La maladie de Crohn (MC) est une maladie inflammatoire chronique intestinale (MICI). Son incidence et sa prévalence ont augmenté en Europe au cours des dix dernières années (150 pour 100000 habitants) constituant ainsi un problème de santé majeur. L’inflammation chronique dans la MC favorise la mise en place d’une angiogenèse pathophysiologique. Inflammation et angiogenèse sont deux réponses cellulaires suspectées dans la survenue des cancers coliques associés au MICI. Même si les facteurs favorisant la mise en place de la MC restent non élucidés, la contribution des bactéries exogènes est fortement suspectée. Parmi ces bactéries, les E.coli adhérentes et invasives (AIEC), isolées à partir de la muqueuse iléale de patients porteurs de la MC, sont un bon candidat. Les objectifs de mon projet de thèse étaient de caractériser les mécanismes moléculaires induits par les AIEC et impliqués dans la mise en place des réponses pro inflammatoire et pro angiogénique des cellules intestinales épithéliales. Le facteur de transcription hypoxique (HIF-1) est au cœur de l’immunité innée et de l’angiogenèse. J’ai émis l’hypothèse que les AIEC pouvaient moduler le niveau d’expression de HIF-1α et ainsi contrôler les réponses pro inflammatoire et pro angiogénique. Dans mon premier article, j’ai montré que HIF-1α est maximalement exprimé au niveau de l’épithélium iléal des patients porteurs de la MC. Ensuite, j’ai montré sur un modèle murin compétent pour l’infection par les AIEC, les souris CEABAC10, que les bactéries induisent l’augmentation du niveau protéique de HIF-1 α ainsi que l’activation de la voie de signalisation du VEGF, le facteur angiogénique le plus puissant. / Non communiqué.

This thesis is concerned with the role of iron in modulating right ventricular (RV) afterload during exercise in healthy people aged between 50 and 80 years. This is predicated on the requirement of the hypoxia-inducible factor (HIF) pathway for ferrous iron. A secondary objective is to examine the reactive oxygen species (ROS) hypothesis in human hypoxic pulmonary vasoconstriction (HPV) using exposure to hyperoxia. Chapters 3 and 4 describe basal relationships that may affect the HIF pathway and exercise capacity during ageing. These were explored in 113 participants using blood tests and exercise tests. Age and inflammatory factors, C-reactive protein, and ferritin were associated with impaired exercise capacity. In addition, ageing did not significantly affect haematological variables or iron status indicators. Chapters 5 and 6 describe the effect of a single intravenous iron infusion on the haematological variables in 32 participants in a randomised, placebo-controlled and double-blinded study. The effects of iron infusion on RV afterload during light exercise, and exercise capacity during heavy exercise, were examined in these participants. With iron infusion, erythropoietin production, and the increase in RV afterload during light exercise were blunted, potentially indicating involvement of the HIF pathway. However, blunting of RV afterload neither influenced the cardiac output during light exercise nor exercise capacity. Chapter 7 describes a study of 11 healthy volunteers, which investigated the ROS hypothesis in HPV using acute isocapnic hypoxia following an 8-hour exposure to hyperoxia. This sustained hyperoxic exposure did not influence the hypoxic behavior of the pulmonary vasculature. This thesis demonstrates the complex relationship between iron status and exercise capacity in older adults. It shows that the decrease in RV afterload during exercise caused by intravenous iron supplementation does not lead to an augmented cardiac output or exercise capacity. Finally, it calls into question the role of ROS in HPV.

