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.

Parkinson's disease (PD) is the most common neurodegenerative motor disorder, whose clinical features are rest tremor, bradykinesia, muscular rigidity and postural instability. Although most reported cases are sporadic, a handful of familial cases and their causative genes have been identified. Loss-of-function mutations in DJ-1, one of these genes, are responsible for 1% of familial PD cases. Our laboratory has previously reported that DJ-1- lacking neurons are sensitive to oxidative stress, induced by hydrogen peroxide or the neurotoxin MPTP. To investigate the possible mechanisms through which DJ-1 protects against oxidative stress, we performed a proteomic screen and identified Von Hippel Lindau (VHL) and Paraoxonase2 (PON2) as potential DJ-1 interacting partners. VHL is an E3 ubiquitin ligase which, in normal conditions, poly-ubiquitinates HIF-1 , a subunit of a master hypoxic/oxidative stress transcription factor, whose function is protective in oxidative and hypoxic stresses. In the present study, we provided further evidence of interaction of DJ-1 with VHL. We also demonstrated that HIF-1 protein level, as an indicator of VHL activity, is lower in cells lacking DJ-1, suggesting the inhibitory role of DJ-1 on VHL. Our in vitro studies also showed that DJ-1 inhibits ubiquitin ligase activity of VHL on HIF-1 by reducing the VHL-HIF-1 interaction. Importantly, accumulation of HIF-1 protects embryonic cortical neurons against MPP+ induced neuronal death. Finally, we confirmed the impairment of HIF-1 response to oxidative stress in human lymphoblastoids of DJ-1-linked PD cases. In the second part of this study, we demonstrated the interaction of DJ-1 and PON2. Interestingly, PON2 lactonase activity is reduced in DJ-1 deficient cells which could be rescued by re-introduction of DJ-1, suggesting a modulating role of DJ-1 on PON2 activity. In addition, PON2 deficiency, like DJ-1 deficiency, hypersensitizes neurons to MPP+, which could be rescued by over-expression of PON2 in both cases. Taken together, our data provide evidence that DJ-1 exerts its protective role by inhibiting VHL activity, enhancing HIF-1 stability, and increasing PON2 pro-survival function in PD models.

