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M.Med. Faculty of Health Sciences, University of the Witwatersrand, 2008 / Objectives: Characterization of HIV-1 from slow progressors (SP) is important to facilitate vaccine and antiviral drug development. In order to identify virus attenuations that may contribute to slower rates of disease progression, the full viral genomes from primary isolates of six slow progressing HIV-positive children were sequenced. Methods: Primary virus biological phenotypes were determined by growth in CCR5- and CXCR4-expressing U87.CD4 cell lines. Proviral DNA was isolated from co-cultured PBMCs, and the near full-length genomes and LTR regions were PCR amplified, sequenced and analysed. Predicted amino acid (aa) sequences for all the HIV-1 proteins were extensively analyzed. Results: All primary HIV-1 isolates utilized CCR5, and were determined to be HIV-1 subtype C by phylogenetic analysis. Predicted aa sequence analysis revealed open reading frames for all HIV-1 genes which encoded for proteins of the expected length, with several exceptions. For example, isolate LT5 had a 2 aa insertion in the Vpr mitochondriotoxic domain. Isolate LT21 contained an additional 5aa in the C-terminus of tat exon 2, while the integrase enzyme in isolate LT39 had an additional 3aa at the Cterminus. Rev from isolates LT45 and LT46 did not have the characteristic subtype C 16aa truncation, and in addition, had a further 3aa. In addition, altered functional domains was noted in several isolates, such as the cAMP-dependent kinase PKA phosphorylation site in Nef (LT5), a Vpr mutation involved in decreasing pro-apoptotic activity (LT42), and the Nef ExxxLL motif involved in the interaction with AP-1 and AP-2 (LT46). Conclusions: The slower HIV disease progression in these six children may be attributed to altered protein functions. For example, LT46 Nef is unable to bind AP-1 and AP-2 and therefore inactive on CD4 endocytosis. The biological relevance of these findings requires further investigation.

Thesis (M.Sc.(Med.)(Epidemiology and Biostatistics)), Faculty of Health Sciences, University of the Witwatersrand,2009 / BACKGROUND Studies suggest an association between Bacterial Vaginosis (BV) and HIV infection; however, its temporal effect has not been greatly investigated. METHODS This is a secondary data analysis of a cohort study: set out to describe the association between BV infection and HIV acquisition. There were 750 participants enrolled in the primary cohort study. The main exposure, BV, was measured from a gram stain slide prepared from a vaginal swab. The slide was read in a laboratory qualitatively and scored by Nugents scoring. A score of 7 or above was considered positive for BV. The outcome variable (HIV) was determined by dual rapid tests and confirmed in the laboratory by a third generation ELISA. Descriptive statistics was done to describe demographic characteristics and the prevalence of BV and STIs. HIV incidence rate was calculated. Kaplan Meier survival time analysis and log rank test for significance were performed. Cox regression (univariate and multivariate) was done to determine association of BV with HIV infection. RESULTS The baseline prevalence of BV was 52 %, 95 % CI; 45 – 59. There were 21 HIV seroconversions experienced of which 7 had BV results missing and were excluded in the analysis. The remaining 14 seroconversions were followed for a mean time of 0.40 of a year and accumulated follow up time at risk of 286 person years, this represented an HIV incidence rate of 4.9 per 100 person years of follow up, 95 % CI: 2.9 – 8.27. Kaplan Meier curves revealed a higher risk of HIV-1 acquisition among women who were BV positive than the women who were BV negative. A log rank test showed that the v probability of seroconversion was different among the women depending on BV status, chi-square value 3.8, p 0.05. Controlling for confounding variables, seroconversion was high, but not significant, among BV positive women, adjusted hazard ratio 3.21; 95 % CI; 0.85-12.12, p value 0.08. CONCLUSION This study suggests that BV increases HIV seroconversion risk though statistical significance was not achieved. Vaginal cleansing education, screening and treating women with BV could maintain normal vaginal flora and reduce their susceptibility to HIV.