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / IntroduÃÃo: HipÃxia constitui em uma caracterÃstica comum de tumores sÃlidos, como em cÃnceres de cabeÃa e pescoÃo, e nessas condiÃÃes, uma via de sinalizaÃÃo envolvendo um regulador de resposta a oxigÃnio, chamado de Fator Induzido por HipÃxia-1 (HIF-1) tem-se destacado, na tentativa de permitir uma melhor compreensÃo sobre a biologia tumoral do cÃncer. Objetivo: Investigar o papel do HIF-1 em carcinoma epidermÃide oral (CEO) e em lesÃes potencialmente malignas que apresentam displasia epitelial oral (DEO). MÃtodos: Estudo observacional, analÃtico e transversal, atravÃs do diagnÃstico e anÃlise imuno-molecular de lesÃes malignas e potencialmente malignas. Foram incluÃdos 10 casos biopsiados com diagnÃstico histopatolÃgico de CEO e 10 casos de DEO. Utilizou-se a tÃcnica imunohistoquÃmica da Estreptoavitina-Biotina com o anticorpo HIF-1 (marca Abcam, diluiÃÃo 1:200, 60 minutos, recuperaÃÃo antigÃnica com citrato pH6 Pascal), analisando 05 campos de cada caso no aumento de 400X. O nÃmero de cÃlulas em cada um dos cinco campos foi somado e considerou-se como unidade amostral o percentual de cÃlulas imuno-positivas para HIF-1α conforme marcaÃÃo nuclear e citoplasmÃtica, bem como intensidade desta Ãltima. Os resultados foram obtidos e comparados entre grupos por meio dos testes t de Student e ANOVA multifatorial, seguido do pÃs-teste de Bonferroni, tomando como base os nÃveis de significÃncia de 5%. Resultados: Foi observado uma expressÃo citoplasmÃtica de HIF-1α em 58.4Â6.0% das cÃlulas epiteliais das DEO e em 77.8Â3.9% das cÃlulas dos CEO, com diferenÃa significante (p=0.022). O percentual de cÃlulas positivas para HIF-1α com marcaÃÃo nuclear tambÃm mostrou diferenÃa significante (p=0.021) entre DEO (0.2Â0.1%) e CEO (2.4Â0.8). NÃo houve diferenÃa significante entre o percentual de cÃlulas com imunomarcaÃÃo positiva fraca em citoplasma entre DEO e CEO (p=0.337), no entanto, houve aumento significante no percentual de cÃlulas com marcaÃÃo citoplasmÃtica moderada (p=0.029) e forte (p=0.031) entre DEO e CEO. ConclusÃo: Foi observada uma aumento da expressÃo, nuclear e citoplasmÃtica, de HIF-1α de DEO para CEO, sugerindo seu envolvimento em estÃgios iniciais da carcinogÃnese oral. RelevÃncia ClÃnica: Buscar o entendimento das complexas vias de sinalizaÃÃo, bem como do microambiente tumoral, e diferentes comportamentos biolÃgicos no cÃncer oral. / Introduction: Hypoxia is a common feature in solid tumors, such as head and neck cancers, and in these conditions a signaling pathway involving a response regulator oxygen, called hypoxia-inducible factor-1 (HIF-1) we have highlighted in an attempt to provide a better understanding of tumor biology of cancer. Objective: To investigate the role of HIF-1 in oral squamous cell carcinoma and premalignant lesions presenting oral epithelial dysplasia. Methods: Observational, analytical and cross through the diagnosis and immuno-molecular analysis of malignant and premalignant lesions. 10 biopsied cases with histopathological diagnosis of the CEO and DEO 10 cases were included. It was used the technique of immunohistochemistry Estreptoavitina-Biotin with HIF-1 antibody (Abcam mark, 1:200 dilution, 60 min pH6 citrate antigen retrieval Pascal) analysis of 05 fields each case at 400X magnification. The number of cells in each of five fields was added and the sample was considered as a unit the percentage of positive cells immuno-HIF-1α nuclear and cytoplasmic as well as intensity thereof. The results were obtained and compared between groups by the Student and multifactor ANOVA followed by Bonferroni post-test t test, based on the significance levels of 5%. Results: Cytoplasmic expression of HIF-1α was observed in 58.4 Â 6.0% of the epithelial cells of the DEO and 77.8 Â 3.9% of the cells of the CEO, with a significant difference (p = 0.022). The percentage of cells positive for HIF-1α nuclear staining also showed significant difference (p = 0.021) between DEO (0.2 Â 0.1%) and CEO (2.4 Â 0.8). There was no significant difference between the percentage of cells with weak positive immunostaining in the cytoplasm between DEO and CEO (p = 0.337), however, a significant increase in the percentage of cells with moderate cytoplasm staining (p = 0.029) and strong (p = 0.031) between DEO and CEO. Conclusion: One increasing the expression, nuclear and cytoplasmic HIF-1α DEO to CEO was observed, suggesting their involvement in the early stages of oral carcinogenesis. Clinical Relevance: Seek understanding of the complex signaling pathways, as well as the tumor microenvironment, and different biological behaviors in oral cancer.