SENA, C.M. Imunoexpressão do fator induzido por hipóxia (hif-1) em carcinoma basocelular. 2016. 50 f. Dissertação (Mestrado em Ciências da Saúde) - Campus de Sobral, Universidade Federal do Ceará, Sobral, 2016 / Submitted by Mestrado Ciências da Saúde (ppgcsufcsobral@gmail.com) on 2017-02-01T18:53:24Z No. of bitstreams: 1 2016_dis_cmsena.pdf: 10501307 bytes, checksum: 0f78ffaad25bbfc3a02edff35006d19c (MD5) / Approved for entry into archive by Ana Márcia Sousa (marciasousa@ufc.br) on 2017-02-02T11:45:40Z (GMT) No. of bitstreams: 1 2016_dis_cmsena.pdf: 10501307 bytes, checksum: 0f78ffaad25bbfc3a02edff35006d19c (MD5) / Made available in DSpace on 2017-02-02T11:45:40Z (GMT). No. of bitstreams: 1 2016_dis_cmsena.pdf: 10501307 bytes, checksum: 0f78ffaad25bbfc3a02edff35006d19c (MD5) Previous issue date: 2016-03-28 / BACKGROUND: Skin cancer (non-melanoma) is a lesion with high prevalence, with the Basal Cell Carcinoma (BCC) is the most common type of this malignancy. Recent research has sought molecular biomarkers for cancer, with a focus on energy metabolism of cells, highlighting the factor induced by hypoxia, HIF-1. To evaluate the immunohistochemical expression of HIF-1α in CBC; correlate the immunoreactivity of HIF-1α with normal tissue, histologic subtypes and clinical parameters of CBC. METHODS: The sample consisted of 27 cases of BCC in the head and neck region, classified as histologic subtype. Was performed immunohistochemical assessment by the technique of streptoavidin-Biotin with HIF-1α antibody (clone EP1215Y, ABCAM®, 1: 100 dilution, antigen retrieval with citrate pH6, Pascal Pan, System Envision Flex, overnight incubation). five fields each case in 400x magnification were assessed, using the Image J program Statistical tests were performed based on significance level of 5% using the Mann-Whitney test / Kruskal-Wallis test. RESULTS: Obtained immunoreactivity of HIF-1α in all cases of BCC, 100% (n = 27), whichever is the nodular subtype in males and elderly face. Observed was weak cytoplasmic immunostaining in cells of higher side lesions (51.3 ± 20.3) (p = 0.018) and no cytoplasmic immunostaining of cells in lesions in non-face (68.7 ± 22.6) (p = 0.015). Strong immunostaining was higher in nuclear BCC cells compared to that observed in normal skin cells (6.9 ± 7.4) (p = 0.036); greater immunoreactivity weak cytoplasmic HIF-1α in the lesions of the cells with positive margins (61.1 ± 10.4) (p = 0.004); higher percentage of cells with no cytoplasmic immunostaining in lesions with clear margins (56.0 ± 23.2) (p = 0.004); and greater weak cytoplasmic immunostaining in AML cells with positive margins compared to the normal skin cells and free margins (61.1 ± 10.4) (p = 0.042). There was no statistical difference between the immunostaining of HIF-1α between cells of different subtypes and between cells ulcerated and non-ulcerated. CONCLUSION: There was immunoreactivity of HIF-1α in all cases of CBC sample analyzed, with no statistical difference in the histological subtype, less immunostaining in normal epithelial tissue cells. The immunoreactivity of HIF-1α, both as nuclear cytoplasmic may be indicative of the involvement of this protein in these tumors, suggesting a role of HIF-1α in the CBC carcinogenesis. / INTRODUÇÃO: O Câncer de pele (não melanoma) é uma lesão com elevada prevalência, sendo o Carcinoma Basocelular (CBC) o tipo mais frequente dessa neoplasia maligna. Pesquisas recentes têm buscado biomarcadores moleculares para câncer, com enfoque no metabolismo energético das células, destacando-se o Fator Induzido por Hipóxia, HIF-1. OBJETIVOS: Avaliar a expressão imuno-histoquímica do HIF-1αem CBC; correlacionar a imunoexpressão do HIF-1αcom tecido normal, subtipos histológicos e parâmetros clínicos de CBC. METODOLOGIA: A amostra foi constituída por 27 casos de CBC na região de cabeça e pescoço, classificados conforme subtipo histológico. Realizou-se a avaliação imunohistoquímica pela técnica da Estreptoavidina-Biotina, com anticorpo HIF-1α(clone EP1215Y, ABCAM ® , diluição 1:100, recuperação antigênica com citrato pH6, Panela Pascal, Sistema Envision Flex, incubação overnight). Foram analisados cinco campos de cada caso no aumento de 400x, utilizando o programa Image J. Os testes estatísticos foram realizados com base nos níveis de significância de 5%, utilizando-se os testes de Mann-Whitney/Kruskall-Wallis. RESULTADOS: Obteve-se imunoexpressão do HIF-1α em todos os casos de CBC, 100% (n=27), prevalecendo o subtipo nodular em idosos do sexo masculino e em face. Observaramse imunomarcação citoplasmática fraca maior nas células das lesões em face (51,3±20,3) (p=0,018) e ausência de imunomarcação citoplasmática nas células das lesões em não face (68,7±22,6) (p=0,015). Houve maior imunomarcação nuclear forte em células de CBC comparativamente ao que foi observado nas células de pele normal (6,9±7,4) (p=0,036); maior imunoexpressão citoplasmática fraca do HIF-1α nas células das lesões com margens comprometidas (61,1±10,4) (p=0,004); maior percentual de células sem imunomarcação citoplasmática em lesões com margens livres (56,0±23,2) (p=0,004); além de maior imunoexpressão citoplasmática fraca em células de CBC com margens comprometidas, comparando-se às células de pele normal e margens livres (61,1±10,4) (p=0,042). Não houve diferença estatística entre a imunomarcação do HIF-1αentre as células dos diversos subtipos, bem como entre células de lesões ulceradas e não ulceradas. CONCLUSÃO: Houve imunoexpressão do HIF-1α em todos os casos de CBC da amostra analisada, sem diferença estatística quanto ao subtipo histológico, com menor imunomarcação em células de tecido epitelial normal. A imunoexpressão do HIF-1α, tanto nuclear quanto citoplasmática pode ser indicativo da participação dessa proteína nessas neoplasias, sugerindo possível papel do HIF-1αnas carcinogênese do CBC.

