Hepatitis C virus (HCV) is a serious global health problem with an estimate of 170 million carriers worldwide. Most individuals exposed to this blood-borne pathogen develop chronic infection, which may result in severe liver complications as well as end-stage liver diseases including cirrhosis and hepatocellular carcinoma. Current treatment options are suboptimal with no effective vaccines available to date. Development of a readily accessible mouse model that is permissive to natural HCV infection is important to facilitate drug and vaccine discovery, and also to better understand the viral pathogenesis. The inherent difficulty is that HCV displays very limited tropism, infecting only livers from humans or chimpanzees. An attempt was made to elucidate the key determinants in rendering the murine intracellular environment permissive to HCV replication. The results revealed that deletion of the interferon regulatory factor-3 and overexpression of microRNA-122 can independently enhance viral subgenomic replication in murine fibroblasts, with microRNA-122 being the stronger determinant. Interestingly, the phenotype established by these genetic manipulations was insufficient to support full-length HCV genome replication. Murine hepatic cell lines, with or without microRNA-122 expression, were also non-permissive to genomic HCV replication, despite the fact that translation of viral RNA was observed. These results suggest that additional host-specific factor(s) are required to support replication of full-length HCV RNA. These studies provide insight on the essential factors capable of influencing permissiveness of rodent cells to HCV replication, and also suggest genetic modifications to be considered when modeling the complete viral life cycle in a rodent animal model.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18881号 / 医博第3992号 / 新制||医||1009(附属図書館) / 31832 / 京都大学大学院医学研究科医学専攻 / (主査)教授 小柳 義夫, 教授 松岡 雅雄, 教授 朝長 啓造 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

The human immunodeficiency virus type-1 (HIV-1) is the major etiological agent of acquired immunodeficiency syndrome (AIDS), the cause of over 30 million deaths worldwide. Highly active antiretroviral therapy (HAART) has demonstrated great efficacy at suppressing viral load and is therefore the standard therapeutic treatment for HIV-1 infection. Noncompliance due to severe HAART-associated side effects significantly undermines therapeutic efficacy. Dronabinol, the synthetic form of the cannabinoid THC found in marijuana, is FDA-approved for countering some of these side effects. Studies have reported that cannabinoids restrict HIV-1 replication, although no mechanism has yet been proposed. Thus the purpose of this study was to characterize the effects of cannabinoids on HIV-1 infection and to determine the molecular basis of cannabinoid-induced viral suppression. By transcriptomic sequencing of T cells treated with cannabinoids, we have found that the expression of BAG6, a protein uncharacterized within the context of HIV-1 infection, was downregulated. To identify the role of this protein during infection, we knocked down BAG6 and were able to recapitulate the protective effects of cannabinoids by observing reduced severity of viral challenge. Moreover, we have also identified BAG6 to be a binding partner of two HIV-1 viral accessory proteins, Vif and Vpr. Importantly, we have discovered that Vpr mediates targeted degradation of BAG6 by leveraging the host proteasome during the early stages of the viral lifecycle, revealing a hitherto unknown function of this poorly-understood viral protein. We thus establish modulation of BAG6 expression as a novel mediator of the effects of cannaninoids on HIV-1 infection. / Microbiology and Immunology

Two thirds of plant viruses are positive-strand RNA viruses including the family Tombusviridae. One of the best-studied members of this family is Tomato bushy stunt virus (TBSV). Like many other viruses, TBSV has much fewer genes when compared to its hosts’ genome. Nevertheless, TBSV utilizes its genome very judiciously. To compensate for a lack of many proteins of its own, it codes for multi-functional replication protein p33 and also co-opts host factors to facilitate its replication. By using recombinant replication proteins p33 and p92 containing single amino acid changes in protein-protein interaction domains (S1 and S2), I demonstrated that the replication proteins are required in sequential steps during virus replication. The in vitro cell-free extract(CFE) based TBSV replication assays revealed that mutations in S1 and S2 domains affected RNA template selection, recruitment and assembly of replicase complex. TBSV replicates on the cytosolic surface of peroxisomal membranes. To identify the host factor involved in this process of transporting viral replication proteins to peroxisome, I tested the peroxisomal transporter proteins for their ability to bind to p33 in vitro, which led to the discovery of Pex19p. Pull-down and co-purification experiments revealed transient nature of p33-Pex19p binding as expected from a transporter. When pex19p was retargeted to mitochondria, a large fraction of p33 was also re-distributed to the mitochondria validating the importance of Pex19p in p33 localization. TBSV also utilizes its genomic RNA for non-template activities during its replication. Accordingly, TBSV RNA serves as a platform for the assembly of replicase complex. To further characterize the regulatory cis-elements involved in this process, I utilized CFE and different TBSV RNA mutants together with recombinant p33 and p92 in vitro replication assays. These experiments revealed the role of RNA recruitment element [RIISL(+)] and 3’ non-coding regions as minimal cis-elements required to assemble functional replicase complex. The experiments also indicated that the RIISL(+) and 3’ non coding regions could be physically separated on two different RNA molecules to assemble TBSV replicase, suggesting insights into viral evolution.