Orientador: Andre Luís Berteli Ambrósio / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T16:57:05Z (GMT). No. of bitstreams: 1 Fala_AngelaMaria_M.pdf: 4666864 bytes, checksum: 3bcb145c1dc353b15b43b75e5723a310 (MD5) Previous issue date: 2013 / Resumo: A adaptação das células cancerosas ao microambiente é o ponto central que leva ao fenótipo invasivo e metastático, e é garantida principalmente através do controle preciso da expressão gênica. A resposta às necessidades energéticas e biossintéticas e principalmente à disponibilidade de oxigênio intracelular, por exemplo, é em grande parte mediada pelo fator de transcrição induzido por hipóxia 1 (HIF-1). HIF-1 é um heterodímero composto pelas subunidades ? e ?, que respondem a sequência consenso (5'-RCGTG-3') e ativam a transcrição de mais de 100 genes envolvidos em diversos aspectos cruciais da biologia tumoral, incluindo angiogênese, metabolismo de glicose, diferenciação celular, apoptose e resistência a radio e quimioterapias. São conhecidas três isoformas da subunidade ? (1 a 3) e todas se heterodimerizam com a subunidade ?. No geral, HIFs são constituídas de diferentes domínios funcionais, como de ligação ao DNA, de heterodimerização, transativação e degradação. Atualmente, pouco se sabe sobre os mecanismos estruturais e funcionais dos domínios da HIF- 1, deste modo este trabalho objetivou o estudo estrutural destes domínios. Os domínios bHLH, Pas-1 e Pac de HIF-1? e HIF-? em diferentes combinações entre si e o domínio Pac da HIF-3? foram clonados, as proteínas foram expressas em sistema bacteriano e purificadas por diferentes técnicas cromatográficas. Diversas destas construções se mostraram insolúveis ou suscetíveis a degradação, enquanto outras foram purificadas com sucesso. As construções Pac, um exemplo de sucesso na produção, foram submetidas a ensaios de anisotropia de fluorescência e ressonância magnética nuclear, o que nos permitiu a caracterização dos perfis de interação entre as várias combinações de heterodimerização. Neste contexto, os resultados mostram que o equilíbrio dinâmico da interação entre Pac-1? com a subunidade -1? é alcançado imediatamente, enquanto que para a interação entre Pac-3? e -1?, são necessários pelo menos 30 horas de incubação. O mesmo pode ser extraído da caracterização da interação direta entre Pac-1? e Pac-3?. Nos experimentos de RMN, foi possível identificar a região de interação entre as subunidades -1? e -3? com a subunidade ?, separadamente. Ambas as subunidades ? interagem com a Pac-1? na região das fitas-beta 1 e 5 e no loop entre as fitas 4 e 5. Em conjunto, estes resultados impactam no mecanismo de antagonização de HIF-3? na atividade transcricional de HIF-1?. Houve ainda a formação de monocristais da subunidade Pac-3?, que foram submetidos a experimentos preliminares de difração de raios X, que apesar de resultar em dados anisotrópicos e insuficientes para resolução estrutural, permitiram a caracterização dos parâmetros cristalinos, incluindo a presença de um alto conteúdo de solvente. Adicionalmente, são também apresentados os resultados obtidos visando a expressão e cristalização das Prolilhidroxilases (PHDs) isoformas 1 a 4, durante estágio de seis meses no Structural Genomics Consortium (SGC), da Universidade de Oxford, na Inglaterra. Foram expressas de maneira solúvel e purificadas, diversas construções das isoformas 1 e 3 das PHDs humanas. Cristais foram obtidos, porém estes foram determinados como sendo de compostos inorgânicos presentes na condição de cristalização. Como resultado final, está sendo estabelecida uma colaboração entre o nosso grupo e o SGC para que os estudos estruturais com PHDs se estendam e sejam realizados em nosso laboratório aqui no Brasil / Abstract: The adaptation process of cancer cells to the microenvironment is the central point leading to the invasive and metastatic phenotypes, and is guaranteed mainly through the precise control of gene expression. The cell response to the energetic and biosynthetic needs and especially to the availability of intracellular oxygen is mediated by the hypoxia inducible transcription factor 1, or HIF-1. HIF-1 functions as a heterodimer composed by subunits ? and ?, binding to responsive elements with the consensus sequence 5'-RCGTG-3 ', thus activating the transcription of more than 100 genes involved in many crucial aspects of tumor biology, including angiogenesis, metabolism glucose, cell differentiation, apoptosis, and resistance to radiotherapy and chemotherapy. There are three known isoforms of the ? subunit (1, 2 and 3) and all heterodimerize with the ? subunit. HIFs are composed of different functional domains, such as the DNA binding domain, the heterodimerization, transactivation and the oxygen-dependent degradation domains. Currently, little is known about the mechanisms of structural and functional domains of HIF-1, thus this work was to study these structural domains. The domain (bHLH, Pas-1 and Pac) of HIF-1? and HIF-? in different combinations with each other and Pac domain of HIF-3? were cloned, the proteins were expressed in bacterial system and purified by various chromatographic techniques. Several of these constructs proved insoluble or susceptible to degradation, while others were purified successfully. The constructs Pac, an example of success in production, were tested for fluorescence anisotropy and nuclear magnetic resonance, which allowed us to characterize the profiles of the interaction between the various combinations of heterodimers. In this context, the results show that the dynamic equilibrium of the interaction between the Pac-1? and -1? subunits is achieved immediately, whereas for the interaction between Pac-3? and -1?, it takes at least 30 hours of incubation. The same can be observed from the characterization of direct interaction between Pac-1? and -3?. From the NMR experiments, it was possible