PhD, Faculty of Health Sciences, University of the Witwatersrand / The vast potential of the RNA interference (RNAi) pathway as a new tool for the development of therapeutic modalities has been quickly realised since its discovery in 1998. RNAi effector mimics have been developed to successfully silence an array of disease-causing genetic elements. However, because of the rapidly mutating genome of viruses such as the human immunodeficiency virus (HIV), inhibition of replication cannot be sustained with single RNAi effector mimics. Instead, a combinatorial approach is required, analogous to the cocktail of drugs necessary for successful highly active antiretroviral therapy (HAART). Pioneering studies utilizing long hairpin RNAs (lhRNAs) showed that the long double-stranded RNA stem region acts as a Dicer substrate and is processed into multiple siRNA species. This intrinsic combinatorial property of lhRNAs was exploited in this thesis by attempting to incorporate three non-contiguous potent siRNA sequences within a single lhRNA stem expressed from an RNA Pol III promoter. Although significant knockdown of three independent HIV target sequences was possible, the limitations of this approach became apparent when it was observed that human Dicer does not function efficiently as a multiple turnover enzyme. The generation of siRNA products therefore occurred in a gradient, with higher levels of siRNA produced from the base of the hairpin stem and decreasing quantities generated towards the loop. Modifications to the configuration of integrated siRNA sequences within the stem region enabled augmented RNAi activity of siRNAs in the second position of the hairpin stem. This led to the notion that further manipulation of the structural design of the stem duplex may improve efficacy of up to two siRNAs. Dual-targeting anti-HIV lhRNAs encoding only two highly effective siRNAs targeted against non-contiguous sites within the tat, nef, LTR and int viral genes were therefore propagated. The spatial arrangement of two siRNA sequences was extensively characterised within dual-targeting lhRNAs by inserting up to three random base pairs at the junctions of siRNA encoding sequences and 5 bp preceding the terminal loop sequence. A universally optimal hairpin design was identified which contained a single mismatched base pair between two 19 bp + 2 nt siRNA sequences, as well as a terminal extension. Two powerful dual-targeting lhRNA species, lhRNA-tat-nef +1 and lhRNA-LTR-int +1, each capable of producing two potent anti-HIV siRNA products in equal quantities were selected for incorporation into a combinatorial RNAi system. These two effective dual-targeting lhRNAs were combined, adjacent to one another within a single RNA Pol III-expressed transcript to create a novel lhRNA-based combinatorial RNAi structure. This double lhRNA (dlhRNA) construct served as a precursor for four discrete highly functional RNAi effector sequences which were capable of simultaneously silencing four unique HIV target sites within the tat, nef, LTR and int genes. Furthermore, the ectopic expression of dlhRNAs did not elicit activation of the interferon response, nor did it cause saturation of the endogenous miRNA biogenesis pathway in vitro. In conclusion, the inherent combinatorial RNAi properties of long hairpin RNAs were evaluated and the detailed analysis is presented in this thesis. Structurally optimised dualtargeting lhRNAs subsequently formed the core components of a novel dlhRNA precursor which meets all the requirements for an effective combinatorial RNAi strategy and therefore holds great promise for mediating an effective and sustained gene therapy against HIV.