xi, 52 p. ; ill. (some col.) A print copy of this title is available through the UO Libraries under the call number: SCIENCE QH445.2 .B35 2008 / Activation of Heat shock proteins (Hsps) is critical to adaptation to low oxygen levels (hypoxia) and enduring the oxidative stress of reoxygenation. Hsps are known to be regulated by Heat shock factor (Hsf), but my results demonstrate an unexpected regulatory link between the oxygen sensing and heat shock pathways. Hsf transcription is upregulated during hypoxia due to direct binding by Hypoxia-inducible Factor-1 (HIF-1) to HIF-1 response elements in an Hsf intron. This increase in Hsf transcripts is necessary for full Hsp induction during hypoxia and reoxygenation. The HIF-1-dependent increase in Hsps has a functional impact, as reduced production of Hsps decreases viability of adult flies exposed to hypoxia and reoxygenation. Thus, HIF-1 control of Hsf transcriptional levels is a regulatory mechanism for sensitizing heat shock pathway activity in order to maximize production of protective Hsps. This cross-regulation represents a mechanism by which the low oxygen response pathway has assimilated complex new functions by regulating the heat shock pathway's key transcriptional activator. Beyond studying the regulation of specific genes. I have also developed a method to identify small, yet important, changes within entire genomes. Genetic variation is the foundation of phenotypic traits, as well as many disease states. Variation can be caused by inversions, insertions, deletions, duplications, or single nucleotide polymorphisms (SNPs) within a genome. However, identifying a genetic change that is the cause of a specific phenotype or disease has been a difficult and laborious task for researchers. I developed a technique to quickly and accurately map genetic changes due to natural phenotypic variation or produced by genetic screens. I utilized massively parallel, high-throughput sequencing and restriction site associated DNA (RAD) markers, which are short tags of DNA adjacent to the restriction sites. These RAD markers generate a genome-wide signature of fragments for any restriction enzyme. Taken together with the fact that the vast majority of organisms have SNPs that disrupt restriction site sequences, the differences in the restriction fragment profiles between individuals can be compared. In addition, by using bulk segregant analysis, RAD tags can be used as high-density genetic markers to identify a genetic region that corresponds to a trait of interest. This dissertation includes both previously published and unpublished co-authored materials. / Adviser: Eric Johnson

xiii, 56 p. : ill. (some col.) / Correct regulation of insulin/insulin-like growth factor signaling (IIS) is essential for proper development and growth. More recently, proper regulation of IIS has been shown to be important for adaptation and survival under stressful conditions. Despite the importance of IIS, the mechanism underlying IIS regulation under various environmental stresses remains to be elucidated. One mechanism of regulating IIS involves the binding of insulin and insulin-like growth factors by insulin-like growth factor binding proteins (IGFBPs), which prevent the factors from interacting with the insulin receptor (InR). The only identified IGFBP in <italic>Drosophila</italic> to date is imaginal morphogenesis protein late 2 (Imp-L2), which was previously implicated in the regulation of IIS during starvation. Here, we investigate whether Imp-L2 is required to regulate IIS under low oxygen stress (hypoxia). The ability to tolerate hypoxia requires cellular adaptations that decrease the need for oxygen and increase the supply of it. In a wide variety of organisms many of these adaptations are either directly or indirectly regulated by the transcription factor hypoxia-inducible factor-1 (HIF-1). Our results reveal a regulatory link between HIF-1, Imp-L2, and IIS during hypoxia. We demonstrate that Imp-L2 transcript abundance is increased during hypoxia in a HIF-1 dependent manner resulting in inhibition of IIS and increased hypoxia tolerance. This dissertation includes unpublished co-authored material. / Committee in charge: Dr. Victoria Herman, Chairperson; Dr. Eric Johnson, Advisor; Dr. Bruce Bowerman, Member; Dr. Christopher Doe, Member; Dr. Kenneth Prehoda, Outside Member