to identify the region of interaction between the subunits -1? and -3? with the -1? subunit. Both ? subunits interact with Pac-1? via betastrands 1 and 5 and the loop between the strands 4 and 5. Overall, these results impact in the mechanism of HIF-3? antagonizing the transcriptional activity of HIF-1?. We also obtained single crystals for Pac-3? subunit, which were subjected to preliminary experiments of X-ray diffraction. Although resulting in anisotropic, insufficient data for and structural resolution, it has allowed the characterization of crystalline parameters, including the presence of a high solvent content. Additionally, we also present the results targeting the expression and crystallization of Prolyl-hydroxylases (PHDs) human isoforms 1-4, during the six-month period at the Structural Genomics Consortium (SGC), the University of Oxford in England. Several construct from of isoforms 1 and 3 were successfully expressed and purified in the soluble form. Likewise, crystals were obtained, but these were determined to be composed by inorganic compounds present in the crystallization conditions. At the end, a collaboration was established between our and the SGC group for the structural studies with the PHDs to extend and carry out the experiments in our lab here in Brazil / Mestrado / Clinica Medica / Mestra em Clínica Médica

Abstract Oxygen deprivation (hypoxia) is related to many disease conditions, such as anemia, but is also a critical regulatory signal during normal development. Cellular responses to hypoxia are largely mediated through alterations in gene regulation brought about by the transcription factor known as hypoxia inducible facor (HIF). One of the most extensively studied systemic consequences of hypoxia is the induction of red blood cell production, erythropoiesis, which occurs through a HIF-dependent increase in erythropoietin (EPO) gene expression. The amount of HIF in cells is regulated by three HIF prolyl 4-hydroxylases (HIF-P4Hs) while a fourth P4H possessing a transmembrane domain (P4H-TM) is able to act on HIF at least in vitro. The putative role of P4H-TM in regulating erythropoiesis is studied here by administering a HIF-P4H inhibitor, FG-4497, to P4h-tm null and wild-type mice. By comparing the observed effects with those seen in FG-4497 treated hypomorphic Hif-p4h-2 and Hif-p4h-3 null mice, it is demonstrated for the first time that P4H-TM is involved in the regulation of Epo production in the mammalian kidney, but not in the liver. Long bones are formed via endochondral ossification, in which a cartilaginous template, the growth plate, is first laid down and then replaced with bone. The growth plate is rich in extracellular matrix (ECM) and contains a hypoxic central region in which HIF has been shown to regulate chondrocyte function. Importantly, growth plate chondrocytes are highly active in collagen synthesis. Collagen prolyl 4-hydroxylases (C-P4Hs I-III) provide collagen molecules with thermal stability and are thus necessary for the formation of a proper ECM. Through an in vitro approach it is demonstrated that hypoxia increases the amount and activity of C-P4H in primary mouse epiphyseal growth plate chondrocytes in a HIF-1-dependent manner. Lastly, it was set out to characterize mouse lines with complete inactivation of C-P4H-II with or without partial inactivation of C-P4H-I. A significant reduction in the total amount of C-P4H and its activity was found to result in mild chondrodysplasia and altered bone properties. The above mouse models provided new information on the specific in vivo roles of the C-P4H isoenzymes I and II. / Tiivistelmä Kudosten alentunut happipitoisuus (hypoksia) liittyy osana moniin elimistön patologisiin tiloihin, kuten anemiaan. Lisäksi se on tärkeä säätelytekijä normaalin yksilönkehityksen aikana. Jotta solut havaitsisivat hypoksian ja reagoidakseen siihen, on niille kehittynyt säätelyjärjestelmä, jossa hypoksiassa indusoituva transkriptiotekijä, HIF, on tärkeässä asemassa. Yksi merkittävin HIF:n indusoima systeeminen vaikutus elimistössä on punasolujen tuotannon, erytropoieesin, kiihtyminen. Sitä tapahtuu erytropoietiinia koodittavan geenin (EPO) lisääntyneen ilmentymisen kautta. HIF-tekijän määrää soluissa säätelee kolme HIF-prolyyli-4-hydroksylaasientsyymiä (HIF-P4Ht 1-3). Transmembraanisen prolyyli-4-hydroksylaasin (P4H-TM) tiedetään myös vaikuttavan HIF-tekijän määrään soluissa in vitro, mutta sen vaikutusta nisäkkään erytropoieesiin ei ole aiemmin tutkittu. Käyttämällä hyväksi kolmea eri transgeenista hiirilinjaa (P4h-tm-/-, Hif-P4h-2gt/gt, Hif-p4h-3-/-) ja HIF-P4H entsyymeitä inhiboivaa lääkeainetta, FG-4497, tässä työssä osoitettiin ensimmäistä kertaa, että P4H-TM osallistuu nisäkkään Epo-hormonin tuoton säätelyyn. Pitkät luut muodostuvat endokondraalisen luutumisen kautta. Siinä ensin muodostuu rustoinen malli, kasvulevy, joka vähitellen korvaantuu luukudoksella. Kasvulevyn sisin kerros on sen soluille, kondrosyyteille, hypoksinen kasvuympäristö. HIF:illä on todettu olevan tärkeä rooli kondrosyyttien toiminnan säätelijänä. Kasvulevyn soluvälitila sisältää runsaasti kollageeneja. Kollageenin prolyyli-4-hydroksylaasit (C-P4Ht I-III) ovat avainasemassa kollageenien biosynteesissä ja siten niiden toiminta on välttämätöntä kestävän soluvälitilan muodostumiselle. Käyttämällä in vitro menetelmiä, tässä työssä osoitettiin, että hiiren epifyseaalisten kasvulevyjen kondrosyyteissä hypoksia lisää C-P4H:n määrää ja aktiivisuutta HIF-tekijästä riippuvalla mekanismilla. Eri C-P4H-isoentsyymeiden toiminnasta ja merkityksestä in vivo tiedetään vain vähän. Tässä työssä karakterisoitiin hiirilinja, jossa C-P4H-II on täysin inaktiivinen, ja hiirilinja, jossa lisäksi C-P4H-I on osittain inaktiivinen. Merkittävästi alentuneen C-P4H:n aktiivisuuden todettiin aiheuttavan hiirimallissa lievän kondrodysplasian sekä heikentyneet luun ominaisuudet.

