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abstract: Pathogenic Gram-negative bacteria employ a variety of molecular mechanisms to combat host defenses. Two-component regulatory systems (TCR systems) are the most ubiquitous signal transduction systems which regulate many genes required for virulence and survival of bacteria. In this study, I analyzed different TCR systems in two clinically-relevant Gram-negative bacteria, i.e., oral pathogen Porphyromonas gingivalis and enterobacterial Escherichia coli. P. gingivalis is a major causative agent of periodontal disease as well as systemic illnesses, like cardiovascular disease. A microarray study found that the putative PorY-PorX TCR system controls the secretion and maturation of virulence factors, as well as loci involved in the PorSS secretion system, which secretes proteinases, i.e., gingipains, responsible for periodontal disease. Proteomic analysis (SILAC) was used to improve the microarray data, reverse-transcription PCR to verify the proteomic data, and primer extension assay to determine the promoter regions of specific PorX regulated loci. I was able to characterize multiple genetic loci regulated by this TCR system, many of which play an essential role in hemagglutination and host-cell adhesion, and likely contribute to virulence in this bacterium. Enteric Gram-negative bacteria must withstand many host defenses such as digestive enzymes, low pH, and antimicrobial peptides (AMPs). The CpxR-CpxA TCR system of E. coli has been extensively characterized and shown to be required for protection against AMPs. Most recently, this TCR system has been shown to up-regulate the rfe-rff operon which encodes genes involved in the production of enterobacterial common antigen (ECA), and confers protection against a variety of AMPs. In this study, I utilized primer extension and DNase I footprinting to determine how CpxR regulates the ECA operon. My findings suggest that CpxR modulates transcription by directly binding to the rfe promoter. Multiple genetic and biochemical approaches were used to demonstrate that specific TCR systems contribute to regulation of virulence factors and resistance to host defenses in P. gingivalis and E. coli, respectively. Understanding these genetic circuits provides insight into strategies for pathogenesis and resistance to host defenses in Gram negative bacterial pathogens. Finally, these data provide compelling potential molecular targets for therapeutics to treat P. gingivalis and E. coli infections. / Dissertation/Thesis / M.S. Biology 2013

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In DNA immunization a plasmid expressing an antigen of interest is inoculated into an animal and antigen-specific humoral and cellular immune responses are raised. In this dissertation we sought to further our understanding of antibody responses raised following DNA inoculation. Specifically, we examined the role of lymphoid tissue in the initiation and maintenance of the long-term antibody response, the role of CD4+ and CD8+ T cells in the DNA-raised antibody response, the longevity of functional antigen expression, and the nature of the antigen presenting cell. In all of these studies mice were immunized with an influenza hemagglutinin-expressing plasmid and plasmid was delivered by either the gene gun or intramuscular routes of inoculation. To examine the role of lymphoid tissue in the initiation and maintenance of the long-term antibody response, responses raised in gene gun immunized mice were compared to responses raised in mice primed with an influenza infection. Antibody and antibody secreting cell (ASC) responses were analyzed at various times following immunization or sublethal infection for as long as 1.5 years. We found that the antibody response raised with a single gene gun immunization was similar in longevity to that raised in infection-primed mice. The long-term maintenance of the antibody response was associated with the localization of the majority of antibody secreting cells to the bone marrow. The kinetics of ASC bone marrow localization was 4-to-8 weeks slower in DNA-immunized than infection primed mice. This corresponded to a slower rise in the antibody response to plateau levels in DNA-immunized mice. We hypothesize that it is possible that the difference in kinetics may be linked to differences in the time course and dose of antigen expression following DNA immunization and a natural infection. Antibody and ASC responses were also compared following a challenge influenza virus infection. We found that DNA-immunized and infection-primed mice responded similarly in the acute post challenge phase with increases in antibody secreting cells in the mediastinal lymph nodes. While only DNA-immunized mice had post challenge increases in antibody, the antibody response remained 3-to-4 fold lower than post challenge responses in infection primed mice. We suggest that despite post challenge increases in these responses in DNA-immunized mice that the immune response raised with DNA immunization efficiently limited replication of the challenge virus and thus limited the post challenge antibody response. We also addressed the role that CD4+ and CD8+ T cells played in the ability to prime and boost the DNA-raised antibody response. To answer this question mice were in vivo depleted of CD4+ or CD8+ T cells for 3 weeks prior to through 2 weeks following DNA immunization or boost. Antibody responses were measured 4 and 8 weeks after DNA prime and 2 weeks after DNA boost. For both the gene gun and intramuscular routes of inoculation, the antibody response was independent of CD8+ T cells, but dependent on CD4+ T cells. The presence of CD4+ T cells was required at the time of DNA immunization, but not at the time of DNA boost. The absence of CD4+ T cells at the time of DNA delivery resulted in a four week delay in the appearance of antibody. Since influenza hemagglutinin has been characterized as a T-dependent antigen the requirement for CD4+ T cells at the time of DNA prime was not surprising, but the appearance of a delayed H1-specific antibody response suggested that DNA-expressed antigen had continued to be available to prime CD4+ T cells as they reappeared following the disappearance of depleting antibody. The independence of the antibody response on the presence of CD8+ T cells suggested that DNA-primed H1-specific CD8+ T cells did not limit the plateau level of response or the ability to boost a suboptimal response. The results from our CD4+ T cell depletion experiment suggested that DNA-expressed antigen continued to be available for an extended period of time following immunization. To examine the duration of functional antigen expression for raising an antibody response, mice lacking α/β T cells (TCR-/-) were immunized with DNA or immunized with hemagglutinin protein. Naive T cells from TCR+/+ mice were transferred into the immunized TCR-/- mice on various days post DNA or protein immunization. The results from these studies show that antigen is available to raise antibody longer following DNA immunization than following a protein immunization. This result is likely due to continued expression of plasmid DNA. We found differences in the longevity of antigen expression following gene gun and intramuscular routes of inoculation. For gene gun immunizations, not intramuscular immunizations, approximately 90% of functional antigen was lost within one week of immunization. We suggest that this is consistent with a role for antigen expression by transfected cells within the target site, the epidermis, which is largely lost by 1-2 weeks following gene gun immunization. We also found that following intramuscular immunization the dominant IgG isotype changed with time of TCR+/+ T cell transfer. By contrast, there was no change in the dominant isotype following gene gun immunizations. These results suggest that the factor(s) that contribute to the development of the Th1-bias seen following intramuscular DNA immunizations are lost early. To examine the nature of the antigen presenting cell following DNA immunization, dendritic cells were sorted from the inguinal lymph nodes and spleens of gene gun or intramuscularly immunized mice on various days following DNA delivery. The dendritic cell (CD11c+) and non-dendritic cell (CD11c-) populations were used in restimulation assays with H1-specific T cell clones. Despite similar titers of raised antibody in gene gun and intramuscularly immunized mice, H1-specific antigen presenting dendritic cells were isolated from the inguinal lymph nodes and spleens of gene gun, but not intramuscularly immunized mice. Antigen presentation by dendritic cells was detected for as long as 21 days following gene gun delivery. We hypothesize that the inability to detect dendritic cell presentation of antigen following intramuscular DNA delivery may be due to a more broad distribution of antigen presenting cells, different properties of antigen presenting cells, and/or the contribution of other non-dendritic cells to antigen presentation following intramuscular, but not gene gun, immunizations. We present our results within a model for the initiation and maintenance of DNA-raised antibody responses. Within this model our data specifically contribute to understanding the initiation and generation of the DNA-raised antibody response within lymphoid tissue and the maintenance of the DNA-raised antibody response.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2004. / Vita. Bibliography: 150-177.

Lectinas são proteínas que possuem pelo menos um domínio não catalítico que se liga reversível e especificamente a um monossacarídeo ou oligossacarídeo. A capacidade de ligação a diferentes tipos de açúcares torna essas moléculas ferramentas úteis no estudo de diversos processos celulares específicos. Embora as lectinas de plantas sejam amplamente estudadas, aquelas referentes à família Apocynaceae ainda são pouco exploradas. Resultados prévios obtidos pelo nosso grupo de pesquisa mostraram que extratos brutos de súber do caule da apocinácea Tabernaemontana hystrix Steud apresenta atividade hemaglutinante. Além de aglutinar eritrócitos do sistema ABO, a putativa aglutinina foi capaz de estimular a síntese de RNAm de IL-6 e TGF- beta em células esplênicas de camundongos. À vista disso, no presente projeto tivemos como objetivo identificar, caracterizar bioquimicamente e avaliar o possível potencial imunoestimulador da aglutinina de T. hystrix. Os extratos de T. hystrix obtidos por meio da farinha de raspas do súber apresentaram atividade hemaglutinante, o que não foi observado no extrato do caule destituído de súber e no extraído das folhas. Para comprovar que se tratava da atividade observada anteriormente, obtivemos a inibição da hemaglutinação com a glicoproteína fetuína, mas não houve inibição por monossacarídeos. Foi determinado um protocolo de isolamento da hemaglutinina com precipitação do extrato do súber com sulfato de amônio, cuja atividade foi recuperada no material precipitado na faixa de 30 a 60% de saturação, seguido de cromatografias sequenciais por (1) interação hidrofóbica (HiTrap Octyl), (2) troca catiônica (HiTrap SP), (3) fase reversa (EC Nucleosil C18) e (4) afinidade (Blue Sepharose). Nessas colunas a atividade foi recuperada do (1) material não retido e dos eluatos (2 e 4) com 1M e 0,5M de NaCl, respectivamente, e (3) 83% de acetonitrila. Esse protocolo produziu uma preparação homogênea contendo um peptídeo cuja análise eletrofóretica revelou massa molecular (MM) aproximada de 3kDa e concentração hemaglutinante mínima de 50g/mL. A fim de determinar se esse peptídeo formava estrutura quaternária (dímeros, tetrâmetros, etc.), característica da maioria das lectinas de plantas, submeteu-se a preparação a uma eletroforese em gel nativo (PAGE), não sendo observadas mudanças na MM do peptídeo e nem a presença de outras moléculas com MM maiores que pudessem estar associadas a ele, o que sugere que a aglutinina de T. hystrix (denominado aqui de Tabhys) é um peptídeo de MM aproximada de 3kDa. O fato da heveína, um dos peptídeos lectínicos com atividade antifúngica mais estudado, ter especificidade por quitina nos motivou a tentar o isolamento do peptídeo em coluna desse polissacarídeo. Observou-se atividade hemaglutinante e presença de peptídeo com MM de 3kDa no material eluído com Ácido acético a 0,1M da coluna de quitina. Curiosamente, nenhuma de nossas preparações foram capazes de inibir o crescimento do fungo Trichophyton rubrum. O peptídeo purificado foi testado quanto a sua capacidade em induzir a proliferação celular e a produção de citocinas em células esplênicas murinas. Os resultados dos ensaios de RT-PCR em tempo real e citometria de fluxo demonstraram que o a aglutinina de T. hystrix não foi capaz de estimular a proliferação de linfócitos, entretanto, induziu o aumento de mensagem para a citocina TGF-beta, cujo pico de produção ocorreu em célula estimuladas com 37ng/mL. Neste estudo, relatamos a presença de um peptídeo no extrato de T. hystrix com atividade hemaglutinante, o que é relativamente raro e novo. Devido a isso, este estudo pode proporcionar novas perspectivas e paradigmas nos estudos das lectinas a nível molecular e estrutural. / Lectins are proteins that have at least one non-catalytic domain that binds specifically and reversibly to a monosaccharide or oligosaccharide. This ability to bind to different types of sugars makes these molecules useful tools in the study of various specific cellular processes. Although the plant lectins are widely studied, those belong to Apocynaceae family are still little explored. Previous results obtained by our research group showed that bark crude extracts from Tabernaemontana hystrix Steud (Apocynaceae) had hemagglutination activity. Besides to agglutinate erythrocytes from ABO blood group system, the putative agglutinin induced the synthesis of IL-6 and TGF-beta mRNA in mouse spleen cells. Here we aim to identify, characterize biochemically and evaluate the possible immunostimulatory potential of T. hystrix agglutinin. The haemagglutination activity was obtained from crude extracts of bark flour, but not of flours of stems without bark and leaves. The activity of the bark extract was similar to that from the previous study, since the haemagglutination was inhibited by the glycoprotein fetuin, but not by monosaccharides. An isolation protocol was determined by using ammonium sulfate precipitation, with haemagglutination activity recovered in the range of 30-60% of saturation, and sequential chromatography procedures: (1) hydrophobic interaction (HiTrap Octyl), (2) cation-exchange (HiTrap SP), (3) reverse phase (EC Nucleosil) and (4) affinity (BlueSepharose) chromatography. From these columns the activity was recovered in the (1) unbound material, and eluates (2 and 4) with 1M and 0,5M of NaCl, respectively, and (3) 83% acetonitrile. On the basis of electrophoresis analysis, the protocol produced a preparation comprised of only band corresponding a peptide with molecular weight (MW) of about 3-kDa, with minimum haemagglutination concentration of 50g/ml. To determine if this molecule arrangement had a quaternary structure arrangement, a feature of most known lectins, we submitted the preparation to a native electrophoresis. Because there was neither change in migration pattern nor presence of molecules of higher molecular mass, we suggested that T. hystrix peptide (Tabhys) is a peptide with MW of about 3-kDa. Since hevein, which is a most studied lectin-like peptide with antifungal activity, binds specifically to chitin, we performed an affinity chromatography in the chitin column with bark extract. We observed haemagglutination activity and the presence of peptide with MW of 3-kDa in the material bound to column and eluted with 0,1M acetic acid. Curiously, this peptide was not able to inhibit the growth of the fungus Trichophyton rubrum. Thereafter, when the purified peptide was used to stimulate murine spleen cells, we detected the expression of TGF-beta message, with a peak production obtained in cell stimulated with 37 ng/mL of Tabhys. In the current study, we isolated a peptide from crude extract of T. hystrix bark with haemagglutination activity, providing new perspectives in molecular and structural researches of peptide lectins.

Lectins and hemagglutinins are carbohydrate binding proteins present in a diversity of organisms including humans, vertebrate and invertebrate animals, plants, fungi, and bacteria. They are usually the abundant storage proteins in leguminous plants. They display a host of biological activities such as antitumor, antifungal, antiviral, insecticidal, and antibacterial activities. / The biological properties of isolated proteins, including hemagglutinating, antifungal, anti-tumor and HIV-1 reverse transcriptase inhibitory activities, were examined. Their biochemical and biological properties were compared with other purified proteins. / The seeds contain an abundance of proteins, some of which are storage proteins but may play a role of protection from pathogenic microbes and phytophagous insects. Antifungal peptides/proteins, antiviral proteins, ribosome-inactivating proteins, proteinase inhibitors, chitinases, proteinases, and defensins, are some examples of the myriad of seed proteins. The aforementioned proteins are collectively called plant defense proteins in view of their antipathogenic activities. These antifungal proteins exhibit a wide range of molecular masses and amino acid sequences. / Two lectins with potentially exploitable activities were purified from Capparis spinosa seeds and Hibiscus mutabilis seeds, respectively. A hemagglutinin was isolated from Phaselous vulgaris , cultivar "French bean 35", and detailed apoptotic pathway in breast cancer cells, MCF-7 cells, was investigated. A novel dimeric beta-lactoglobulin-like antifungal protein and an antifungal amidase were purified from Passiflora edilus seeds and Peltophorum pterocarpum, respectively. / Lam, Sze Kwan. / Adviser: Tsi Bun Ng. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 188-204). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Measles (MV) virions, like those of other enveloped viruses, enter cells by fusing their lipid membranes with those of the target host cells. Additionally, infected tissues often possess giant multinucleate cells, known as syncytia, which are formed by fusion of infected cells with uninfected neighbors. Expression of both the MV attachment (H) and fusion (F) proteins is required for membrane fusion. MV H mediates receptor binding in order to bring the two membranes into close proximity prior to F activation and is thought to trigger F activation through a specific interaction between the two proteins. Although measles H and F are efficiently transported to the cell surface when expressed independently, evidence has been reported in support of an intracellular interaction between the two proteins that can be detected using an ER co-retention approach. However, it was not determined if the putative co-retention was specific to the two measles glycoproteins, as is their ability to complement each other for efficient fusion promotion. Thus, in this thesis, the formation of an intracellular complex between MV H and F was re-examined. Consistent with the formation of an intracellular complex, cell surface expression and receptor binding of untagged wt MV H is slightly reduced by co-expression of an excess of ER-tagged MV F compared to co-expression with wt F. However, the reduction in surface expression is non-specific in that it can also be induced with heterologous proteins of NDV, which lack significant homology with those of MV. Although this approach did not detect a specific intracellular interaction between MV H and F, it cannot be ruled out that there is a weak association of the proteins that is undetectable by this method. This led to the use of an alternative approach to investigate the cellular site(s) of interaction between the measles H and F proteins. Consistent with a cell surface interaction between MV H and F, the combination of surface biotinylation and co-immunoprecipitation detects formation of a virus-specific H-F complex. Approximately, 21% of the total amount of MV H at the cell surface can be captured with MV F using an antibody against the latter protein. Two complementary approaches were used to address the relationship between this cell surface interaction and receptor recognition by MV H. First, the proteins were co-immunoprecipitated from the surface of Chinese hamster ovary (CHO) cells, which do not express either MV receptor, CD46 or CD150. Similar levels of MV H can be co-immunoprecipitated with F from the surfaces of parental CHO cells and stably transfected cells that express, human CD46 (CHO-CD46), indicating that binding to CD46 is not the trigger for the H-F interaction. Second, MV H proteins, carrying mutations that dramatically reduce CD46 binding, were shown to co-immunoprecipitate efficiently with F from the surface of HeLa cells. Significantly, these results indicate that MV H and F interact in the absence of, and thus prior to, receptor binding. This is in direct contrast to the NDV HN-F cell surface interaction, which is thought to be triggered by receptor binding. Identification of the domains of the para myxovirus attachment and fusion proteins that mediate membrane fusion activities is an essential part of understanding the mechanism of fusion. As a result of the H-F interaction prior to receptor binding, MV H attachment to its cellular receptor must result in conformational changes that trigger activation of the F protein. Site-directed mutagenesis analyses of two regions of MV H indicate that a HR domain in the stalk of the attachment protein is essential to the ability of H to activate F. However, either it is not the only region of H that interacts with F or it is indirectly involved in F activation because mutations in the HR do not disrupt MV H-F complex formation at the cell surface. Additionally, the functional interaction between MV H and F may be mediated, at least in part, by Loop 1 of the amino terminus of the C-rich region of the fusion protein. However, the exact role of this region of the F protein in fusion promotion remains to be determined. Importantly, the cell surface interaction between MV H and F proteins appears to be mediated by more that one region of each protein. In contrast to NDV, in no case has a definitive link between any single amino acid difference in MV H or F and an inability to form the cell surface H-F complex been established. In conclusion, the data presented in this dissertation support a model of measles membrane fusion in which the Hand F proteins form a complex prior to receptor recognition. This complex may hold F in its meta-stable pre-fusion state until binding of H to receptors at the cell surface triggers dissociation of the complex, releasing F to assume its fusogenic form. Importantly, these data also indicate that, although paramyxoviruses may all use the same general process. for promotion of membrane fusion, the mechanism may vary in multiple aspects. A more complete understanding of the means by which measles promotes membrane fusion may direct the development of specific strategies aimed at interfering with the early stages of infection.

Lectinas são proteínas que possuem pelo menos um domínio não catalítico que se liga reversível e especificamente a um monossacarídeo ou oligossacarídeo. A capacidade de ligação a diferentes tipos de açúcares torna essas moléculas ferramentas úteis no estudo de diversos processos celulares específicos. Embora as lectinas de plantas sejam amplamente estudadas, aquelas referentes à família Apocynaceae ainda são pouco exploradas. Resultados prévios obtidos pelo nosso grupo de pesquisa mostraram que extratos brutos de súber do caule da apocinácea Tabernaemontana hystrix Steud apresenta atividade hemaglutinante. Além de aglutinar eritrócitos do sistema ABO, a putativa aglutinina foi capaz de estimular a síntese de RNAm de IL-6 e TGF- beta em células esplênicas de camundongos. À vista disso, no presente projeto tivemos como objetivo identificar, caracterizar bioquimicamente e avaliar o possível potencial imunoestimulador da aglutinina de T. hystrix. Os extratos de T. hystrix obtidos por meio da farinha de raspas do súber apresentaram atividade hemaglutinante, o que não foi observado no extrato do caule destituído de súber e no extraído das folhas. Para comprovar que se tratava da atividade observada anteriormente, obtivemos a inibição da hemaglutinação com a glicoproteína fetuína, mas não houve inibição por monossacarídeos. Foi determinado um protocolo de isolamento da hemaglutinina com precipitação do extrato do súber com sulfato de amônio, cuja atividade foi recuperada no material precipitado na faixa de 30 a 60% de saturação, seguido de cromatografias sequenciais por (1) interação hidrofóbica (HiTrap Octyl), (2) troca catiônica (HiTrap SP), (3) fase reversa (EC Nucleosil C18) e (4) afinidade (Blue Sepharose). Nessas colunas a atividade foi recuperada do (1) material não retido e dos eluatos (2 e 4) com 1M e 0,5M de NaCl, respectivamente, e (3) 83% de acetonitrila. Esse protocolo produziu uma preparação homogênea contendo um peptídeo cuja análise eletrofóretica revelou massa molecular (MM) aproximada de 3kDa e concentração hemaglutinante mínima de 50g/mL. A fim de determinar se esse peptídeo formava estrutura quaternária (dímeros, tetrâmetros, etc.), característica da maioria das lectinas de plantas, submeteu-se a preparação a uma eletroforese em gel nativo (PAGE), não sendo observadas mudanças na MM do peptídeo e nem a presença de outras moléculas com MM maiores que pudessem estar associadas a ele, o que sugere que a aglutinina de T. hystrix (denominado aqui de Tabhys) é um peptídeo de MM aproximada de 3kDa. O fato da heveína, um dos peptídeos lectínicos com atividade antifúngica mais estudado, ter especificidade por quitina nos motivou a tentar o isolamento do peptídeo em coluna desse polissacarídeo. Observou-se atividade hemaglutinante e presença de peptídeo com MM de 3kDa no material eluído com Ácido acético a 0,1M da coluna de quitina. Curiosamente, nenhuma de nossas preparações foram capazes de inibir o crescimento do fungo Trichophyton rubrum. O peptídeo purificado foi testado quanto a sua capacidade em induzir a proliferação celular e a produção de citocinas em células esplênicas murinas. Os resultados dos ensaios de RT-PCR em tempo real e citometria de fluxo demonstraram que o a aglutinina de T. hystrix não foi capaz de estimular a proliferação de linfócitos, entretanto, induziu o aumento de mensagem para a citocina TGF-beta, cujo pico de produção ocorreu em célula estimuladas com 37ng/mL. Neste estudo, relatamos a presença de um peptídeo no extrato de T. hystrix com atividade hemaglutinante, o que é relativamente raro e novo. Devido a isso, este estudo pode proporcionar novas perspectivas e paradigmas nos estudos das lectinas a nível molecular e estrutural. / Lectins are proteins that have at least one non-catalytic domain that binds specifically and reversibly to a monosaccharide or oligosaccharide. This ability to bind to different types of sugars makes these molecules useful tools in the study of various specific cellular processes. Although the plant lectins are widely studied, those belong to Apocynaceae family are still little explored. Previous results obtained by our research group showed that bark crude extracts from Tabernaemontana hystrix Steud (Apocynaceae) had hemagglutination activity. Besides to agglutinate erythrocytes from ABO blood group system, the putative agglutinin induced the synthesis of IL-6 and TGF-beta mRNA in mouse spleen cells. Here we aim to identify, characterize biochemically and evaluate the possible immunostimulatory potential of T. hystrix agglutinin. The haemagglutination activity was obtained from crude extracts of bark flour, but not of flours of stems without bark and leaves. The activity of the bark extract was similar to that from the previous study, since the haemagglutination was inhibited by the glycoprotein fetuin, but not by monosaccharides. An isolation protocol was determined by using ammonium sulfate precipitation, with haemagglutination activity recovered in the range of 30-60% of saturation, and sequential chromatography procedures: (1) hydrophobic interaction (HiTrap Octyl), (2) cation-exchange (HiTrap SP), (3) reverse phase (EC Nucleosil) and (4) affinity (BlueSepharose) chromatography. From these columns the activity was recovered in the (1) unbound material, and eluates (2 and 4) with 1M and 0,5M of NaCl, respectively, and (3) 83% acetonitrile. On the basis of electrophoresis analysis, the protocol produced a preparation comprised of only band corresponding a peptide with molecular weight (MW) of about 3-kDa, with minimum haemagglutination concentration of 50g/ml. To determine if this molecule arrangement had a quaternary structure arrangement, a feature of most known lectins, we submitted the preparation to a native electrophoresis. Because there was neither change in migration pattern nor presence of molecules of higher molecular mass, we suggested that T. hystrix peptide (Tabhys) is a peptide with MW of about 3-kDa. Since hevein, which is a most studied lectin-like peptide with antifungal activity, binds specifically to chitin, we performed an affinity chromatography in the chitin column with bark extract. We observed haemagglutination activity and the presence of peptide with MW of 3-kDa in the material bound to column and eluted with 0,1M acetic acid. Curiously, this peptide was not able to inhibit the growth of the fungus Trichophyton rubrum. Thereafter, when the purified peptide was used to stimulate murine spleen cells, we detected the expression of TGF-beta message, with a peak production obtained in cell stimulated with 37 ng/mL of Tabhys. In the current study, we isolated a peptide from crude extract of T. hystrix bark with haemagglutination activity, providing new perspectives in molecular and structural researches of peptide lectins.

Antiviral drug resistance is a major problem in the treatment of viral infections, including influenza and hepatitis C virus (HCV). Influenza neuraminidase (NA) is a viral sialidase on the surface of the influenza virion and a primary antiviral target in influenza. Two subtypes of NA predominate in humans, N1 and N2, but different patterns of drug resistance have emerged in each subtype. To provide a framework for understanding the structural basis of subtype specific drug resistance mutations in NA, we used molecular dynamics simulations to define dynamic substrate envelopes for NA to determine how different patterns of drug resistance have emerged in N1 and N2 NA. Furthermore, we used the substrate envelope to analyze HCV NS3/4A protease inhibitors in clinical development. In addition, influenza hemagglutinin (HA) is a primary target of neutralizing antibodies against influenza. Novel broadly neutralizing antibodies (BnAbs) against the stem region of HA have been described and inhibit several influenza viral subtypes, but antibody neutralization escape mutations have emerged. We identified potential escape mutations in broadly neutralizing antibody F10 that may impact protein dynamics in HA that are critical for function. We also solved crystal structures of antibody fragments that are important for understanding the structural basis of antibody binding for influenza BnAbs. These studies can inform the design of improved therapeutic strategies against viruses by incorporating an understanding of structural elements that are critical for function, such as substrate processing and protein dynamics, into the development of novel therapeutics that are robust against resistance.