A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand , in fulfillment of the requirements for the degree of Master of Science in Medicine, 2013 / Sequence analysis from HIV-1 (human immunodeficiency virus type 1) subtype B and more recently subtype C infected patients has revealed that mutations in the HIV-1 protease region that confer drug resistance to boosted protease inhibitor (PIs) are rarely detected at the time of virological failure. Mutations in the HIV-1 subtype B gag-pol cleavage sites are thought to be compensatory mutations which arise as a result of PI use. This study investigated the presence of compensatory mutations in the HIV-1 subtype C gag-pol cleavage sites and matched pol genotypes from South African patients failing a boosted PI-based regimen, as compared to antiretroviral drug naïve patients. A new amplification protocol encompassing the near full-length gag, PR and partial RT was established and used to sequence the HIV-1 gag-pol cleavage sites from 23 proviral DNA samples (p24 antigen cultured peripheral blood mononuclear cells; PBMCs), and 51 patient samples (23 antiretroviral drug-naïve, 26 failing second-line lopinavir/ritonavir containing regimens), all attending the Charlotte Maxeke Johannesburg Hospital. Nucleotide sequences were aligned and codon positions S373Q, A431V, I437T/V, L449P or P453L associated with known gag-pol cleavage site mutations were analysed and compared. The pol genotypes were established using an in house assay. Antiretroviral drug resistant primary virus isolates were grown from samples from patients enrolled on the CIPRA-SA study, and propagated in coculture with PHA-activated, IL-2 stimulated PBMCs. HIV-1 gag-pol cleavage sites and pol genotypes for all primary virus isolates were established as described above. Fifty one of 74 patient samples, used to establish the in-house gag-pol cleavage site assay, were successfully amplified and sequenced. Detailed analysis of the five known gag-pol cleavage sites revealed that 5 patient samples (4 PI-exposed, 1 unknown regimen) encoded for the previously described mutations that impact on gag-pol cleavage in the absence of any major PR mutations. A further five samples from patients on the failing PI-based regimen had major PR mutations. No known mutations in the gag-pol region were identified in patients failing a first line regimen. The pol mutations described in this study were similar to the findings reported for treatment failures in South African HIV-1 subtype C infected patients. Primary virus was grown from only 25 of the 91 PBMC CIPRA samples. None of the 25 CIPRA-SA primary virus isolates had gag-pol cleavage site mutations, and only 9 harboured known RT antiretroviral drug resistant mutations. Overall, the presence of HIV-1 gag-pol cleavage site mutations may account for virological treatment failure in 5 of the South African patient samples analysed. Although the gag-pol cleavage site mutations detected in the current study are only present in a small proportion of treatment-experienced South African patients, this may increase due to more patients accessing second line PI-containing regimens. Thus, future genotyping work incorporating the analysis of the gag-pol cleavage sites in addition to the PR and RT regions is warranted. The antiretroviral drug resistant primary viruses obtained provide valuable reagents for future phenotyping studies.

Mouse cytomegalovirus (MCMV), a β-herpesvirus, encodes the m145 family of glycoproteins. Several members of this family are predicted to adopt the MHC-I fold although their amino acid sequences exhibit less than 30% identity to classical MHC-I (MHC-Ia) proteins. Our aim was to determine how related the viral proteins are to MHCIa and characterize them in terms of cellular expression, structure and function. We studied the cellular localization of FLAG-tagged m17, M37, m145, m151, m152, m153 and m155 in transfected mouse fibroblasts. Flow cytometry analysis of transfected cells showed that M37, m145, m151 and m153 localize predominantly to the cell surface, whereas the majority of m17, m152 and m155 remain inside the cell. MHC-Ia proteins require assembly with β2-microglobulin (β2m) and peptide for stable cell surface expression. Transient transfection studies with β2m- or transporter associated with antigen (TAP)-deficient cell lines revealed that M37, m145, m151 and m153 could be expressed stably at the cell surface in the absence of β2m or TAP expression. To generate protein