Positive strand RNA viruses are intracellular parasites, and their genome replication and infection involves complex virus-host interactions. Therefore, identification of host factors and dissection of their functions during virus replication could facilitate our understanding of the mechanism of virus infection. Those host factors may also provide new targets for viral disease control. Tomato bushy stunt virus (TBSV) has recently become one of the model viruses to study positive strand RNA virus replication and hostvirus interactions. To identify host factors involved in TBSV replication we used yeast as a model host. Co-expression of the replication proteins and a replicon RNA (DI RNA) via plasmids in yeast resulted in robust replication of the viral RNA. Previous work using a yeast single gene deletion library (YKO) revealed 96 yeast genes affecting virus replication. The essential yeast genes could not be deleted so we used the Yeast Tet Promoters Hughes Collection (yTHc) where the original promoter was replaced by Tetracyclin-titratable promoter. I tested the 800 essential host genes available in yTHc. In total, we found 30 new host genes whose down-regulated expression either increased or decreased the accumulation of a TBSV repRNA. The identified essential yeast genes fall into different categories on the basis of the cellular processes they are involved in, such as RNA transcription/metabolism, protein metabolism/transport etc. Detailed analysis of the effects of some of the identified yeast genes revealed that they might affect RNA replication by altering (i) the amounts of p33 and p92(pol) viral replication proteins, (ii) the activity of the tombusvirus replicase complex, and (iii) the ratio of plus- versus minus-stranded RNA replication products. Altogether, this and previous YKO screening of yeast led to the identification of 126 host genes (out of ~5,600 genes that represent ~95% of all the known and predicted yeast genes) that affected the accumulation of tombusvirus RNA. In the YKO screening, we found NSR1 (homologous to plant nucleolin) gene, whose deletion led to increased TBSV repRNA accumulation. Nucleolin is an abundant RNA binding protein, which shuttles between the nucleolus, the nucleoplasm and the cytoplasm. This protein is involved in rRNA maturation, ribosome assembly and regulation of cellular RNA metabolism.We found that over-expression of Nsr1p in yeast or nucleolin in Nicotiana benthamiana inhibited the accumulation of tombusvirus RNA by ~10-fold. Temporal regulation of Nsr1p over-expression revealed that the inhibitory effect of Nsr1p was more profound when it was expressed at early stages of viral replication. In vitro binding experiments showed that Nsr1p binds preferably to the RIII in the repRNA (which is derived from 3’ UTR of viral genome). Consistent with its RIII specific binding, over-expression of Nsr1p only reduced 40% of the accumulation of TBSVΔRIII repRNA in yeast. The purified recombinant Nsr1p inhibited the in vitro replication of the viral RNA in a yeast cell-free assay when pre-incubated with the viral RNA before the in vitro replication assay. Our data suggest that Nsr1p/nucleolin inhibits tombusvirus replication by interfering with the recruitment of the viral RNA for replication.