Abstract Prolyl hydroxylases catalyze the post-translational formation of 3- and 4-hydroxyprolines in polypeptides. To date, two prolyl 4-hydroxylase families are known to exist: collagen prolyl 4-hydroxylases (C-P4Hs) which reside in the endoplasmic reticulum, and hypoxia-inducible factor prolyl 4-hydroxylases (HIF-P4Hs) which reside in either the cytoplasm or nucleus. C-P4Hs and HIF-P4Hs belong to the 2-oxoglutarate dioxygenase family and require Fe2+, 2-oxoglutarate, O2 and ascorbate in their reaction. C-P4Hs are critical enzymes in collagen biosynthesis since the formation of 4-hydroxyproline residues stabilizes the collagen triple helix at body temperature. HIF-P4Hs regulate, through hypoxia-inducible factor HIF, the expression of genes that are essential for the survival of cells during hypoxia. This thesis reports the cloning and characterization of two novel P4Hs, from human and a plant, which show some distinct features when compared to previously characterized P4Hs. The human P4H was found to have a unique transmembrane domain, with its catalytic region residing inside the lumen of the endoplasmic reticulum, its subcellular localization thus being identical to that of the C-P4Hs. However, unlike C-P4Hs, it hydroxylated HIF-α both in vitro and in cellulo. Furthermore, its expression level was induced in hypoxic conditions in most of the cell lines studied. The Arabidopsis thaliana P4H had distinct differences in its substrate specificity when compared to another previously characterized A. thaliana P4H. Interestingly, the putative peptide binding regions of the two new P4Hs characterized in this study shared some homology. Three prolyl 3-hydroxylase (P3H) isoenzymes are known to exist in vertebrates and they also belong to the 2-oxoglutarate dioxygenases. It is known that 3-hydroxyprolines are found only in collagens, being most abundant in type IV collagen. The function of this modification is as yet poorly understood, but its absence in collagen I has recently been shown to lead to recessive lethal osteogenesis imperfecta. The human P3H isoenzymes were cloned during these thesis studies, and were expressed as recombinant proteins. The kinetic properties of one of them, P3H2, which was found to be expressed in structures rich with basement membranes, was studied in detail. / Tiivistelmä Prolyylihydroksylaasit ovat entsyymejä, jotka katalysoivat 3- ja 4-hydroksiproliinien muodostumisen valkuaisaineissa. Nykyisin tunnetaan ainakin kaksi prolyyli-4-hydroksylaasien (P4H) entsyymiperhettä: endoplasmakalvostossa sijaitsevat kollageeni prolyyli-4-hydroksylaasit (kollageeni-P4H:t) sekä vähähappisissa olosuhteissa aktivoituvaa transkriptiotekijää, hypoksiaindusoituvaa faktoria (HIF), hydroksyloivat prolyyli-4-hydroksylaasit (HIF-P4H:t). HIF-P4H:t sijaitsevat sytoplasmassa ja tumassa. Sekä kollageeni-P4H:t että HIF-P4H:t kuuluvat 2-oksoglutaraattidioksygenaasien laajaan entsyymiperheeseen. Nämä entsyymit tarvitsevat kosubstraateikseen rautaa, 2-oksoglutaraattia, happea sekä C-vitamiinia. Kollageeni-P4H:t hydroksyloivat kollageenien proliinitähteitä ja ovat avainasemassa kollageenisynteesissä, sillä muodostuneet 4-hydroksiproliinitähteet ovat ehdoton vaatimus stabiilille kollageenirakenteelle. HIF-P4H:t säätelevät puolestaan niiden geenien ilmenemistä, jotka ovat välttämättömiä elimistön selviytymiselle vähähappisissa olosuhteissa. HIF-P4H:t hydroksyloivat HIF-transkriptiotekijän α-alayksikön tiettyjä proliinitähteitä hapen läsnä ollessa, joka ohjaa α-alayksikön proteasomaaliseen hajotukseen eikä aktiivista HIF transkriptiotekijää siten muodostu. Alentuneessa happipitoisuudessa HIF-P4H entsyymien toiminta estyy, HIF stabiloituu ja aktivoi kohdegeeniensä toiminnan. Kollageeni-P4H entsyymejä pidetään erityisen sopivina lääkekehityksen kohteina fibroottisten ja HIF-P4H entsyymejä iskeemisten sairauksien hoitoon. Tässä väitöskirjatyössä on karakterisoitu aiemmin tuntematon ihmisen transmembraaninen P4H entsyymi (P4H-TM). Entsyymi osoittautui indusoituvan vähähappisissa olosuhteissa useissa solulinjoissa ja hydroksyloivan HIF-transkriptiotekijää muistuttaen siten HIF-P4H entsyymejä. Kuitenkin P4H-TM:n