material for crystallization screening we evaluated both bacterial and insect cell expression systems. Although most m145 family members could be expressed in bacteria in insoluble inclusion bodies, none of the proteins could be accurately refolded. Since M37, m151 and m153 are cell surface molecules with the potential to bind receptors on host cells, we focused our structure determination efforts on them and evaluated their expression in Drosophila S2 insect cells. The extracellular domains of all three proteins expressed at significant levels, however, m151 tended to aggregate and precipitate over time. M37 and m153 were stable and could easily be purified to homogeneity. Size exclusion chromatography and SDS-PAGE analysis of m153 suggested that it forms a non-covalent homodimer. Analytical ultracentrifugation experiments confirmed this observation and provided an estimated molecular mass of 78.8 kDa. Enzymatic and mass spectrometry analyses showed that insect-expressed m153 is highly glycosylated. We tested a wide range of crystallization conditions for m153. It formed very fragile crystals and after optimization we obtained several that diffracted to 2.3 Å. To determine the structure of m153, we prepared a seleno-methionine derivative in insect cells, collected data on a single crystal and solved the phases by the single anomalous dispersion method. The m153 model was refined at 2.4 Å resolution to final Rcryst and Rfree of 23% and 27.9%, respectively. The m153 homodimer is formed by two MHC-I-like heavy chains, each consisting of two α-helices arranged on a platform of seven β-strands and an Ig-like α3 domain. The monomers are arranged “head-to-tail”, with the α1α2 platform domain of one chain interacting with the Ig-like α3 domain of the other. The α1 and α2 helices are closely juxtaposed and do not form a peptide binding groove. Three N-linked carbohydrate residues were visualized in the crystal structure. Major deviations from the MHC-I fold include an extended N-terminus, which originates next to the α3 domain, and an elongated α2 helix (designated H2b) that reaches down towards the α3 domain. In addition, m153 has two unique disulfide bonds, one between strands of the platform domain and another that links the extended N-terminus to the H2b helix. Both unique disulfide bonds were verified by mass spectrometry. The canonical Ig-fold disulfide bond is present in the α3 domain. Alanine mutation of four amino acids involved in interface hydrogen bonds abolished m153 dimer formation, validating the dimer interface visualized in the structure. The crystal structure of m153, together with the recently reported m157 structure, confirms the MHC-I fold for the MCMV m145 family and highlights shared structural features in the m145 family. We have demonstrated dimerization of full-length m153 in mammalian cells by bimolecular fluorescence complementation and co-immunoprecipitation studies. Further, we have shown that m153 is expressed at the surface of MCMV-infected cells at early times after infection. To initiate a search for host ligands of m153, we generated a reporter cell line by introducing an m153-human zeta chain fusion protein into 43.1 cells that contain an NFAT-driven GFP construct. While a variety of mouse cell lines were unable to stimulate the m153-reporter cells, coculture with mouse splenocytes specifically induced GFP production in m153-reporters but not in the parental or control reporter cell lines. We identified conventional CD11c+ and plasmacytoid dendritic cells (DCs) as the most potent m153-reporter cell stimulating populations in the spleen. The stimulation was shown to be m153-specific, dose- and cell contact-dependent. DCs derived from bonemarrow cultures also potently stimulated the m153-reporter cells. Macrophages and NK cells exhibited weaker stimulation of the reporter cells, indicating lower levels of ligand or that only small subsets of the cells express a ligand. DCs from several mouse strains, but not from the rat, stimulated m153-reporter cells. We evaluated DC surface phenotype and migratory capacity after coculture with m153-reporter cells or on m153-coated plates, but could not detect any changes induced specifically by the presence of m153.