Orientador: Marcelo Brocchi / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-11T15:02:44Z (GMT). No. of bitstreams: 1 Mendes_GuilhermeMartinesTeixeira_M.pdf: 1976901 bytes, checksum: 9e7c5547a60ab82c824934c40b10ce27 (MD5) Previous issue date: 2008 / Resumo: O gênero Salmonella spp é formado por bacilos gram-negativos, que podem ser divididos em 3 espécies: S. enterica, S. bongori e S. subterranea. A maioria das sorovariedades patogênicas para o homem está incluída no subgrupo I da espécie S. enterica. A infecção por S. enterica inicia-se com a ingestão de água ou alimentos contaminados. Estes microrganismos são patógenos intracelulares facultativos e, uma vez ingeridos, apresentam a capacidade de aderir e invadir células da mucosa intestinal, preferencialmente células M. Uma vez ultrapassada a mucosa intestinal, S. enterica invade, persiste e prolifera no interior de vacúolos de células do sistema retículo endotelial podendo assim, alcançar diferentes órgãos e tecidos do hospedeiro, causando infecção sistêmica. Sendo assim, linhagens mutantes avirulentas, mas ainda capazes de causar infecção transitória, são boas candidatas a potenciais vacinas vivas orais. Tais mutantes são potenciais carreadores de proteínas heterólogas, compondo, assim, as chamadas vacinas multi-valentes. Que seja de nosso conhecimento, não existem mutantes atenuados de S. enterica desenvolvidos inteiramente no Brasil, havendo necessidade de pagamento de patentes a grupos estrangeiros para sua utilização. Em eucariotos, o DNA cromossômico bacteriano está associado a proteínas (histonas) formando o núcleo, enquanto em procariotos, estas proteínas são denominadas histona-like, formando um nucleóide. Dentre essas proteínas podemos citar a IHF (integration host factor), um heterodímero que controla ou influencia vários processos celulares, como a duplicação e recombinação do DNA, além de regular positiva ou negativamente a expressão de vários genes. Neste estudo, mutantes nulos para os genes himA e himD de IHF foram criados pela técnica de recombinação homóloga mediada pelo sistema ? Red (Datsenko e Wanner, 2000) e testados quanto a atenuação da virulência e capacidade de desencadear resposta imune efetiva e protetora contra a salmonelose murina. Os mutantes também foram caracterizados quanto a diversas características biológicas, como a capacidade de invasão e sobrevivência intracelular, resistência a radicais reativos de oxigênio e nitrogênio, ente outras, sendo os resultados comparados com as respectivas linhagens selvagens. Os mutantes himA e himD de S. enterica foram atenuados e capazes de induzir resposta imune protetora quando desafiados com doses elevadas da linhagem selvagem, indicando que estas linhagens recombinantes são potenciais candidatas a vacinais vivas orais / Abstract: The genus Salmonella sp is formed by gram-negative bacilli, which can be divided into 3 species: S. enterica, S. bongori and S. subterranea. The majority of the serovars pathogenic to humans is included in the subgroup I of the S. enterica species. The infection with S. enterica starts either the ingestion of contaminated water or food. These microorganisms are facultative intracellular pathogens and, once ingested, they have the capacity to adhere and invade cells of the intestinal mucosa, with preference for M cells. Then, S. enterica can invade and proliferate within vacuoles of immune cells, particularly macrophages, achieving different organs and tissues of the host, causing systemic infection. Mutant strains of S. enterica with attenuation of the virulence but that are still able to cause a transient infection, are good candidates for potential live oral vaccines. These mutants are also good carriers of heterologous antigens to cells of the immunological system, been able to induce an effective immunological response. To the best of our knowledge, no mutants of this type were developed in Brazil leading to the needed to pay royalties to foreign groups for their use. In prokaryotes the genomic DNA are associated with a number of proteins, the so called histone-like proteins, with structural and regulatory properties, forming the nucleoid. The IHF (Integration Host Factor) is one of the more abundant histone-like in prokaryotes. IHF is a heterodimeric DNA-binding protein that controls a number of cellular processes, such as DNA duplication and DNA recombination and also modulates the expression of different genes. In this work we constructed recombinant strains of S. enterica mutants for the himA and himD genes that encode for the IHF subunits using the ? Red system (Datsenko and Wanner, 2000) and tested for attenuation and immunogenicity. The mutant strains were also characterized and compared to the parental strains for other biological characteristics such as the capacity to invade and proliferate into eukaryotic cells and to survive to different stress conditions. The S. enterica himA and himD mutant strains were attenuated for virulence and able to induce a protective immunity against the wild type strain of S. enterica indicating that these recombinant strains are candidates to formulate a new live oral vaccine / Mestrado / Ciencias Basicas / Mestre em Clinica Medica

