Os substitutos valvares possuem grande importância pela freqüência da sua utilização e porque de seu bom desempenho depende o sucesso do procedimento cirúrgico realizado. Vários substitutos valvares têm sido empregados, todos com complicações inerentes ao material utilizado, como trombose, calcificação, degeneração, dificuldade de esterilização, custo, complexidade de produção, etc. Buscando disponibilizar homoenxertos preservados de maneira mais simples e econômica, avaliamos uma nova forma de preservação utilizando o polietileno-glicol, método L-Hydro. Este método consiste na extração controlada de substâncias antigênicas e de incorporação de um agente antinflamatório e anti-trombótico. Em dez carneiros jovens substituímos o Tronco Pulmonar, em sete por homoenxertos pulmonares tratados pelo processo L-Hydro e em três por homoenxertos pulmonares a fresco, implantados ortotopicamente e seguidos por 320 dias. Os carneiros foram avaliados por exames laboratoriais e ecocardiográficos. Ao cabo dos 320 dias foram sacrificados, quando se procedeu à avaliação hemodinâmica, radiológica, macroscópica e por microscopia óptica e eletrônica, de varredura e transmissão. Os resultados foram analisados pelo teste t de Student de amostras independentes para os dados contínuos, pela análise de variância para as medidas repetidas e pelo teste exato de Fischer para os dados categóricos. Na evolução clínica e nos exames laboratoriais não conseguimos estabelecer diferenças significativas entre os dois grupos. O ecocardiograma revelou diferença quanto ao gradiente médio pulmonar, significativa aos 10 meses de seguimento, maior no grupo controle do que no grupo L-Hydro. A avaliação radiológica e macroscópica não estabeleceu diferenças. Na avaliação microscópica, óptica e eletrônica, células de revestimento e intersticiais foram encontradas nos dois grupos igualmente. O porcentual de revestimento celular calculado nos dois grupos foi semelhante. Nódulos de celularidade foram observados somente no grupo de homoenxertos a fresco. Em conclusão, estes dados indicam que os dois grupos apresentaram desempenho clínico e hemodinâmico semelhante. Ao ecocardiograma o grupo L-Hydro apresentou melhor desempenho; apresentou também evidências histológicas de repopulação celular intersticial e endotelial. Na análise macro e microscópica, óptica e eletrônica, o grupo L-Hydro apresentou macroscopia, estrutura histológica e ultraestrutural semelhante ao homoenxerto afresco, à exceção de nódulos de maior celularidade intersticial, presentes apenas no homoenxerto a fresco / Valve substitutes are highly important in account of their frequent use and since the success of a surgical procedure depends on their good performance. A variety of valve substitutes have been used, all presenting complications pertaining to their materials, such as thrombosis, calcification, degeneration, sterilization difficulties, cost, production complexity, etc. In an effort to make available homografts preserved in a simpler and less costly way, we evaluated a new preservation form using polyethyleneglycol, the LHydro method. This method consists in the controlled extraction of antigenic substances and the incorporation of an anti-inflammatory and anti-thrombotic agent. We substituted the pulmonary trunk in ten ovines, seven received LHydro treated pulmonary homografts and three received pulmonary homografts fresco, orthotopically implanted and followed-up for 320 days. Ovines where evaluated by means of laboratory tests and echocardiographic exams. At the end of the 320 days, they were euthanized, and hemodynamic, radiology, macroscopic, optic and electronic microscopic, scanning and transmission evaluations were performed. Results were analyzed by Student t test of independent samples for continuous data, by variance analysis of repeated measures, and by Fischer exact test for categorical data. We couldnt establish relevant differences in clinical evolution and laboratory tests between both groups. Echocardiogram revealed a difference in the pulmonary medium gradient, which was significant at the 10 months followup, higher in the control group than in the L-Hydro group. Radiologic and macroscopic evaluations didnt established differences. In the optic and electronic microscopic evaluation, liner and interstitial cells were equally found in both groups. The cell liner percent calculated in both groups was similar. Cellularity nodules were observed only in the homograft fresco group. In conclusion, these data indicate that both groups presented similar clinical and hemodynamic performances. The L-Hydro groups echocardiogram presented a better performance. It also presented histological evidences of interstitial and endothelial cell repopulation. In the macro and optic and electronic microscopic analysis, group L-Hydro presented macroscopy, histological structure and ultra-structural similar to the homograft fresco group, with the exception of nodules with higher interstitial cellularity, present only in the homograft fresco group.