Durante a endotoxemia, observa-se alteração no eixo hormônio do crescimento(GH)/fator de crescimento semelhante à insulina (IGF)-1. Acredita-se que o aumento de citocinas pró-inflamatórias seja responsável por essa alteração, apesar do mecanismo para essa alteração ainda não estar completamente elucidado. A ghrelina é um hormônio peptídico que apresenta propriedades anti-inflamatórias, e, portanto, pode contribuir para a manutenção da integridade do eixo GH/IGF-1. O objetivo do presente estudo foi avaliar o efeito do tratamento sistêmico de ghrelina sobre o eixo GH/IGF-1 em ratos Wistar submetidos à endotoxemia. Para a indução da endotoxemia, foi administrado lipopolissacarídeo (LPS; 5mg/kg intraperitoneal) sistemicamente. Os animais foram tratados com ghrelina (15nmol/kg; endovenoso) concomitantemente à administração de LPS e tiveram o sangue e o fígado coletados após 2h,6h ou 12h. Foram quantificadas a concentração sanguínea do fator de necrose tumoral alfa (TNF-?), interleucina (IL)-1?, IL-6, nitrato, corticosterona, GH, IGF-1 e ghrelina endógena, assim como a concentração hepática de TNF-?, IL-1? e IL-6. O TNF-?, IL-1?, IL-6, GH, IGF-1 e ghrelina endógena foram quantificados pela técnica de ELISA. A corticosterona foi quantificada pela técnica de radioimunoensaio. O Nitrato foi quantificado pela técnica de quimioluminescência. Também foram quantificadas a expressão proteica hepática do receptor do hormônio secretador do GH (GHSR-1a) e do receptor do GH (GHR) pela técnica de Western Blott, bem como a expressão gênica hepática de IGF-1 e GHR pela técnica de PCR-RT. Os ratos submetidos à endotoxemia apresentaram redução sérica de IGF-1 e de GH, caracterizando a alteração do eixo GH/IGF-1. Os animais endotoxêmicos e tratados com ghrelina apresentaram menor redução dos níveis circulantes de IGF-1, além de apresentarem menores níveis de TNF-?, IL-1?, IL-6 e nitrato após administração de LPS. A menor redução de IGF-1 circulante após o tratamento com ghrelina não foi relacionada a alterações na expressão proteica de GHSR-1a ou GHR, nem relacionada a alterações na expressão gênica de IGF-1 ou GHR nos intervalos de tempo analisados. Portanto, a propriedade anti-inflamatória da ghrelina levou à redução do aumento dos mediadores pró-inflamatórios e contribuiu para a manutenção da integridade do eixo GH/IGF-1 ao atenuar a queda na concentração sanguínea de IGF-1. Endotoxemia. Inflamação. Eixo GH/IGF-1 / During the endotoxemia it is possible to observe a change on the growth hormone (GH) /insulin-like growth factor (IGF)-1 axis. It is believed that the pro inflammatory cytokines increase is responsible for this change even not having the mechanism for this change completely elucidated. The ghrelin is a peptidic hormone which has antiinflammatory properties and, because of that, can contribute to the GH/IGF-1 axis integrity maintenance. This research goal is to evaluate the ghrelin systemic treatment effect on the GH/IGF-1 axis on Wistar rats submitted to endotoxemia. To induct the endotoxemia it was given systemically to the rats a lipopolysaccharide (LPS; 5mg/kg intraperitoneal). The animals were treated with ghrelin (15nmol/kg; intravenous) while receiving the LPS and they had their blood and liver collected after 2h, 6h or 12h. Blood concentration of alfa tumoral necrose (TNF-?), interleukin (IL)-1?, IL-6, nitrate, corticosterone, GH, IGF-1 and endogenous ghrelin were quantified as well as their TNF-?, IL-1? e IL-6 hepatic concentration. The TNF-?, IL-1?, IL-6, GH, IGF-1 and the endogenous ghrelin were quantified through the ELISA technique. The corticosterone was quantified through the radioimmunoassay technique. The nitrate was quantified through the chemiluminescence technique. The hepatic protein expression from the growth hormone secretagogue receptor (GHSR)-1a and the receptor of the GH (GHR) were quantified through the Western Blott technique and the IGF-1 and the GHR hepatic gene expression through the PCR-RT technique. The rats submitted to the endotoxemia presented an IGF-1 and a GH serum decrease characterizing a change on the GH/IGF-1 axis. The endotoxemic animals treated with ghrelin showed a smaller reduction of the IGF-1 circulating levels besides presenting a smaller TNF-?, IL-1?, IL- 6 and nitrate levels after receiving the LPS. The smallest IGF-1 circulating decrease, after the treatment with ghrelin, was related neither to the changes on the GHSR-1a or GHR protein expressions nor to the IGF-1 or GHR gene expressions during the analyzed time intervals. Therefore, the ghrelin anti-inflammatory property inflected a reduction of the pro-inflammatory mediators increase and contributed for the GH/IGF- 1 axis integrity maintenance while mitigating the IGF-1 blood concentration fall.