La grippe est une maladie infectieuse qui se traduit par des épidémies saisonnières et pandémies occasionnelles et qui a des conséquences importantes sur la santé publique. Elle est causée par le virus influenza, un virus à ARN négatif segmenté. Chaque segment du génome est transcrit et repliqué dans le noyau de la cellule hôte par la polymérase virale, une ARN polyméraseARN-dépendente. Afin de pouvoir produire un ARN messager (ARNm) viral qui peut être reconnu par la machinerie de traduction de la cellule, la polymérase de la grippe se sert d’un mécanisme appelé ‘cap-snatching’ (mécanisme de ‘vol de coiffe’). La polymérase virale intéragit avec un ARNm coiffé, produit par l’ARN polymérase II de la cellulle hôte, coupe cet ARN à proximité de la coiffe et l’utilise comme amorce pour l’initiation de la transcription de son propre matériel génétique. Par conséquence, la transcription virale est fonctionnellement liée à la transcription cellulaire. L’objet des recherches présentées dans cette thèse portait sur l’intéraction entre la polymérase de la grippe et la machinerie de transcription de la cellule hôte et plus précisément, la caractérisation structurale et fonctionelle de l’association entre la polymérase de la grippe et le domain C-terminal de l’ARN polymérase II (CTD), qui sert de support pour la coordination des processus de synthèse et les modifications post-transcriptionelles de l’ARNm. Les structures cristallographiques obtenues de la polymérase d’influenza A et B, associées aux peptides dérivés du domain CTD de l’ARN polymérase II, ont permis d’élucider comment la polymérase virale reconnait spécifiquement la polymérase cellulaire. La perturbation de cette intéraction mène à l’inhibition de l’amplification du virus, ce qui confirme que cette association est essentielle. Dans la seconde partie de la thèse, un complexe actif de ‘cap-snatching’ a été assemblé in vitro et caractérisé fonctionellement, constitué du complexe d’élongation contenant l’ARN polymérase II avec un ARNm coiffé et son domain CTD phosphorylé, lié à la polymérase virale elle-même transcriptionnellement active. De plus, l’intéraction de la polymérase virale avec deux facteurs impliqués dans la régulation de l’élongation par l’ARN polymérase II, DRB sensitivity-inducing factor et Tat stimulatory factor 1, a été caractérisée. En conclusion, le travail de thèse présenté ici a contribué à élucider le mécanisme de recrutement de la polymérase d’influenza à la machinerie de transcription cellulaire et a montré que l’association entre les polymérases virale et cellulaire est essentielle pour la réplication du virus. La perturbation de cette intéraction est une piste prometteuse pour la conception de nouveaux médicaments contre le virus influenza. / Influenza is an infectious disease causing seasonal epidemics and occasional pandemics, which are a significant health burden for the global human population. The causative agent, influenza virus, is a negative-strand segmented RNA virus. Each segment of the viral genome is transcribed and replicated by a virally-encoded RNA-dependent RNA polymerase in the nucleus of the infected cell. In order to produce a functional viral messenger RNA (mRNA) that can be processed by the cellular translation machinery, influenza polymerase employs a mechanism called‘cap-snatching’. The viral polymerase binds to a nascent, capped transcript, produced by the cellular RNA polymerase II, cleaves it shortly after the cap and uses it as a primer for the transcription of its own genomic segments. Viral transcription is therefore functionally dependent on cellular transcription. The studies described in this thesis aimed to investigate the interaction between influenza polymerase and the cellular transcription machinery. The main focus was to characterize structurally and functionally the association of influenza polymerase with the C-terminal domain (CTD) of RNA polymerase II, which serves as a scaffold for coordinating co-transcriptional events during mRNA synthesis and processing. Crystal structures of influenza A and B polymerase bound to phosphorylated peptides mimicking the CTD of RNA polymerase II gave new insight on how the viral polymerase directly recognizes the transcribing cellular polymerase. Moreover, disrupting this interaction was found to be severely detrimental to viral replication, confirming the essentiality of this association. In the second part of this work, an active cap-snatching complex was assembled in vitro and characterized functionally. This comprised a reconstituted RNA polymerase II elongation complex with emerging capped transcript and phosphorylated CTD with bound and transcriptionally active influenza polymerase. Additionally, the interaction of the viral polymerase with two factors involved in the regulation of transcription elongation by RNA polymerase II, DRB sensitivity-inducing factor and Tat stimulatory factor 1, was analyzed biochemically. Overall, the work presented here gives insights into the mechanism of recruitment of the influenza polymerase to the cellular transcription machinery and shows that the association of the viral and cellular polymerase is essential for the viral replication. Targeted disruption of this interaction is therefore a promising avenue for the design of novel anti-influenza drugs.