Abstract The maintenance of integrity of the genome is essential for a cell. DNA repair and faithful DNA replication ensure the stability of the genome. DNA polymerases (pols) are the enzymes that synthesise DNA, a process important both in DNA replication and repair. In DNA replication DNA polymerases duplicate the genome during S phase prior to cell division. Pols α, δ, and ε are implicated in chromosomal DNA replication, but their exact function in replication is not yet completely clear. The mechanisms of different repair pathways and proteins involved are not yet completely characterised either. The deeper understanding of DNA repair and replication mechanisms is crucial for our understanding on the function of the cell. The mechanism of repair of DNA double strand breaks (DSBs) by non-homologous end joining (NHEJ) was studied with an in vitro assay. DNA polymerase activity was found to be involved in NHEJ and important in stabilising DNA ends. Antibodies against pol α, but not pol β or ε, decreased NHEJ significantly, which indicates the involvement of pol α in NHEJ. In addition, the removal of proliferating cell nuclear antigen (PCNA) slightly decreased NHEJ activity. The division of labour between pols α, δ, and ε during DNA replication was studied. Results from UV-crosslinking, chromatin association, replication in isolated nuclei, and immunoelectron microscopy (IEM) studies showed that there are temporal differences between the activities and localisations of the pols during S phase. Pol α was active throughout S phase, pol ε was more active at early S phase, whereas the activity of pol δ increased as S phase advanced. These results suggest that pols δ and ε function independently during DNA replication. Pol ε could be crosslinked to nascent RNA, and this labelling was not linked to DNA replication, but rather to transcription. Immunoprecipitation studies indicated that pol ε, but not pols α and δ, associated with RNA polymerase II (RNA pol II). Only the hyperphosphorylated, transcriptionally active RNA pol II was found to associate with pol ε. A large proportion of pol ε and RNA pol II colocalised in cells as determined with immunoelectron microscopy. The interaction between pol ε and RNA pol II suggests that they are involved in a global regulation of transcription and DNA replication.

Abstract Analysis of the primary structure of DNA polymerase ε B subunit defined similarities to B subunits of eukaryotic DNA polymerases α, δ and ε as well as the small subunits of DNA polymerase DI of Euryarchaeota. Multiple sequence alignment of these proteins revealed the presence of 12 conserved motifs and defined a novel protein superfamily. The members of the B subunit family share a common domain architecture, suggesting a similar fold, and arguing for a conserved function among these proteins. The contribution of human DNA polymerase ε to nuclear DNA replication was studied using the antibody K18 that specifically inhibits the activity of this enzyme in vitro. This antibody significantly inhibited DNA synthesis both when microinjected into nuclei of exponentially growing human fibroblasts and in isolated HeLa cell nuclei, but did not inhibit SV40 DNA replication in vitro. These results suggest that the human DNA polymerase ε contributes substantially to the replicative synthesis of DNA and emphasises the differences between cellular replication and viral model systems. The human DNA polymerases ε and δ were found capable of gap-filling DNA synthesis during nucleotide excision repair in vitro. Both enzymes required PCNA and the clamp loader RFC, and in addition, polymerase δ required Fen-1 to prevent excessive displacement synthesis. Nucleotide excision repair of a defined DNA lesion was completely reconstituted utilising largely recombinant proteins, only ligase I and DNA polymerases δ and ε provided as highly purified human enzymes. This system was also utilised to study the role of the transcription factor II H during repair. Human non-homologous end joining of model substrates with different DNA end configurations was studied in HeLa cell extracts. This process depended partially on DNA synthesis as an aphidicolin-dependent DNA polymerase was required for the formation of a subset of end joining products. Experiments with neutralising antibodies reveal that DNA polymerase α but not DNA polymerases β or ε, may represent this DNA polymerase activity. Our results indicate that DNA synthesis contributes to the stability of DNA ends, and influences both the efficiency and outcome of the end joining event. Furthermore, our results suggest a minor role of PCNA in non-homologous end joining.