A galectina-1 (Gal-1) é uma lectina homodimérica multifuncional capaz de reconhecer e se ligar a beta-galactosídeos por meio de um domínio denominado carbohydrate recognition domain (CRD). A Gal-1 humana (Gal-1h) e a Gal-1 de camundongo (Gal-1c) mantêm 88,15% de homologia e, apesar de não existirem mutações em aminoácidos-chave do CRD, há substituições próximas a esses resíduos. Considerando as implicações dessas diferenças em estrutura e função, e que é comum a utilização de modelos murinos para estudar a função Gal-1, o presente trabalho objetiva analisar comparativamente a Gal-1c e a Gal-1h por meio de ensaios de cristalização e determinação estrutural da Gal-1c, além da avaliação comparativa da atividade lectínica da Gal-1h e da Gal-1c por glycan array e hemaglutinação. Também foi avaliada a capacidade de ambas as Gal-1 em induzir a exposição de fosfatidilserina (FS) em neutrófilos ativados provenientes de medula de camundongos normais ou deficientes de ?-2 integrina (Mac-1), de modo a investigar se a interação Gal-1/Mac-1 estaria envolvida nesse processo. Preparações homogêneas e ativas de Gal-1c e Gal-1h foram utilizadas nos ensaios. Os cristais de Gal-1c foram obtidos em 20% de polietilenoglicol 3350 e 0,2 M de fluoreto de amônio. Os dados de difração de raios X foram coletados e processados, obtendo-se uma estrutura com resolução de 2,4 Å. Observou-se que substituições de aminoácidos entre a Gal-1c e a Gal-1h estão localizadas em regiões expostas ao solvente, próximas do CRD e distantes da interface de dimerização. A análise comparativa entre Gal-1c e Gal-1h mostrou que estas substituições conferem a Gal-1c um caráter mais polar, com consequente aumento da distribuição de volume molecular. Nos ensaios de hemaglutinação, pode-se observar que é necessária uma concentração 2 vezes maior de Gal-1c para aglutinar eritrócitos humanos, de carneiro e de coelho na mesma proporção que a Gal-1h. Por meio do glycan array, pode-se determinar o perfil de ligação a glicanas de ambas as Gal-1. As duas Gal-1 apresentam afinidade por glicanas ramificadas contendo galactose terminal, e a Gal-1h apresentou maior intensidade de ligação às glicanas quando comparada à Gal-1c. Preparações de Gal-1c e Gal-1h induzem níveis semelhantes de exposição de FS na superfície de neutrófilos deficientes ou não de Mac-1, sugerindo que a interação Gal-1/Mac-1 não esteja envolvida no processo de exposição de FS na superfície de neutrófilos ativados. Assim, a diferença sequencial entre a Gal-1c e a Gal-1h é capaz de gerar diferenças estruturais consideráveis que implicam no reconhecimento diferencial de glicanas, o que, entretanto, não se reflete na capacidade de indução de FS na superfície de neutrófilos ativados. Além disso, a interação Gal-1/Mac-1 parece não participar desse processo, o que pode indicar que o papel da Gal-1 no turnover de neutrófilos, via reconhecimento fagocítico, seja um processo complexo e independente dessa interação. / Galectin-1 (Gal-1) is a homodimeric and multifunctional lectin that recognizes and binds to beta-galactoside by a carbohydrate recognition domain (CRD). Human Gal-1 (hGal-1) and mouse Gal-1 (mGal-1) are 88.15% identical, and although there are no mutations in key amino acids within the CRD, there are differences in the amino acids sequence near the CRD. Given the potential of these differences to alter overall structure and function, and the common utilization of murine models to study Gal-1 function, we sought to directly compare key biochemical features of hGal and mGal-1. Thus, we performed crystallization and structure determination assays of mGal-1, and determined the carbohydrate binding specificy of mGal-1 and hGal-1 using a glycan array and using hemagglutination assay. We also evaluated the ability of both Gal-1 to induce exposure of phosphatidylserine (PS) in activated neutrophils from the bone marrow of normal or ?-2 integrin (Mac-1) deficient mice, in order to investigate the involvement of Gal-1/Mac-1 interaction in this process. To accomplish this, homogeneous and active preparations of hGal-1 and mGal-1 were used in the study. mGal-1 crystals were obtained in 20% polyethylene glycol 3350 and 0.2 M ammonium fluoride. Data from X-ray diffraction were collected and processed, yielding a structure with a final resolution of 2.4 Å. The amino acid substitutions found between mGal-1 and hGaI-1 are detected on the solvent-exposed surfaces where the CRDs are located and not on the proteins dimerization surfaces. A comparative structural analysis between mGal-1 and hGal-1 shows that these amino acid substitutions confer to mGal-1 a greater number of ionizable residues, polar character, appearance of the acid regions clustered, and a slight increase of volume distribution. In hemagglutination assays, twice the concentration of mGal-1 was required to cause equivalent agglutination of human, sheep or rabbit erythrocytes as hGal-1. Glycan array analysis demonstrated that both galectins have affinity for branched glycans containing terminal galactose residues. However, hGal-1 appeared to display higher levels of binding that mGal-1. Preparations of mGal-1 and hGal-1 induced similar levels of PS exposure on normal or Mac-1 deficient neutrophils, suggesting that the interaction Gal-1/Mac-1 is not involved in this process. Thus, hGal-1 and mGal-1 appear to possess considerable differences in glycan recognition that likely reflects subtle difference in amino acid sequence. Furthermore, the interaction Gal-1/Mac-1 do not appear to participate in this PS exposure process, which suggest that other Gal-1 receptors are likely important in this process.