Parkinson's disease (PD) is the most common neurodegenerative motor disorder, whose clinical features are rest tremor, bradykinesia, muscular rigidity and postural instability. Although most reported cases are sporadic, a handful of familial cases and their causative genes have been identified. Loss-of-function mutations in DJ-1, one of these genes, are responsible for 1% of familial PD cases. Our laboratory has previously reported that DJ-1- lacking neurons are sensitive to oxidative stress, induced by hydrogen peroxide or the neurotoxin MPTP. To investigate the possible mechanisms through which DJ-1 protects against oxidative stress, we performed a proteomic screen and identified Von Hippel Lindau (VHL) and Paraoxonase2 (PON2) as potential DJ-1 interacting partners. VHL is an E3 ubiquitin ligase which, in normal conditions, poly-ubiquitinates HIF-1 , a subunit of a master hypoxic/oxidative stress transcription factor, whose function is protective in oxidative and hypoxic stresses. In the present study, we provided further evidence of interaction of DJ-1 with VHL. We also demonstrated that HIF-1 protein level, as an indicator of VHL activity, is lower in cells lacking DJ-1, suggesting the inhibitory role of DJ-1 on VHL. Our in vitro studies also showed that DJ-1 inhibits ubiquitin ligase activity of VHL on HIF-1 by reducing the VHL-HIF-1 interaction. Importantly, accumulation of HIF-1 protects embryonic cortical neurons against MPP+ induced neuronal death. Finally, we confirmed the impairment of HIF-1 response to oxidative stress in human lymphoblastoids of DJ-1-linked PD cases. In the second part of this study, we demonstrated the interaction of DJ-1 and PON2. Interestingly, PON2 lactonase activity is reduced in DJ-1 deficient cells which could be rescued by re-introduction of DJ-1, suggesting a modulating role of DJ-1 on PON2 activity. In addition, PON2 deficiency, like DJ-1 deficiency, hypersensitizes neurons to MPP+, which could be rescued by over-expression of PON2 in both cases. Taken together, our data provide evidence that DJ-1 exerts its protective role by inhibiting VHL activity, enhancing HIF-1 stability, and increasing PON2 pro-survival function in PD models.

Dendritic cells (DCs) are antigen presenting cells (APCs), a subtype of immune cells that present cellular information to T cells in the immune system. Hypoxia inducible factor 1 alpha (HIF-1 alpha) is an important transcription factor that facilitates dendritic cell metabolism by upregulating glycolysis in activated DCs. In this project, we examined the effects of HIF-1 alpha inhibition on metabolic processes of dendritic cells. Using techniques such as flow cytometry, western blotting, and extracellular flux analyzers, we used a selective inhibitor of HIF-1 alpha to test the hypothesis that HIF-1 alpha promotes glycolytic dependent processes such as glucose production, survival, and maturation. The results revealed that HIF-1 alpha impacts oxygen consumption rates in DCs, but does not affect survival, maturation rates, and glycolytic rates under the conditions studied. Dendritic cell secretion of IL-12, a proinflammatory cytokine upregulated during metabolism, decreased in a dose dependent manner under HIF-1 alpha inhibition. Understanding the effects of HIF-1 alpha can provide insight on how dendritic cells utilize their fuel source to facilitate immunological tasks and how in the future, we can optimize these sources to improve immune system functionality.

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第20314号 / 薬科博第83号 / 新制||薬科||9(附属図書館) / 京都大学大学院薬学研究科医薬創成情報科学専攻 / (主査)教授 掛谷 秀昭, 教授 高須 清誠, 教授 大野 浩章 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

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