Trypanosoma brucei est un parasite protozoaire responsable de la trypanosomiase humaine africaine. Il présente un cycle de vie complexe alternant entre des hôtes mammifères et un vecteur insecte, la mouche tsé-tsé. Au cours de ce cycle, il rencontre des environnements radicalement distincts auxquels il s’adapte en régulant son métabolisme. Nous avons étudié le métabolisme intermédiaire et énergétique de la forme procyclique évoluant dans le tractus digestif de l’insecte vecteur. Dans cet environnement dépourvu de glucose, la néoglucogenèse est cruciale pour la croissance et la survie des parasites car elle permet la synthèse d’hexoses phosphates et en particulier du glucose 6-phosphate qui alimente plusieurs voies de biosynthèse essentielles. Nos travaux confirment ce flux néoglucogénique alimenté par la proline mais aussi par le glycérol. Nous montrons que le glycérol est une source de carbone efficacement métabolisée et préférentiellement utilisée par la forme procyclique à défaut de la proline et même du glucose pour alimenter son métabolisme intermédiaire. Cette situation qu in’a jamais été décrite auparavant met en évidence la répression du glycérol sur le métabolisme du glucose. Nous montrons également que l’enzyme fructose 1,6-biphosphatase(FBPase), spécifique de la néoglucogenèse, n’est pas essentielle à la survie du parasite en conditions dépourvues de glucose indiquant qu’il existe une alternative à cette enzyme.Toutefois, FBPase joue un rôle important dans la virulence de T. brucei dans l’insecte.De plus, nous avons mis en évidence une autre stratégie d’adaptation de T. brucei basée sur des réarrangements génomiques qui peuvent mener à la synthèse de gènes chimères. / Trypanosoma brucei is a protozoan parasite responsible for human African trypanosomiasis. His complex life cycle alternates between mammalian hosts and the insect vector, the tsetsefly. During this cycle, the parasite encounters dissimilar environments and adapts to the sechanging conditions by regulating his metabolism. We have studied intermediate and energetic metabolism of the procyclic form living in the midgut of the insect vector. In this glucose-depleted environment, gluconeogenesis is crucial for growth and viability of the parasites. Indeed, it allows the synthesis of hexoses phosphates and in particular glucose 6-phosphate which feeds several essential biosynthetic pathways. Our work has confirmed the existence of a gluconeogenic flux fed by proline and glycerol. We have shown that glycerol is an efficiently metabolized carbon source and is preferentially used by the procyclic form rather than proline or even glucose. This situation never described before highlights glycerol repression on glucose metabolism. We have also showed that the enzyme fructose 1,6-biphosphatase (FBPase), specific of the gluconeogenesis, is not essential for the viability ofthe parasite in glucose-depleted conditions, suggesting that there is an alternative to this enzyme. However, FBPase plays an important role for virulence of T. brucei in the insect. Moreover, we have showed another adaptation strategy developed by T. brucei which is basedo n genomic rearrangements leading to the synthesis of chimeric genes.

La diversité des génomes de virus infectant les bactéries, les bactériophages (ou phage en abrégé), est telle qu’il est difficile de les classer de manière satisfaisante, la notion d’espèce elle-même ne faisant pas accord dans la communauté scientifique. A la racine de cette diversité, un des facteurs clé est la recombinaison de l’ADN, qui est élevée chez les bactériophages, et permet des échanges de gènes entre entités parfois fort différentes. Mes travaux se sont centrés sur la recombinaison homologue chez les bactériophages, et en particulier sur la protéine centrale de ce processus, la recombinase. J’ai montré pour deux grands types de recombinases phagiques, de type Rad52 et Sak4, que celles-ci étaient beaucoup moins fidèles dans le processus de recombinaison, comparées à la recombinase bactérienne RecA. De plus, pour Sak4, j’ai observé que cette recombinaison se produisait par appariement simple brin, et qu’elle dépendait entièrement in vivo d’une SSB phagique, dont le gène est situé à proximité du gène sak4 sur le chromosome du phage. Les échanges génétiques sont donc grandement facilités pour les phages contenant ce type de recombinases, mais ils ne sont pas non plus anarchiques : la recombinaison s’observe jusque 22% de divergence, mais deux séquences à 50% de divergence ne peuvent recombiner. Tout se passe donc comme si la notion d’espèce devait être élargie chez les phages par rapport aux bactéries, pour inclure dans un même groupe des génomes portant des traces d’échanges récents de matériel génétique par recombinaison homologue (ce que l’on appelle le mosaïcisme). / The diversity of the viruses infecting bacteria (bacteriophages, or phages for short) is so important that it is difficult to classify them in a pertinent way, and the species notion itself is a matter of debate among specialists. At the root of this diversity, one of the key factors is DNA recombination, which occurs at high levels among phages, and permits gene exchanges among entities that are sometimes very distant. My research has focused on homologous recombination in phages, and in particular on the protein that is key to the process, the recombinase. I have shown, for two different types of recombinases, Rad52-like and Sak4-like, that their fidelity was relaxed, compared to the bacterial recombinase, RecA. Moreover, for Sak4, a protein that had not been studied before, I showed that recombination occurs by single strand annealing, and that it is strictly dependent in vivo on the co-expression of its cognate SSB protein, whose gene is often encoded nearby in phage genomes encoding sak4. Genetic exchanges are therefore greatly facilitated for phages encoding these types of recombinases. Nevertheless, exchanges are not anarchical: recombination is seen up to 22% diverged substrates, but 50% diverged DNA sequences will not recombine. It may be that the species notion should be enlarged for phages, so as to include into a same group all phages exhibiting traces of recent exchanges of genetic material (the so-called mosaicism).