solulokalisaatio poikkesi HIF-P4H entsyymeistä, sillä sen havaittiin sijaitsevan endoplasmakalvostossa, katalyyttinen keskus kalvoston sisällä. Näiden tutkimustulosten valossa on oletettavaa, että tällä ihmisentsyymillä on HIF:n lisäksi toinen toistaiseksi tuntematon fysiologinen substraatti. Väitöskirjassa karakterisoitiin toinen lituruohon, Arabidopsis thalianan, P4H (At-P4H-2), joka poikkesi katalyyttisiltä ominaisuuksiltaan aiemmin karakterisoidusta lituruohon P4H:sta. Näiden kahden kasvientsyymin substraattivaatimusten poiketessa selvästi toisistaan, on niillä solussa todennäköisesti spesifiset tehtävät. At-P4H-2:n oletetulla substraatin sitomisalueella on jakso, joka on 37-prosenttisesti identtinen ihmisen P4H-TM:n kanssa, minkä vuoksi At-P4H-2:n karakterisoinnin uskottiin olevan tärkeä apuväline ihmisen P4H-TM:n tutkimuksissa. Selkärankaisilla prolyyli-3-hydroksylaaseja (P3H) tiedetään olevan kolme, ja myös ne kuuluvat 2-oksoglutaraattidioksygenaaseihin. 3-Hydroksiproliinia esiintyy ainoastaan kollageeneissa, erityisesti tyypin IV kollageenissa, joka on tyvikalvojen tärkeä rakennekomponentti. 3-Hydroksiproliinin merkitystä ei tunneta vielä tarkoin, mutta tyypin I kollageenissa 3-hydroksiproliinin puutoksen on osoitettu johtavan vaikeaan luustosairauteen, osteogenesis imperfectaan. Väitöskirjatyössä ihmisen P3H:t kloonattiin ja tuotettiin rekombinanttiproteiineina. Yhden isoentsyymin (P3H2) katalyyttiset ominaisuudet määritettiin ja sen osoitettiin ilmenevän erityisesti kudoksissa, joissa on paljon tyvikalvorakenteita.

Abstract Breast cancer is the most common cancer affecting the female population of the Western world. It is a heterogeneous disease entity that encompasses tumors with remarkably different forms of behaviour, and it is therefore vital to distinguish patients with good and poor prognoses. The classical prognostic and predictive factors for breast cancer serve as tools for clinical oncologists when planning treatment, but the growing awareness of breast cancer biology is bringing about a need for novel prognostic and predictive biomarkers. This thesis examines the prognostic significance of hypoxia response and cell cycle regulators in ductal breast cancer and in triple-negative breast cancer (negative for hormone receptors and human epidermal growth factor receptor 2), concluding that PHD2 and PHD3 are associated with a good prognosis, while the role of PHD1 is controversial, as it is associated with proliferation in ductal breast cancer but with node-negative status in triple-negative breast cancer. In our experiments HIF-1α redeemed its role as a marker of an adverse prognosis, whereas the role of HIF-2α appeared to be the opposite. Our data suggest that PHDs can have other targets than the HIF-αs, and that triple-negative breast tumors express more HIF-1α and less HIF-2α and PHD3 than those with a good prognosis. Furthermore, we identified cyclin D1 as a biomarker with independent prognostic significance in ductal breast cancer, being associated with good prognostic factors and a better outcome, whereas the opposite was seen in triple-negative breast cancer. CDK4 was associated with high proliferation in triple-negative breast cancer. In addition, high levels of p16 correlated with increased survival in breast cancer patients independently of receptor status. / Tiivistelmä Rintasyöpä on naisten yleisin syöpä läntisessä maailmassa. Rintasyöpä on heterogeeninen tautiryhmä, jossa kasvaimet vaihtelevat biologiselta käyttäytymiseltään huomattavasti. Tästä syystä on tärkeää erottaa hyvä- ja huonoennusteiset potilaat. Syöpälääkärit käyttävät klassisia ennustetekijöitä hoitopäätöksiä tehdessään, mutta lisääntynyt tieto rintasyövän biologiasta on saanut aikaan tarpeen löytää uusia ennustetekijöitä. Tässä väitöskirjatyössä tutkimme hypoksiavasteen ja solusyklin säätelijöiden ennusteellisuutta duktaalisessa rintasyövässä sekä kolmoisnegatiivisessa (ei ilmennä hormonireseptoreita eikä epidermaalikasvutekijäreseptoria) rintasyövässä. PHD2 ja PHD3:n vahva ilmentyminen liittyi parempaan ennusteeseen, mutta PHD1:n esiintymisen vaikutus oli ristiriitainen. PHD1:n ilmentyminen liittyi