Brome mosaic virus (BMV) belongs to a viral class called positive-strand RNA [(+)RNA] viruses. This is the largest class of viruses and includes numerous important pathogens. BMV infects monocotyledonous plants and its replication can be recapitulated in the baker's yeast (Saccharomyces cerevisiae) under laboratory conditions to use yeast as an experimental model system. BMV generally does not infect dicotyledonous plants including the model plant Arabidopsis thaliana. One important shared feature of (+)RNA viruses is that they all make use of host proteins to aid in their own viral replication. In particular, (+)RNA viruses use host intracellular membranes for their replication and lipid composition of these membranes is crucial for viral replication. I show here that BMV replication protein 1a causes redistribution of host Lam5 (Lipid transfer protein Anchored at a Membrane contact site 5) and that Lam5 is necessary for BMV replication in yeast. Furthermore, in the absence of Lam5, BMV 1a affects the distribution of lipid droplets throughout yeast cells. Host factors also play critical roles in defense against viruses. Although wild-type Arabidopsis is not a host for BMV, the Arabidopsis cpr5 (Constitutive expression of Pathogenesis-Related genes 5) mutant can support systemic infection of BMV. I performed screens in Arabidopsis and have identified four genes that contribute to defense against BMV. These include two RNA-binding proteins, a lectin superfamily protein, and an alternative oxidase. My results also contribute to the growing evidence that reactive oxygen species play a key role in BMV replication. In summary, my work provided new insights into BMV replication in hosts and plant defense against BMV infection. The information gained from these projects aids in our understanding of (+)RNA virus biology in general and may identify targets for developing broad-spectrum antiviral strategies. / Doctor of Philosophy / Viruses are important pathogens that can cause devastating diseases not only in humans, but also in animals and plants. It is important to study viruses and their interactions with their hosts to develop antiviral drugs and engineer plant resistance to viruses. Positive-strand RNA viruses are the largest class of viruses and are responsible for numerous human, animal, and plant diseases. Brome mosaic virus (BMV) belongs to this class of viruses and has a simple genome organization. Furthermore, the baker's yeast can support BMV replication. BMV serves as a model to study (+)RNA virus replication and virus-host interactions. I show here that BMV depends on the lipid transport protein Lam5 for proper replication in yeast. Furthermore, although much is known about BMV replication in yeast, more information is needed on BMV replication in plants. To aid in this goal, I performed screens in the model plant Arabidopsis thaliana to identify Arabidopsis mutants that could allow for systemic BMV infection. This project has uncovered four new genes that contribute to defense against BMV. This research aids in our understanding of how BMV replication works and also how plants defend themselves against viruses. This work is important for understanding of the biology of (+)RNA viruses and the plant immune system in general.