Les cassures double brin de l’ADN sont des dommages pouvant engendrer la mort cellulaire. Deux mécanismes majeurs sont impliqués dans leur réparation chez les bactéries : la recombinaison homologue et le Non-Homologous End Joining (NHEJ). Streptomyces est une bactérie modèle pour étudier l'impact relatif des mécanismes de recombinaison sur la structure du génome et son évolution ; le chromosome est en effet caractérisé par sa linéarité, son organisation génétique compartimentée et sa plasticité génomique remarquable. L'objectif de cette recherche est d'identifier les acteurs impliqués dans les mécanismes de réparation des cassures double brin qui restent inconnus chez Streptomyces à ce jour. Concernant la recombinaison homologue, la première étape consiste en une maturation des extrémités d’ADN générées par la cassure. Cette première étape est assurée par un complexe à activité hélicase-nuclease : RecBCD (chez Escherichia coli), AddAB (chez Bacillus subtilis) ou AdnAB (chez les mycobactéries). Une analyse in silico des génomes disponibles de Streptomyces a permis d’identifier chez ces organismes, deux gènes conservés et adjacents, nommés adnA et adnB en raison de leur homologie avec les gènes adnAB récemment identifiés chez les mycobactéries. Les tentatives visant à déléter ces gènes chez Streptomyces ambofaciens et Streptomyces coelicolor ont été infructueuses. Cependant, le fait que leur délétion soit rendue possible par l’ajout d’une copie ectopique du locus sauvage nous a amené à conclure au caractère essentiel d’adnA et adnB chez Streptomyces. La trans-complémentation d’un mutant [delta]recB d’E. coli par le locus adnAB de S. ambofaciens restaure l’activité nucléase cellulaire et la survie en présence ou non d’agent génotoxique, suggérant qu’adnAB code l’homologue fonctionnel de RecBCD d’E. coli. Le rôle central d’adnAB dans la recombinaison homologue et la réplication est discuté. Le mécanisme NHEJ montre une distribution sporadique chez les bactéries et implique les deux protéines Ku et LigD. La protéine Ku se fixe sur les extrémités de l’ADN et recrute la ligase LigD. Cette dernière est une protéine multifonctionnelle présentant, outre une activité ligase, une activité polymérase et parfois une activité nucléase. L’analyse des génomes de Streptomyces a révélé un nombre variable d’homologues de ku (1-3) et d’homologues codant pour l’une ou l’autre des trois activités de LigD. Ces différents gènes définissent deux loci conservés entre espèces de Streptomyces. Chez S. ambofaciens, trois homologues de ku (nommés kuA, kuB et kuC) et deux ligases ATP-dépendantes (nommés ligC et ligD) ont été identifiés. L’exposition de souches déficientes pour ces différents gènes aux agents endommageant l’ADN (la mitomycine C, l’irradiation par faisceau d’électrons) a démontré l’implication de kuA et ligC, deux acteurs conservés, mais aussi des gènes variables kuC et ligD, dans la réparation de l’ADN. Ces résultats ouvrent de nouvelles perspectives pour comprendre le rôle du NHEJ dans l'évolution du génome et la biologie Streptomyces. / Double strand breaks (DSB) constitute the most deleterious form of DNA damage that a bacterial cell can encounter. Two major pathways can carry out DSB repair in bacteria: homologous recombination and Non-Homologous End Joining (NHEJ). Streptomyces is a model bacterium to explore the relative impact of these recombination mechanisms on genome structure and evolution; the chromosome is indeed typified by its linearity, its compartmentalized genetic organization and its remarkable genomic plasticity. The objective of this research is to identify actors involved in DSB repair mechanisms which remain mostly elusive in Streptomyces up to now. The first step of DSB repair by homologous recombination is the resection of broken DNA ends by a multisubunit helicase-nuclease complex exemplified by Escherichia coli RecBCD, Bacillus subtilis AddAB and Mycobacterium tuberculosis AdnAB. In silico analysis of Streptomyces genomes allowed to identify homologues for adnA and adnB which constitute a highly conserved locus within the genus. Attempts to disrupt these two genes were unsuccessful in Streptomyces ambofaciens as well as in Streptomyces coelicolor, unless an extra copy of adnAB was inserted in the chromosome. This indicates that AdnA and AdnB are both essential for Streptomyces growth. Complementation of an E. coli [delta]recB mutant by S. ambofaciens adnAB locus restored nuclease activity and cell survival in the presence or absence of DNA damaging