lisääntyneeseen solujakautumiseen duktaalisessa rintasyövässä, mutta kolmoisnegatiivisessa rintasyövässä sen esiintyminen liittyi vähentyneeseen imusolmukemetastasointiin. Tutkimuksessamme HIF-1α osoittautui huonon ennusteen merkiksi. Sitä vastoin HIF-2α:n ilmentymisen vaikutus näytti liittyvän parempaan ennusteeseen. Tuloksemme osoittavat, että PHD-entsyymeillä on mahdollisesti muitakin kohteita kuin HIF-α:t. Osoitimme myös, että HIF-1α:n ilmentyminen on yleisempää ja HIF-2α:n sekä PHD3:n ilmentyminen vähäisempää kolmoisnegatiivisessa kuin duktaalisessa rintasyövässä. Lisäksi totesimme, että sykliini D1 on itsenäinen ennustetekijä liittyen parempaan ennusteeseen duktaalisessa rintasyövässä. Huomioitavaa on kuitenkin, että kolmoisnegatiivisessa rintasyövän alaryhmässä sykliini D1:n esiintyminen oli huonon ennusteen merkki. CDK4 osoittautui voimakkaan proliferaation merkiksi kolmoisnegatiivisessa rintasyövässä. Lisäksi osoitimme, että p16:n ilmentyminen liittyy parempaan ennusteeseen sekä duktaalisessa rintasyövässä että kolmoisnegatiivisessa rintasyövässä.

Abstract Oxygen homeostasis is critical to all animals, as both excess (hyperoxia) and reduced (hypoxia) levels of oxygen can result in pathological changes and ultimately in the loss of cellular and organismal viability. Complex systems have evolved to sense and adapt to changes in cellular oxygen availability, and the hypoxia-inducible factor HIF plays a pivotal role in this elaborate molecular network. In normoxic conditions the α-subunit of HIF becomes hydroxylated by HIF prolyl 4-hydroxylases (HIF-P4Hs 1-3), earmarking HIF-α for proteasomal degradation. Additionally, in the presence of oxygen the hydroxylation of an asparagine residue by the HIF asparaginyl hydroxylase FIH inhibits the transactivation of HIF-target genes by blocking the interaction of HIF-α with a transcriptional coactivator. In addition to being a feature of an organism’s normal life, hypoxia is also characteristic of many common diseases such as severe anemia and myocardial infarction, and it notably decreases these hydroxylation reactions, as HIF-P4Hs and FIH have an absolute requirement for oxygen as a cosubstrate. HIF-α thus escapes degradation and translocates into the nucleus, where it dimerizes with HIF-β and recruits transcriptional coactivators to the hypoxia-response elements of target genes, inducing their transcription and triggering the hypoxia response aimed at restoring cellular oxygen homeostasis. In this study we generated a genetically modified HIF-P4H-2 hypomorphic mouse line that expresses only 8% of the wild-type HIF-P4H-2 mRNA in the heart. We showed that chronic cardiac HIF-P4H-2 deficiency leads to stabilization of HIF-1α and HIF-2α and protects the heart against acute ischemia-reperfusion injury without causing any adverse effects. Furthermore, we identified and cloned a novel human transmembrane prolyl 4-hydroxylase P4H-TM and showed that it regulates HIF-1α protein levels in cellulo and hydroxylates HIF-1α in vitro similarly to the HIF-P4Hs, but may also have other physiological substrates. Using forward genetic tools we showed that lack of P4H-TM during development leads to basement membrane defects and compromised kidney function in zebrafish embryos. Finally, we demonstrated that FIH displays substrate selectivity in terms of hydroxylation and binding of HIF-1α and novel substrates Notch1-3. We showed that FIH has higher affinity for oxygen with Notch1 than with HIF-1α as a substrate, implying that FIH-mediated hydroxylation of Notch can continue in oxygen concentrations where HIF-1α hydroxylation would be markedly reduced. / Tiivistelmä Happitasapainon ylläpito on edellytys elimistön normaalille toiminnalle, koska sekä liian korkea (hyperoksia) että liian matala (hypoksia) happipitoisuus ovat elimistölle stressitiloja ja johtavat pitkittyessään haitallisiin seurauksiin. Happipitoisuuden muutosten havaitsemiseksi ja niihin reagoimiseksi onkin elimistössä kehittynyt monimutkainen säätelyjärjestelmä, jossa avainasemassa on hypoksia-indusoituva tekijä HIF. Solun happipitoisuuden ollessa normaali yksi kolmesta HIF prolyyli 4-hydroksylaasi-isoentsyymistä (HIF-P4Ht 1-3) katalysoi kahden proliinitähteen hydroksylaation HIF-α-alayksikössä. 