Orientador: Cássio Luís Zanettini Riccetto, Benedicto de Campos Vidal / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-18T18:21:20Z (GMT). No. of bitstreams: 1 Maciel_LuizCarlos_D.pdf: 8260525 bytes, checksum: 6e55aa9d959f06cd42ce61de61d4ac5e (MD5) Previous issue date: 2011 / Resumo: Introdução: O aumento da expectativa de vida elevou o risco da necessidade de cirurgia para o tratamento dos prolapsos de órgãos pélvicos (PO) em mulheres, principalmente no período pós-menopausa. Os distúrbios ocasionados pelos POP afetam a qualidade de vida, ao estabelecer alterações na sexualidade, função urinaria e intestinal. Neste cenário, o uso das telas ganhou popularidade. Embora as telas de polipropileno sejam consideradas um bom material para restaurar o assoalho pélvico, diminuindo o tempo cirúrgico e a dor pós-operatória, existem preocupações quanto à infecção e exposição da tela. Objetivos: Estudar as características da deposição colágena associada aos implantes subcutâneos de telas de polipropileno revestidas por biomembrana de colágeno de submucosa intestinal suína (BMSS) em ratas, e descrever as características da reação inflamatória local. Material e Métodos: 30 ratas fêmeas foram divididas em três grupos conforme a data de eutanásia (7º; 14º, e 28º pós-operatório). Foram implantadas no subcutâneo abdominal destes animais um segmento de tela de polipropileno revestida com colágeno à direita e outro segmento de tela não revestida no lado esquerdo. O material foi fixado em HE e analizado no microscópio de polarização Olympus BX51 e acoplado ao software image Pro plus 6.0 para estudar as propriedades anisotrópicas do colágeno e as características da reação inflamatória local. Resultados: 7ºPO: A biomembrana induziu intensa proliferação vascular, vasodilatação, infiltração de linfocitos e proliferação fibroblástica. No subgrupo da tela de polipropileno isolada notaram-se predomínio de células com aspecto histiocitário e células gigantes. 14ºPO: No subgrupo da biomembrana persistiu o infiltrado predominantemente linfocitário semelhante ao 7ºPO. Não ocorreram alterações expressivas na intensidade da deposição colágena, persistiu o aspecto de congestão vascular. No subgrupo do polipropileno isolado, observou-se alguma proliferação fibroblástica em torno dos filamentos de polipropileno, além de células gigantes achatadas pelo envoltório de colágeno e fibroblastos, mantendo pouco infiltrado linfocitário, em relação à tela recoberta pela biomembrana. 28º PO: No subgrupo da biomembrana de colágeno observou-se intensa deposição colágena com diminuição do infiltrado linfocitário e presença de mononucleares histiocitários. No subgrupo da tela de polipropileno isolada evidenciaram-se células gigantes as quais apresentavam tendência de envolver o polímero, além de menor infiltrado fibroblástico em comparação ao grupo recoberto com a biomembrana. Conclusão: A tela de polipropileno revestida pela BMSS evidenciou resolução precoce da inflamação, melhor neoangiogênese e deposição colágena melhor organizada do que a tela não revestida. Isto pode representar um vantagem em segmentos clínicos se estes resultados experimentais puderem ser reproduzidos em outros estudos / Abstract: Introduction: The raise of expectance of life has brought an increase of women which will underwent pelvic organ prolapse (POP) surgery, mainly in the post menopause period. The distress caused by POP affects women's quality of life, as it alters their sexuality, urinary and bowel functions. In this scenario, the use of meshes is gaining popularity worldwide. Although polypropylene mesh (PM) is considered a good material to restore the pelvic floor, decreasing operative time and post-operative pain, there are concerns about infection and vaginal mesh exposure. Objetives: To study the characteristics of collagen deposition related to covering subcutaneous implants of monofilament polypropylene mesh in rats with purified suine collagen biomembrane and to describe the characteristics of the local inflammatory reaction. Methods: Thirty female rats were shared in three groups according to the euthanasia's day (7º; 14º, 28º post-operative day). Polypropylene meshes were implanted at abdominal subcutaneous, on the right side the mesh covered by a collagen biomembrane and on the left side a non covered PM. The material were stained with HE and analyzed with Olympus BX51 polarized microscope and image Pro plus 6.0 software to study the collagen anisotropic properties and inflammatory reaction. Results: In the 7º PO the CM group showed vascular proliferation and intense infiltration of lymphocytes and fibroblasts. The PM without CM group presented a intense histiocytary infiltrate and a mild fibrosblastic reaction and angiogenesis. At the 14º PO, the group A maintained a similar lymphocytic infiltrate which was observed at the 7º PO, without expressive alterations in the degree of fibroblastic reaction and collagem deposition. In the group B, there was a intense fibroblastic reaction surrounding the PM. Also, there were multinucleated giant cells crushed by a wrap of collagen and fibroblasts. In the 28º PO, the group A revealed an intense fibroblastic reaction process and collagen deposition. In contrast, in the group B, it is observed that the multinucleated giant cells tried to wrap up the polypropylene, and there were a decrease of fibroblastic reaction in comparison to the PM with CM group. Conclusions: PM covered with a purified porcine collagen membrane showed earlier resolution of inflammatory reaction, better neoangiogenesis and more organized host collagen deposition than in pure PM. This can represent an advantage in clinical setting if these experimental results proved to be reproducible in other trials / Doutorado / Fisiopatologia Cirúrgica / Doutor em Ciências da Cirurgia