agent, strongly suggesting that Streptomyces adnAB encodes a functional homologue of E. coli RecBCD. The key role of adnAB in homologous recombination and DNA replication is discussed. The NHEJ mechanism shows a sporadic distribution in bacteria and is known to involve the two proteins Ku and LigD. The Ku protein binds to the ends of the broken DNA and recruits the ATP-dependent ligase LigD which is a multifunctional protein carrying ligase, polymerase and sometimes nuclease activity. In silico analysis of Streptomyces genomes revealed a complex organization with a variable number of ku homologues (1 to 3) and of homologues encoding one of the three distinct LigD activities. These homologues define two conserved loci. S. ambofaciens possesses 3 ku (named kuA, kuB and kuC) and 2 ATP-dependent ligases (named ligC and ligD). Exposure to DNA damaging agents (mitomycin C, electron beam irradiation) of mutant strains got involved kuA and ligC, two conserved actors, but also variable genes such as kuC and ligD in DNA repair. These results open up new prospects to understand the role of NHEJ in the biology and genome evolution of Streptomyces.

Hautement conservé, de la bactérie jusqu'à l’Homme, la recombinaison homologue est indispensable à la survie de tout organisme vivant. Chez l’humain, la protéine hRad51 (human Rad51) y joue un rôle clé en s’autoassemblant au site de cassure sur les extrémités simple-brin d’une molécule d’ADN endommagée pour former le filament nucléoprotéique. Ce filament est capable à lui seul d’effectuer la plupart des opérations nécessaires au bon déroulement de la recombinaison homologue; il va permettre la reconnaissance d’homologie, l’appariement des séquences homologues et l’invasion de brins requise pour la synthèse de l’ADN manquant.La recombinaison homologue est un processus complexe impliquant de multiples partenaires. Pour mieux comprendre le rôle du filament nucléoprotéique au sein de la réaction, on se propose d’étudier ce dernier en l’absence de tout partenaire. Plus précisément, on observe le comportement mécanique de filaments hRad51-ADNdb en fonction des conditions chimiques. La formation du filament nucléoprotéique modifie la conformation de l’ADN sur lequel il s’assemble, l’allongeant de 50% et le déroulant de 43% dans le cas d’une molécule double-brin. Les pinces magnétiques sont un outil permettant de contrôler la force et la torsion appliquées à une unique molécule d’ADN double-brin (ADNdb), elles sont donc l’outil idéal pour sonder les propriétés mécaniques de filaments nucléoprotéiques. Le système des pinces magnétiques a été modifié afin de mesurer des paramètres mécaniques précédemment inaccessibles tel que le couple ressenti ou exercé par le filament. Le but de cette thèse a été d’étudier les propriétés mécano-chimiques des filaments nucléoprotéiques tout en essayant de tracer le paysage énergétique qui régit les transitions de ces systèmes. / Highly conserved throughout the species, homologous recombination is crucial to the survival of any living organism. In humans, the hRad51 protein (human Rad51) plays a key role by self-assembling at the break site on the single stranded extremities of damaged DNA molecules thus forming the nucleoprotein filament. This filament is able by itself to accomplish most of the necessary operations of homologous recombination; it allows the homology search, the pairing of the homologous sequences and the strand exchange.Homologous recombination is a complex process involving many partners. In order to better understand the role of the nucleoprotein filament in this process, we propose to study it in the absence of any partners. We will focus on the study of the mechanical properties of hRad51-dsDNA filaments as a function of chemical conditions. The formation of the nucleoprotein filament modifies the conformation of the DNA molecule on which it assembles, stretching it by 50% and unwinding it by 43% in the case of a double stranded DNA. The magnetic tweezers are a tool allowing the control of the force and torsion applied to a single dsDNA molecule; they are therefore the ideal tool to probe the mechanical properties of nucleoprotein filaments. We modified the magnetic tweezers as to allow the measurement of previously inaccessible mechanical parameters such as the torque applied or felt by the filament. The goal of this thesis has been to study the mechano-chemical properties of nucleoprotein filaments while drawing the energy landscape that governs the various transitions of these systems.

