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

Cancer develops in many organs and tissues in the body through genetic and environmental modifications to acquire the hallmarks of cancer. The hallmarks of cancer allow the cells to become malignant and progress to a tumorigenic state. It has previously been shown in various carcinomas that HIF-2a, a key component in hypoxia adaptation, has a role in autonomous growth, the first hallmark of cancer. Ovarian cancer is the most lethal of the gynecological malignancies and accounts for 3% of new cases in women annually but is the fifth most common cause of death due to cancer. Here, it is shown in two ovarian carcinoma cell lines that HIF-2a is involved in in vitro and in vivo growth. It is also shown that the effect of HIF-2a is due to its role in autonomous growth and not vascularization with the use of in vitro spheroids. From recent findings in the laboratory the oxygen-stimulated translation initiation complex was discovered and HIF-2a is one of its components. In the absence of HIF-2a there is a downregulation in translation in hypoxia in ovarian carcinoma. This is also seen in a HIF-2a translational target, IGF1R and its downstream signaling pathway, which may be involved in autonomous growth as well as other hallmarks of cancer. Taken together, the data in this thesis presents the importance of HIF-2a in autonomous growth and cancer progression in ovarian carcinoma, as well as verifying its role in translation.

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.

L’hypoxie est une caractéristique majeure des glioblastomes (GB). Elle est la cause principale de la résistance aux traitements observée dans ces tumeurs. Les conséquences de la baisse en oxygène au niveau tumoral, sont médiées par les facteurs de transcription Hypoxia Inducible Factors (HIF). Ces facteurs sont des protéines hétérodimériques HIF-α/HIF-1β responsables de la transcription de nombreux gènes cibles. Certaines de ces cibles participent à la progression tumorale et à la mise en place d’un phénotype agressif. L’une des cibles de l’isoforme HIF-2α, est la βIII-tubuline (βIII-t). Cette protéine, qui compose les microtubules, est décrite comme surexprimée dans les gliomes de haut grade, comme les GB. Ces travaux de thèse s’intéressent au rôle de la βIII-t dans la progression tumorale ainsi qu’au développement de stratégies permettant d’inhiber l’expression de HIF-2α. Les résultats obtenus montrent que la βIII-t a une importance centrale dans le développement tumoral. En effet, les tumeurs issues de l’implantation de cellules humaines de GB invalidées pour la βIII-t, se développent significativement moins vite comparées aux tumeurs contrôles. In vitro, nous avons montré que cette protéine est impliquée dans la prolifération, la migration et l’invasion cellulaires. En revanche, nous n’avons pas pu confirmer que la βIII-t est impliquée dans la résistance aux traitements (chimio- ou radiothérapeutiques). Les deux composés testés comme inhibiteurs de HIF-2α (SR2933 et PT2385) ont montré des résultats prometteurs sur la βIII-t, gène cible spécifique de HIF-2α. Cependant, malgré les stratégies développées, nous n’avons pas pu évaluer l’efficacité directe de ces deux composés sur l’hétérodimérisation de HIF-2α avec HIF-1β. / Hypoxia is a major feature of glioblastoma (GB). It is the main cause of the resistance to treatments observed in these tumors. The consequences of the decrease in oxygen at the tumor level, is mediated by the Hypoxia Inducible Factors (HIF). These transcription factors are heterodimeric proteins HIF-α/HIF-1β responsible for the transcription of many target genes. Some of these targets are responsible for setting up an aggressive phenotype and in tumor progression. One of the targets of the HIF-2α isoform is βIII-tubulin (βIII-t). This protein, which is a constituent of microtubules, is described as overexpressed in high grade gliomas, such as GB. In the present thesis, we examined the role of βIII-t in tumor progression and we developed strategies to inhibit the expression of HIF-2α. Our results show that βIII-t is of central importance in tumor development. Indeed, tumors resulting from the implantation of GB human cells invalidated for βIII-t, developed significantly less rapidly compared to control tumors. In vitro, we have shown that this protein is involved in cell proliferation, migration and invasion. However, we could not confirm that βIII-t is involved in resistance to treatments (chemotherapeutic or radiotherapeutic).The two compounds tested as inhibitors of HIF-2α (SR2933 and PT2385) showed promising results on βIII-t, a specific target gene of HIF-2α. However, despite the different tested strategies, we could not evaluate the direct inhibitory action of these two compounds on the heterodimerization of HIF-2α with HIF-1β.

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.

Indiana University-Purdue University Indianapolis (IUPUI) / FGF23 is an osteocyte produced hormone necessary for maintaining systemic phosphate handling, and thus bone structure and function in both rare and common disorders such as chronic kidney disease (CKD). FGF23 is a critical factor in CKD, with elevated levels causing alterations in mineral metabolism and increased odds for mortality. However, the mechanisms directing the production of key modulators of skeletal homeostasis and biomineralization within osteocytes, and how this is altered in chronic kidney disease, remain unclear. The experimental focus of this dissertation was to dissect the molecular systems and role of oxygen sensing in the regulated production of FGF23. In CKD, up to 75% of patients have anemia and concomitant marked elevations in FGF23, increasing mortality odds. Anemia is a potent driver of FGF23 secretion, therefore, current and emerging therapies, including recombinant EPO and the hypoxia inducible factorprolyl hydroxylase inhibitors (HIF-PHI) FG-4592 and BAY 85-3934, were used to improve anemia in the adenine diet-induced mouse model of CKD. In the mice with CKD, iFGF23 was markedly elevated in control mice but was attenuated by 65-85% after delivery of EPO or HIF-PHI, with no changes in serum phosphate. This was associated with improved systemic iron utilization and reductions in mRNA markers of renal fibrosis. In osteocyte-like cell cultures treated with HIF-PHI, integrative RNAseq and ATACseq analysis identified candidate genes upregulated in response to mimicked hypoxia, concomitant with elevated Fgf23 expression. These genes were found to be downregulated in CKD bone, therefore, knock-out cells were generated using CRISPR/Cas9 technology. These cells were found to be functionally similar to in vivo conditional knockout models that have enhanced bone mass and elevated FGF23. Taken together, these results further define novel factors involved in the regulation of FGF23 and identify new therapeutic targets. / 2023-05-26

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

The control of oxygen delivery to cells in the body is the result of a small group of primary oxygen sensors, one of the most important of which is the hypoxia-inducible transcription factor-1 (HIF-1). Two alpha-ketoglutarate dependent non-heme iron dioxygenases are responsible for the regulation of HIF-1 through hydroxylation of residues on the HIF-1a subunit. One of these enzymes, known as the factor inhibiting HIF-1 (FIH-1) is responsible for hydroxylating residue Asn803 on HIF-1a, preventing the transcription of hypoxia related genes controlled by HIF-1. It was hypothesized that there would be a difference in inhibition of FIH-1 from the other HIF-1 regulating enzyme, the prolyl hydroxylase domain-2 (PHD2), when testing a series of ten small molecule inhibitors. The ten inhibitors chosen fell into three classes: pyrones, pyridines, and catechols. Of these inhibitors, it was found that catechols produced a significant inhibitory difference between PHD2 and FIH, and may provide useful in further inhibitor design and synthesis work.