Procurando compreender o envolvimento da aldosterona (Aldo) na injúria renal, o objetivo deste projeto foi avaliar o efeito do tratamento crônico com Aldo sobre a função renal e a histologia das arteríolas renais, procurando correlacionar os achados com a expressão de genes reguladores do processo de fibrose e apoptose. A Aldo não alterou os parâmetros fisiológicos, PA, ritmo de filtração glomerular (estimado pela depuração plasmática de creatinina), proteinúria e a morfologia das arteríolas corticais. No entanto, aumentou a expressão do RNAm para TGF-β1, PAI-1 e BAX no tecido renal, além da contagem de células TUNEL positivas nos glomérulos. O antagonismo ao receptor MR (pelo uso da espironolactona) aboliu o efeito hormonal somente sobre a expressão do RNAm para BAX e a marcação do DNA degradado (TUNEL), enquanto que o antagonismo ao GR (pelo uso do RU 486) reduziu ou aboliu todos os efeitos da Aldo. Os resultados indicam que a Aldo pode induzir respostas precoces sobre o remodelamento do tecido renal, sem ainda comprometer a função renal ou alterar a PA. Essas respostas foram independentes da sobrecarga de sal e ocorreram por um mecanismo que envolveu os receptores MR e GR. / In order to understand the aldosterone (Aldo) involvement in renal injury, the objective of this project was to evaluate the effect of Aldo chronic treatment on renal function and renal arterioles histology, trying to correlate the findings with the regulatory genes of fibrosis and apoptosis. Aldo did not change the physiological parameters, BP, glomerular filtration rate (estimated by creatinina clearance), proteinuria and cortical arterioles morphology. However, Aldo increased mRNA expression for TGF-β1, PAI-1 and BAX in renal tissue, as well as TUNEL-positive cell count in glomeruli. MR receptor antagonism (by spironolactone) abolished only the hormonal effect on the mRNA expression for BAX and degraded DNA labeling (TUNEL); whereas GR antagonism (by RU 486) reduced or abolished all Aldo effects. The results indicated that Aldo can induce early responses on renal tissue remodeling, without altering renal function or BP. These responses were salt independent and involved MR and GR receptors.

Disruption of the integrity of vascular endothelium plays an essential role in the development and the progression of numerous human diseases, including sepsis, atherosclerosis and others. A complex array of transmembrane adhesive proteins located in junctional structures, support endothelial integrity and control vascular permeability. Furthermore, they are able to transmit intracellular signals to coordinate various endothelial biological responses to insure normal vascular function. Immunoglobulin-containing and proline rich receptor-1 (IGPR-1) is a novel cell adhesion molecule that is involved in angiogenesis and in the regulation of endothelial permeability. IGPR-1 is phosphorylated at Ser220, which is required for its ability to mediate actin fibril reorganization. In this study, we demonstrate that the phosphorylation of IGPR-1 at Ser220 is stimulated by cell spreading and cell adhesion in porcine aortic endothelial (PAE) cells. Blocking homophilic trans-dimerization of IGPR-1 by a blocking antibody inhibited cell-density phosphorylation of IGPR-1. More importantly, phosphorylation of IGPR-1 at Ser220 is increased in PAE cells under shear stress, which was essential for IGPR-1-mediated endothelial cell alignment in response to shear stress. Taken together, this study demonstrate that IGPR-1 activity is regulated be endothelial cell spreading and density. And its activity plays an important role in endothelial cell alignment in response to shear stress. / 2020-07-03T00:00:00Z