4-hydroksiproliini toimii signaalina HIF-α:n nopealle proteasomaaliselle hajotukselle. Lisäksi HIF asparaginyyli hydroksylaasi FIH:n katalysoima HIF-α:n asparagiinitähteen hydroksylaatio estää transaktivaatiovaikutuksen. Koska HIF-P4Ht ja FIH tarvitsevat kosubstraatikseen happea, nämä hydroksylaatioreaktiot vähenevät happipitoisuuden laskiessa, jolloin HIF-α stabiloituu ja siirtyy solun tumaan, jossa se muodostaa kompleksin HIF-β-alayksikön kanssa ja houkuttelee paikalle tarvittavat kofaktorit. HIF-kompleksi tehostaa hypoksiavasteessa tarvittavien geenien luentaa sitoutumalla tumassa niiden promoottoreihin ja pyrkii näin palauttamaan solun happipitoisuuden normaaliksi. Tässä työssä luotiin geneettisesti muunneltu HIF-P4H-2 hypomorfi-hiirilinja, jonka sydämissä tuottuu vain 8 % normaalista HIF-P4H-2 lähetti-RNA:n määrästä. HIF-P4H-2:n puutoksen havaittiin johtavan HIF-1α:n ja HIF-2α:n stabiloitumiseen sydämessä ja suojaavan sydäntä kudosvaurioilta iskemian ja reperfuusion aikana aiheuttamatta haitallisia vaikutuksia. Tässä väitöskirjassa karakterisoitiin aiemmin tuntematon ihmisen transmembraaninen prolyyli 4-hydroksylaasi, P4H-TM. Sen osoitettiin säätelevän HIF-1α:n määrää soluissa ja katalysoivan HIF-1α:n kahden proliinitähteen hydroksylaatiota in vitro-olosuhteissa HIF-P4H-entsyymien tavoin. Seeprakalamallin avulla näytettiin, että P4H-TM:n puutos kalan kehityksen aikana aiheuttaa tyvikalvopoikkeavuuksia ja johtaa vakavaan munuaisen toiminnan häiriintymiseen seeprakalan poikasissa. FIH:n katalysoiman hydroksylaatioreaktion kineettisiä ominaisuuksia verrattiin tässä tutkimuksessa ensimmäistä kertaa aiemmin tunnetun HIF-α substraatin ja uusien Notch substraattien kesken. Tulokset osoittivat, että substraatin sitomisessa ja hydroksylaatiossa on merkittäviä eroja eri substraattien välillä.

Persistent organic pollutants (POPs) and adipose tissue hypoxia have been shown to independently affect adipocyte functions. The goals of this study were to (1) determine the effect of PCB-77, PCB-153, and DDE on the differentiation of human preadipocytes, and (2) investigate the cross-talk between PCB-77 and hypoxia in differentiated human adipocytes. First, human preadipocytes were exposed to PCB-77, PCB-153, or DDE during the entire 14-day differentiation period. We found no effect of low POP levels on lipid accumulation. Second, differentiated human adipocytes were exposed to a combination of PCB-77 and hypoxia. We demonstrated gene-specific cross-talk between PCB-77 and hypoxia, showing an additive effect of PCB-77 on VEGF, MCP-1, and adiponectin, as well as an inhibition of PCB-77-induced expression of CYP1A1 by hypoxia. This work has expanded our understanding of the role of POPs and hypoxia in differentiated human adipocytes.

Adaptation to chronic hypoxia is characterized by a variety of functional changes in order to maintain metabolic and energy homeostasis. It has been known for many years that both humans and animals indigenous or adapted to high-altitude hypoxia are more tolerant to an acute ischemic injury of the heart. HIF1α is found as a primary transcriptional regulator of adaptive response to hypoxia in all tissues, whereas HIF2α is more tissue specific. The activity of HIFα is regualted by prolyl hydoxylases (EGLN). The EGLN1 was shown to be more efficient in HIF1α hydroxylation than in HIF2α. As well as the EGLN3 is more specific for HIF2α . Under hypoxic conditions, HIF activates the transcription of about 70 genes. Hence, HIF1α and HIF2α can play an essential role in pathophysiology of ischemic heart disease. The aim of our study was to determine myocardial expression of HIFs and its regulatory hydroxylases in adult male Wistar rats adapted to continuous normobaric hypoxia (H; FIO2 = 0.1) for 3 weeks. Another two groups of rats were exposed to normobaric hypoxia intermittently for either 8 h/day (INH) or 23 h/day (RH) during the 3-week adaptation period. While H induces protective cardiac phenotype, the later regimen (RH) does not. The protein expression of HIFs, PHDs and FIH were then determined by...