La protéine télomérique TRF2 permet de protéger les télomères notamment en régulant leur taille. Dans des cellules humaines, la surexpression de la protéine mutante TRF2ΔB, dont le domaine basique est absent, induit un raccourcissement soudain des télomères. In vitro, ce domaine basique protège des structures d’ADN particulières, appelées Jonctions de Holliday (JH), de la résolution par des endonucléases. Ces JH peuvent être présentes aux télomères d’une part au niveau de la boucle télomérique, une conformation de l’ADN qui ressemble à une structure intermédiaire de la recombinaison homologue (RH), et d’autre part au niveau des fourches de réplication bloquées, fréquentes aux télomères. Nous pensons que le raccourcissement soudain des télomères implique la résolution de JH au cours d’un événement de recombinaison homologue qui doit être étroitement régulé afin d’éviter qu’il ne se réalise de façon inappropriée. Dans le but de mieux caractériser cet événement, j’ai montré que différentes endonucléases capables de résoudre des JH (GEN1, MUS81, SLX1-SLX4) sont impliquées dans le raccourcissement des télomères induit par la surexpression de la protéine TRF2ΔB. Puis j’ai étudié le rôle de la protéine hRAP1 dans la régulation de ce mécanisme et l’implication des protéines de la RH. L’ensemble des résultats obtenus nous ont permis de proposer un nouveau rôle de la protéine TRF2 dans la régulation des événements de recombinaison homologue au cours de la réplication des télomères. / The stability of mammalian telomeres depends upon TRF2 which prevents inappropriate repair and checkpoint activation. In human cells, overexpressing a TRF2 mutant lacking the N-terminal basic domain, TRF2ΔB, induces sudden telomere shortening. In vitro, the basic domain protects particular DNA structures, called Holliday junctions (HJ), of the resolution by endonucleases. These HJ may be present at telomeres in one hand at the t-loop, a DNA conformation looking like a structural intermediate of homologous recombination (HR), and also at the level of stalled replication forks, frequent at telomeres. We believe that the sudden shortening of telomeres involves the resolution of HJ during a HR event that would be tightly regulated to prevent it occurs inappropriately. In order to better characterize this event, I have shown that different proteins harbouring resolving activities (GEN1, MUS81, SLX1-SLX4) are involved in telomere shortening induced by overexpression of TRF2ΔB. Then, I studied the role of hRAP1 in the regulation of this mechanism and involvement of HR proteins. The overall results allowed us to propose a new role of TRF2 in the regulation of HR events during the replication of telomeres.

