to address these issues. HIF-1 is a major regulator of glycolysis and its inhibition would decrease malignant cell metabolism and could lead to a decrease in lactate production. The goal of this pre-clinical study was to evaluate metabolic targeting as a strategy of enhancing radiation response by inhibiting the HIF-1 transcription factor.