Diferentemente do que muitos imaginam, o Brasil é detentor da maior diversidade de espécies de bambus dos países do Novo Mundo. O conhecimento sobre o potencial de aplicações de bambus nativos é extremamente subdesenvolvido em comparação ao de espécies asiáticas. Considerando que as espécies asiáticas são utilizadas na medicina popular e se têm relatos de várias atividades biológicas atribuídas à presença de flavonoides e outras substâncias fenólicas de interesse, espécies de bambu do Neotrópico são potenciais fontes de bioativos. Utilizando-se essa premissa, Merostachys pluriflora, uma espécie nativa de bambu, foi escolhida como modelo de estudo. Este trabalho teve como objetivos quantificar o teor de amido, carboidratos solúveis, lipídeos, fenóis totais, flavonoides, taninos totais e proantocianidinas de folhas e colmos de M. pluriflora; e avaliar o potencial biológico dos seus extratos, fases e substâncias isoladas através de ensaios in vitro da capacidade antioxidante, anti-HIV-1 e antibacteriana. Foi possível observar que o extrato bruto de folhas rendeu o dobro do extrato de colmo e que as fases obtidas com solventes mais polares, como a fase hidrometanólica, apresentaram maiores rendimentos. Dos metabólitos primários quantificados em M. pluriflora, os lipídeos se destacaram em conteúdo em ambos os órgãos estudados. Com relação as substâncias fenólicas, foi possível observar que o extrato bruto dos colmos apresentou uma maior abundância de fenilpropanoides e derivados do ácido clorogênico, enquanto o extrato bruto das folhas apresentou uma maior abundância de flavonoides, quando comparadas aos colmos. Das substâncias fenólicas presentes em M. pluriflora, foram identificadas duas flavonas, a vitexina e a isovitexina; e três fenilpropanoides, o ácido cafeíco, ácido ferúlico e o cafeato de metila. Das fases geradas por partição, a de acetato de etila e de diclorometano, para ambos os órgãos, foram as que apresentaram a maior parte dos constituintes fenólicos, sendo as fases de acetato de etila mais ricas em flavonoides e as de diclorometano em fenilpropanoides. No geral, os extratos brutos, assim como as fases de folhas e colmos de M. pluriflora, apresentaram um grande potencial antioxidante, principalmente antiradicalar e redutor de ferro, apresentando valores de EC50 de 16,30 μg/mL a 94,77 μg/mL no ensaio ABTS, no ensaio FRAP esses valores variaram de 27,92 μg/mL a 145,78 μg/mL. No ensaio antibacteriano, especialmente frente à P. pally, a fase de diclorometano de folhas se mostrou mais ativa, com MIC50 de 126,22 μg/mL o que pode indicar que as substâncias fenólicas de caráter lipofílico, nessa espécie, são promissoras para essa atividade. Embora o ensaio anti-HIV1 mostrou que as amostras não apresentam atividade antirretroviral, este estudo contribui para o conhecimento do potencial antiviral dos extratos de bambus brasileiros. M. pluriflora se mostrou uma espécie promissora para estudos de prospecção, com uma grande quantidade de substâncias fenólicas em sua composição / Brazil is the country with the highest diversity of bamboo species in the New World. Knowledge about the medicinal potential of native bamboos is extremely underdeveloped when compared to Asian species. Some Asian bamboo species are used in folk medicine and have reports of various biological activities attributed to the presence of phenolic compounds, so bamboo species of the Neotropics are potential sources of bioactive substances. Using this assumption, Merostachys pluriflora, a native bamboo species, was chosen as a model for this study. The aimed of this study was to quantify the contents of starch, soluble carbohydrates, lipids, total phenols, flavonoids, total tannins and proanthocyanidins in leaves and culms of M. pluriflora; and evaluate the biological potential of the extracts, phases and isolated substances through in vitro assays: antioxidant activity, anti-HIV1 and antibacterial activity. It was observed that the crude extract of leaves yielded twice more than the culm extract; phases obtained with more polar solvents, such as hydromethanolic phases, had the highest yields. Lipids were the class of primary metabolites that presented higher quantities on both organs studied. Regarding the phenolic substances, it was observed that the crude extract of culms presented higher abundance of phenylpropanoids and chlorogenic acid derivates, but the crude extract from leaves showed higher abundance of flavonoids (all of then derived from apigenin) when compared to culms. Were identified two flavones, vitexin and isovitexin, and three phenylpropanoids, caffeic acid, ferulic acid and methyl caffeate. Phases using ethyl acetate and dichloromethane as extraction solvents were those that retained the majority of phenolic constituents. Ethyl acetate phase presented flavonoids while dichloromethane phase presented phenylpropanoids as major contituients. In general, the crude extracts and phases from leaves and culms of M. pluriflora showed antioxidant activity, especially antiradical and iron reducer capacity, presenting EC50 values of 16.30 mg/mL to 94.77 mg/ml in ABTS assay. For FRAP assay these values ranged from 27.92 mg/mL to 145.78 mg/mL. In the antibacterial assay, especially for P. pally, the dichloromethane phase from leaves was more active, presenting MIC50 of 126.22 mg/mL. This might indicate that the lipophilic phenolic present in this species of bamboo are promising for antibacterial activity. Although the anti-HIV1 assay showed that the samples do not present antiretroviral potential, this study contributes to the knowledge of the antiviral potential of Brazilian bamboo species. M. pluriflora showed to be a promising species for prospecting studies, with a large amount of phenolic substances in its composition

Desde os primórdios da medicina, substâncias derivadas de animais, plantas e microrganismos têm sido utilizadas no tratamento e cura de diversas doenças. Especificamente o uso de plantas medicinais pela população como terapia alternativa no tratamento de doenças, tem sido uma prática comum desde milhares de anos antes da era presente (a.p.). Atualmente, sabe-se que muitos dos metabólitos secundários estão diretamente envolvidos nos mecanismos que permitem a adaptação das plantas ao seu habitat. Dessa forma, e pensando-se em espécies de uso medicinal, é esperado que o potencial biológico também sofra variações de acordo com os fatores bióticos e abióticos. Hyptis (Lamiaceae) constitui um gênero altamente promissor para estudos de prospecção de substâncias farmacologicamente ativas, favorecidos pela grande diversidade de espécies que ocorrem nos domínios fitogeográficos brasileiros, sendo, portanto, escolhido como modelo para responder às questões deste trabalho. O objetivo geral foi a expansão das análises fitoquímicas e do potencial biológico de quatro espécies de Hyptis pertencentes à seção Peltodon , além de verificar uma possível variação fitoquímica (quantitativa e/ou qualitativa) entre as espécies estudadas e coletadas não só em diferentes domínios, mas em diferentes fitofisionomias. Como resultado, Hyptis seção Peltodon apresentou composição química semelhante às espécies da subfamília Nepetoideae em relação à constituição fenólica, destacando a presença do ácido cafeico, ácido rosmarínico e nepetoidinas, corroborando estudos já existentes. Também reportamos que Hyptis seção Peltodon possui flavonoides derivados da flavona apigenina, sendo que identificação de flavonas C-glicosiladas nos extratos brutos sugerem o uso das mesmas como importantes marcadores taxonômicos no nível de seção. As espécies que apresentaram maior potencial antioxidante e anti-HIV-1 foram H. comaroides e H. meridionalis. Com relação às espécies coletadas nos dois domínios fitogeográficos propostos concluímos que para o grupo estudado e para as substâncias analisadas, populações que se encontrem no domínio Tropical Atlântico são as melhores candidatas como fonte de antioxidantes naturais. Entretanto, futuras pesquisas são necessárias a fim de investigar e compreender melhor os efeitos sinérgicos de múltiplos fatores ambientais no metabolismo secundário bem como no potencial biológico de espécies vegetais / Since the beginning of medicine, substances derived from animals, plants, and microorganisms have been used in the treatment and cure of various diseases. The use of medicinal plants by the population as an alternative therapy in the treatment of diseases has been a common practice for thousands of years before the present (b.p.). Nowadays we known that many of the plant secondary metabolites are directly involved in the mechanisms that allow adaptation to their habitat. Considering species of medicinal use, it is expected that the biological potential also suffers variations according to the biotic and abiotic factors. Hyptis (Lamiaceae) is a highly promising genus for prospective studies of bioactive substances, favored by the great diversity of species that occur in the Brazilian phytogeographical domains, being therefore chosen as a model to answer the questions of this work. The objective of this study was to contribute with chemical data and the biological potential of four Hyptis species belonging to the Peltodon section, as well as to verify a possible phytochemical variation (quantitative and/or qualitative) between species naturally occurring in two different phytogeographical domains. As results of this work, Hyptis section Peltodon presented phenolic composition similar to species of the subfamily Nepetoideae, with the presence of caffeic acid, rosmarinic acid, and nepetoidins, corroborating existing studies. We also reported that Hyptis section Peltodon possesses flavonoids derived from the flavone apigenin, and the identification of C-glycosylated flavones in the crude extracts suggest the use of these as important taxonomic markers at the section level. We observed that in general, the species that presented the highest antioxidant and anti-HIV-1 potential were H. comaroides and H. meridionalis. Regarding the influence of the phytogeographic domains we concluded that for the group studied and for the substances analyzed, populations that occurs in the Tropical Atlantic domain are the best candidates as source of natural antioxidants. Future research, however, is necessary to better understand the synergistic effects of multiple environmental factors on secondary metabolism, as well as the biological potential of plant species

Diferentemente do que muitos imaginam, o Brasil é detentor da maior diversidade de espécies de bambus dos países do Novo Mundo. O conhecimento sobre o potencial de aplicações de bambus nativos é extremamente subdesenvolvido em comparação ao de espécies asiáticas. Considerando que as espécies asiáticas são utilizadas na medicina popular e se têm relatos de várias atividades biológicas atribuídas à presença de flavonoides e outras substâncias fenólicas de interesse, espécies de bambu do Neotrópico são potenciais fontes de bioativos. Utilizando-se essa premissa, Merostachys pluriflora, uma espécie nativa de bambu, foi escolhida como modelo de estudo. Este trabalho teve como objetivos quantificar o teor de amido, carboidratos solúveis, lipídeos, fenóis totais, flavonoides, taninos totais e proantocianidinas de folhas e colmos de M. pluriflora; e avaliar o potencial biológico dos seus extratos, fases e substâncias isoladas através de ensaios in vitro da capacidade antioxidante, anti-HIV-1 e antibacteriana. Foi possível observar que o extrato bruto de folhas rendeu o dobro do extrato de colmo e que as fases obtidas com solventes mais polares, como a fase hidrometanólica, apresentaram maiores rendimentos. Dos metabólitos primários quantificados em M. pluriflora, os lipídeos se destacaram em conteúdo em ambos os órgãos estudados. Com relação as substâncias fenólicas, foi possível observar que o extrato bruto dos colmos apresentou uma maior abundância de fenilpropanoides e derivados do ácido clorogênico, enquanto o extrato bruto das folhas apresentou uma maior abundância de flavonoides, quando comparadas aos colmos. Das substâncias fenólicas presentes em M. pluriflora, foram identificadas duas flavonas, a vitexina e a isovitexina; e três fenilpropanoides, o ácido cafeíco, ácido ferúlico e o cafeato de metila. Das fases geradas por partição, a de acetato de etila e de diclorometano, para ambos os órgãos, foram as que apresentaram a maior parte dos constituintes fenólicos, sendo as fases de acetato de etila mais ricas em flavonoides e as de diclorometano em fenilpropanoides. No geral, os extratos brutos, assim como as fases de folhas e colmos de M. pluriflora, apresentaram um grande potencial antioxidante, principalmente antiradicalar e redutor de ferro, apresentando valores de EC50 de 16,30 μg/mL a 94,77 μg/mL no ensaio ABTS, no ensaio FRAP esses valores variaram de 27,92 μg/mL a 145,78 μg/mL. No ensaio antibacteriano, especialmente frente à P. pally, a fase de diclorometano de folhas se mostrou mais ativa, com MIC50 de 126,22 μg/mL o que pode indicar que as substâncias fenólicas de caráter lipofílico, nessa espécie, são promissoras para essa atividade. Embora o ensaio anti-HIV1 mostrou que as amostras não apresentam atividade antirretroviral, este estudo contribui para o conhecimento do potencial antiviral dos extratos de bambus brasileiros. M. pluriflora se mostrou uma espécie promissora para estudos de prospecção, com uma grande quantidade de substâncias fenólicas em sua composição / Brazil is the country with the highest diversity of bamboo species in the New World. Knowledge about the medicinal potential of native bamboos is extremely underdeveloped when compared to Asian species. Some Asian bamboo species are used in folk medicine and have reports of various biological activities attributed to the presence of phenolic compounds, so bamboo species of the Neotropics are potential sources of bioactive substances. Using this assumption, Merostachys pluriflora, a native bamboo species, was chosen as a model for this study. The aimed of this study was to quantify the contents of starch, soluble carbohydrates, lipids, total phenols, flavonoids, total tannins and proanthocyanidins in leaves and culms of M. pluriflora; and evaluate the biological potential of the extracts, phases and isolated substances through in vitro assays: antioxidant activity, anti-HIV1 and antibacterial activity. It was observed that the crude extract of leaves yielded twice more than the culm extract; phases obtained with more polar solvents, such as hydromethanolic phases, had the highest yields. Lipids were the class of primary metabolites that presented higher quantities on both organs studied. Regarding the phenolic substances, it was observed that the crude extract of culms presented higher abundance of phenylpropanoids and chlorogenic acid derivates, but the crude extract from leaves showed higher abundance of flavonoids (all of then derived from apigenin) when compared to culms. Were identified two flavones, vitexin and isovitexin, and three phenylpropanoids, caffeic acid, ferulic acid and methyl caffeate. Phases using ethyl acetate and dichloromethane as extraction solvents were those that retained the majority of phenolic constituents. Ethyl acetate phase presented flavonoids while dichloromethane phase presented phenylpropanoids as major contituients. In general, the crude extracts and phases from leaves and culms of M. pluriflora showed antioxidant activity, especially antiradical and iron reducer capacity, presenting EC50 values of 16.30 mg/mL to 94.77 mg/ml in ABTS assay. For FRAP assay these values ranged from 27.92 mg/mL to 145.78 mg/mL. In the antibacterial assay, especially for P. pally, the dichloromethane phase from leaves was more active, presenting MIC50 of 126.22 mg/mL. This might indicate that the lipophilic phenolic present in this species of bamboo are promising for antibacterial activity. Although the anti-HIV1 assay showed that the samples do not present antiretroviral potential, this study contributes to the knowledge of the antiviral potential of Brazilian bamboo species. M. pluriflora showed to be a promising species for prospecting studies, with a large amount of phenolic substances in its composition

Student Number : 9000987E - PhD thesis - School of Medicine - Faculty of Health Sciences / The generation of human monoclonal antibodies (mAbs) that are able to block HIV-1 infection in vitro would be useful reagents for studying virus neutralization, and assist in identifying neutralizing antibody (NAb) epitopes of HIV-1 envelope glycoprotein. This may provide important information for designing HIV-1 vaccine that aim to induce NAbs. HIV-1 subtype C individuals with high levels of NAb titres were identified, and peripheral blood mononuclear cells (PBMC) from these individuals were isolated and B-cells transformed with Epstein-Barr virus (EBV). Clones specific to HIV-1 gp120 using cell lysate preparations derived from HIV-1 subtype C infected cell lines were generated by performing limiting dilutions. Transformation efficiencies were estimated at over 80% by evaluating EBV-transformation cultures by microscopic visualization. Of these approximately 5% were HIV-1-specific. Five clones derived from the Du23 (1) sample secreting anti-HIV-1 antibodies were generated: 2.3C, 2.9D, 3.2C, 4.12E, and 1.5D. The 1.5D mAb could not be confirmed as anti-HIV-1 clone and it was probably lost during the process of subculturing. The remaining four Du23 mAbs were determined to be of IgG1 isotype lambda (λ) light chain. These mAbs bind to gp120, and 2.9D is probably a polyreactive clone. Clones 2.3C, 3.2C and 4.12E appear to be A32-like, but do not share the same epitope. We have determined that the binding sites for all four Du23 mAbs require at least the C1 region, and they also showed binding sites overlapping with F91 and 1.5E. All four Du23 mAbs required intact gp120 proteins for their binding, and soluble CD4 enhance their binding. Thus, their binding site is discontinuous and conformational. These mAbs are non-neutralizing as they showed limited activity of 30-59% when tested using T-cell line grown viruses or 0-30% when tested against pseudovirions. This activity is rather low when compared to over 80% shown by broadly neutralizing mAbs that have been described in the literature. The challenge in generating mAbs, in particular subtype C-derived, is to find those antibodies capable of suppressing viral replication in vivo and be capable of preventing infection. These reagents could be used to identify epitopes to guiding the design of HIV-1 subtype C envelope immunogens or vaccines. It is also envisaged that neutralizing antibodies used in therapeutic setting or in combination with antiviral drug therapy could reduce viral load and retard disease progression in infected people.

University of Venda / MSc (Microbiology)

Human immunodeficiency virus (HIV) has become a global pandemic over the past few decades, with new infections and related deaths in the millions each year. There is no cure in sight for HIV-1 infection, and there has been little progress in developing an efficacious vaccine. Heterosexual transmission of HIV-1 remains the principal mode of transmission throughout the world and thus measures, such as topical vaginal microbicides, to prevent infection of the female reproductive tract are actively being explored. Recent trials of topical vaginal microbicides have shown that their interaction with the mucosal surfaces of the female reproductive tract as well as semen can hinder microbicide effectiveness against HIV-1 infection. Therefore, understanding the role these fluids play in HIV transmission would be critical towards developing effective antiviral prophylaxes. A recent study from our group demonstrated that human cervicovaginal secretions contained numerous cationic antimicrobial peptides and proteins, which collectively inhibited HIV-1 infection of target cells and tissues. To ascertain if human seminal plasma (SP), the main vector responsible for transmitting HIV-1, exhibited antiviral activity we utilized several antiHIV assays in the presence or absence of minimally manipulated SP. The majority of the intrinsic anti-HIV-1 activity of SP resided in the cationic polypeptide fraction. Antiviral assays utilizing luciferase reporter cells and lymphocytic cells revealed the ability of whole SP to prevent HIV-1 infection, even when SP was diluted 3200-fold. Subsequent fractionation by continuous flow acid-urea (AU)-PAGE and antiviral testing revealed that cationic polypeptides within SP were responsible for the majority of anti-HIV-1 activity. A proteomic approach was utilized to resolve and identify 52 individual cationic polypeptides that contribute to the aggregate anti-HIV-1 activity of SP. One peptide fragment of semenogelin I, termed SG-1, was purified from SP by a multi-step chromatographic approach, protein sequenced, and determined to exhibit anti-HIV-1 activity against HIV-1. Anti-HIV-1 activity was transient, as whole SP incubated for prolonged time intervals exhibited a proportional decrease in anti-HIV-1 activity that was directly attributed to the degradation of semenogelin I peptides. Collectively, these results indicate that the cationic polypeptide fraction of SP is active against HIV-1, and that semenogelin-derived peptides contribute to the intrinsic anti-HIV-1 activity of SP. Conversely, naturally occurring peptidic fragments from the SP-derived prostatic acid phosphatase (PAP) have been reported to form amyloid fibrils called "SEVI" capable of enhancing HIV-1 infection in vitro. In order to understand the biological consequence of this proviral effect, we extended these studies in the presence of human SP. PAP-derived peptides were agitated to form SEVI and incubated in the presence or absence of SP. While PAP-derived peptides and SEVI alone were proviral, the presence of 1% SP ablated their proviral activity in several different anti-HIV-1 assays. The anti-HIV-1 activity of SP was concentration dependent and was reduced following filtration. Supraphysiological concentrations of PAP peptides and SEVI incubated with diluted SP were degraded within hours, with SP exhibiting proteolytic activity at dilutions as high as 1:200. Sub-physiological concentrations of two prominent proteases of SP, prostate-specific antigen (PSA) and matriptase, could degrade physiological and supraphysiological concentrations of PAP peptides and SEVI. While human SP is a complex biological fluid, containing both antiviral and proviral factors, our results suggest that PAP peptides and SEVI may be subject to naturally occurring proteolytic components capable of reducing their proviral activity. Our studies demonstrate the overall antiviral activity of human SP, but there is still a critical need for effective topical vaginal microbicides that can prevent HIV-1 transmission. The synthetic human retrocyclins are cyclic antimicrobial peptides that are remarkably active against HIV-1, and are being developed as topical vaginal microbicides. Herein, we assessed whether the putative proviral SEVI was able to adversely affect the anti-HIV-1 activity of the retrocyclin analog RC-101. While SEVI alone enhanced viral infection, this effect was completely negated in the presence of RC-101. Retrocyclins such as RC-101 are inhibitors of HIV-1 entry, by preventing gp41-mediated viral fusion. Interestingly, using an HIV-1 reverse transcriptase (RT) specific assay, we also determined that RC-101 directly inhibited the activity of RT in a dose dependent manner, suggesting a secondary mechanism of viral inhibition. Our group has determined that RC-101 induces only a modest level of resistance in HIV, which may be due in part to RC-101's dual mechanisms of viral inhibition.

Albert Font Haro ABSTRACT Modulation of plasmacytoid dendritic cell function: role of immunoreceptors TIM-3 and BDCA-2 Plasmacytoid dendritic cells (pDCs) are key players in the antiviral response as well as in linking innate and adaptive immune response. They express endosomal toll-like receptors 7 and 9, which can detect ssRNA and unmethylated CpG DNA, respectively. Due to the constitutive expression of the transcription factor IRF7, pDCs are able to rapidly produce massive quantities of type I (α, β, ω) and type III (1, 2, 3, 4) interferons (IFN-I and IFN-III) as well as pro- inflammatory cytokines such as IL-1, IL-6 and TNF-α. After maturation, they also function as antigen-presenting cells. Despite intense research, the mechanisms of IFN and pro-inflammatory cytokines production and regulation are still poorly understood. Using the pDC cell line GEN2.2 and also primary human pDCs, we shed light on the role of kinases MEK and SYK in IFN-I production and regulation. We found that SYK is not only involved in the regulatory receptor (RR)-mediated BCR-like pathway that represents the negative regulation of IFN-I and IFN-III secretion but also in the positive TLR7/9-mediated signal transduction pathway that leads to IFN-I production, representing the immunogenic function. We also found that MEK plays a...