Orientadores: Afonso Celso Vigorito, Maria Elvira Pizzigatti Corrêa / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-27T23:05:57Z (GMT). No. of bitstreams: 1 Boer_CamilaCominato_D.pdf: 4791570 bytes, checksum: ef421fd26cc41a5841f0e57323bce220 (MD5) Previous issue date: 2015 / Resumo: Recentemente, a quantificação do fluxo salivar e dos componentes bioquímicos da saliva, foram considerados possíveis biomarcadores para a patogênese da doença do enxerto contra o hospedeiro crônica (DECHc) oral em pacientes submetidos ao Transplante de Células Tronco Hematopoiéticas (TCTH). O objetivo deste estudo foi avaliar prospectivamente a composição inorgânica salivar em diferentes períodos do transplante alogênico e no diagnóstico da DECHc e correlacioná-los com as manifestações clínicas orais. A saliva não estimulada foi coletada para determinação de fluxo salivar e das concentrações de cálcio (Ca), fosfato (Pi), cloro (Cl), magnésio (Mg), potássio (K) e sódio (Na), utilizando reações colorimétricas e absorção atómica. Os Índices de saúde bucal, mucosite e DECHc orais foram avaliados por exame clínico odontológico. A hiposalivação foi avaliada por parâmetros visuais e os pacientes responderam a um questionário sobre xerostomia, dor oral e sensibilidade. Foram incluídos no estudo, consecutivamente, 55 pacientes submetidos ao primeiro TCTH alogênico. Destes 55 pacientes, 49 (89%) foram reavaliados entre os dias D+ 8 e D+10 e 6 (11%) pacientes foram a óbito antes desse período. Após a avaliação entre os dias D+ 8 e 10, 2/ 49 (4%) pacientes recaíram, 13/49 (27%) foram a óbito 34/49 (69%) pacientes permaneceram vivos até o final deste estudo. Dos 34 pacientes 28 (82%) foram avaliados entre os dias D+ 80 e D+ 100, sem o diagnóstico de DECHc, e 12/ 34 (35%) pacientes avaliados no momento do diagnóstico da DECHc, com mediana de dias do diagnóstico de 110 (68-412). A avaliação da saúde bucal entre os dias 8 e 10 apresentou mediana do índice gengival (IG) maior quando comparada com os dados do pré-TCTH (p=0,03). Os índices Dentes Cariados, Perdidos e Obturados (CPOD), Índice de Placa (IP) e o (IG) não apresentaram qualquer alterações nos períodos do estudo. De 49 pacientes, 32 (65%) apresentaram mucosite grau 2 a 4 e a frequência foi maior ao condicionamento de altas doses (p=0,03) e nos pacientes mais jovens (p=0,02). Entre os dias 8 e 10 a queixa de dor oral foi maior nos pacientes mais jovens e no condicionamento de altas doses, (p=0,03) e (p=0,02), respectivamente. O fluxo salivar foi significativamente maior entre os dias 8 e 10 (p=0,05) em comparação ao pré-TCTH. O Pi diminuiu entre os dias 8 e 10 dias (p=0,007) e as concentrações de Na e Cl aumentaram entre os dias 8 e 10 (p=0,001 para ambos). O fluxo salivar no mesmo período demonstrou correlação negativa com a concentração de Pi (p=0,02) e positiva com as concentrações de Na e Cl, (p=0.003) e (p=0.001), respectivamente. Pacientes submetidos ao condicionamento de altas doses apresentarem menores concentrações de Pi (p=0,05). Entre os dias 80 e 100, a xerostomia foi mais frequente nos pacientes submetidos ao condicionamento com altas doses, comparada com de baixa dose (p=0,05). O fluxo salivar foi significativamente menor entre os dias 80-100 (p=0,02) em comparação com o pré-TCTH e as concentrações de Na, Cl e K aumentaram entre 80-100 dias (p=0,03), (0,02) e (p=0,003), respectivamente. O fluxo salivar no mesmo período demonstrou correlação negativa com as concentrações de Na e Cl, (p=0.01) e (p=0.013), respectivamente. Pacientes submetidos ao condicionamento de altas doses apresentaram menor concentração de Na (p=0.05), enquanto que os pacientes que receberam condicionamento de baixas doses apresentaram maiores concentrações de Na e Pi (p=0.05),( p=0,04), respectivamente. No momento do diagnostico da DECHc, o fluxo salivar não mostrou diferença estatística significativa entre os períodos do estudo. Foi observada um tendência de maior concentração de Na no diagnóstico da DECHc (p=0,06) e diminuição significativa da concentração de Pi (p=0.004) em comparação com o pré-TCTH. As concentrações de cálcio e magnésio não apresentaram qualquer alteração durante o mesmo período avaliado. O presente estudo mostrou alterações nos componentes salivares inorgânicos em períodos pós-TCTH, principalmente durante a inflamação da cavidade oral, como mucosite e DECHc. Nós podemos especular que o sódio, cloreto, fosfato e, na saliva, pode ser utilizado como um biomarcador potencial de atividade inflamatória oral em estudos posteriores / Abstract: Recent studies have considered the qualitative and quantitative assessment of salivary flow, as well the biochemical components of saliva, as possible biomarkers that might contribute to the pathogenesis of chronic Graft-Versus-host-Disease (cGHVD) in HSCT patients. The aim of this study was to evaluate the inorganic salivary status at different periods of allogeneic HSCT and cGVHD onset, prospectively. Patients were evaluated in pre-HSCT, between the days 8 and 10, 80 and 100 days without cGVHD, and at the onset of oral cGVHD. Oral health indexes, mucositis, and oral cGVHD were prospectively evaluated by oral clinical examination. Hyosalivation was clinically evaluated by visual parameters, and patients answered a questionnaire concerning xerostomia, oral pain and sensitivity. Unstimulated saliva was collected to determine the salivary flow rate and the concentrations of Calcium (Ca), Phosphate (Pi), Chloride (Cl), Magnesium (Mg), Potassium (K), and Sodium (Na) using colorimetric reactions and atomic absorption. Fifty-five consecutive patients undergoing first allogeneic HSCT were included in this study. Forty-nine out of 55 (89%) patients were evaluated between the days 8 and 10, and 6 (11%) had died before the evaluation. After the evaluation between 8 and 10 days, 2 out of 49 (4%) patients relapsed, 13 out of 49 (27%) patients died and thirty-four out of 49 (69%) patients remained alive until the end of the study. Twenty-eight out of 34 (82%) patients were evaluated between days 80 and 100 without cGVHD, and twelve out of 34 (35%) were evaluated at oral cGVHD onset, at a median time of 110 days (68-412). Oral health evaluation between the days 8 and 10 showed a higher median of Gingival Index (GI) compared with baseline data (p=0.03), and the Decayed, Missing and Filled Teeth Index (DMFT) and Plaque Index (PI) presented no statistical difference. Thirty-two out of 49 (65%) patients developed grade 2-4 oral mucositis associated with a high dose conditioning regimen (p=0.03) and younger patients (p=0.02). Between the days 8 and 10 younger patients and those who received high dose conditioning had more oral pain (p=0.03) and (p=0.02), respectively. The salivary flow rate was significant higher compared with the baseline between days 8 and 10 (p=0.05), Pi concentration was decreased, (p=0.007) and Na and Cl were increased (p=0.001), (p=0.001), respectively. The analysis of the salivary flow rate during the same period showed a negative correlation with Pi concentration (p=0.02) and a positive correlation with Na and Cl concentration, (p=0.003) and (p=0.001), respectively. Patients who received a high dose conditioning regimen presented a lower Pi concentration (p=0.05), and regardless of the conditioning regimen, no other biochemical component showed statistical difference. Xerostomia was more frequent between the days 80 and 100 in high dose, compared with reduced dose conditioning (p=0.05). The salivary flow rate was decreased between days 80 and 100 (p=0.02) and Na, Cl and K concentration was increased, (p=0.03), (p=0.02) and (p=0.003), respectively. The analysis of the salivary flow rate during the same period showed a negative correlation with Na and Cl, (p=0.01) and (p=0.013), respectively. Patients who received high dose conditioning regimen presented a lower Na concentration (p=0.05), whereas those who underwent a reduced dose conditioning regimen presented higher Na and Pi concentrations (p=0.05),( p=0,04), respectively. No other statistical difference was detected in the other biochemical components regardless of the conditioning regimen. At cGVHD onset, the salivary flow rate showed no statistical difference compared with the other periods. A trend was observed in the higher Na concentration compared with the baseline (p=0.06) and Pi concentration presented a significant decrease (p=0.004). Ca and Mg concentrations showed no changes during all evaluation periods. The present study showed changes in inorganic salivary components in post-HSCT periods, mainly during oral cavity inflammation, such as mucositis and cGVHD. We speculate that Na, Cl, and Pi in saliva could be used as a potential biomarker for oral inflammatory activity in further studies / Doutorado / Clinica Medica / Doutora em Clínica Médica

付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラム / 京都大学 / 新制・課程博士 / 博士(医学) / 甲第23091号 / 医博第4718号 / 新制||医||1050(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 篠原 隆司, 教授 増永 慎一郎, 教授 小川 誠司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM