published_or_final_version / Microbiology / Master / Master of Philosophy

Thesis (M. Phil.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 61-64). Also available in print.

Influenza virus H1N1 is a huge threat on human health. Influenza occurs with seasonal variations and reaches peak prevalence in winter, with many people killed worldwide every year. In the research of interaction between influenza virus and cells, four major parts were in the range of our consideration, namely the proteins of virus, the proteome of host cell, the method of proteomic and the potencial medicine related with those significant proteins. Hemagglutinin (HA), as an envelope protein, plays an important role in influenza A virus. It was found that HA has a series of isoforms in two dimensional gels in this study. For the investigation of HA, firstly, virus was purified by sucrose density-gradient centrifugation, followed by the separation of virus proteins through electrophoresis method, and then these proteins were digested by different enzymes and analyzed through MALDI-TOF MS and ESI-Q-TOF MS. Database searching was used for identification of sequences. The results of the virus samples digested by different enzymes were compared, and the isoforms of HA were proved to be related with the glycan and their glycosylation sites. A novel strategy of stable-isotope N-phosphorylation labeling was developed for peptide de novo sequencing and protein quantification based on organic phosphorus chemistry. Different from other stable-isotope labeling reagents that needed to be activated in advance for peptide coupling, N-phosphorylation labeling reagents were activated in situ to form labeling intermediates with high activity and selectivity targeting on N-terminus and -amino group of lysine under various reaction conditions. The obtained results showed excellent correlation of the measured ratios to theoretical ratios with errors that ranging from 0.5 to 6.7 % and relative standard deviation of less than 10.6 %, indicating the reproducibility and preciseness of the developed method. The method development based on organic phosphorus chemistry offered a new approach for quantitative proteomics by using novel stable-isotope labeling reagents. A method combining hydrazide chemistry, stable isotope labeling and mass spectrometry analysis was developed and applied to study glycoproteins of H1N1 (A/Purto Rico/8/1934) infected cell line (A549). The result showed that some glycoproteins were significant in influenza virus infected cells. In these glycoproteins, RPC1_HUMAN, RHG25_HUMAN , RPTOR_HUMAN, ARHGC_HUMAN, ROCK1_HUMAN, DOCK3_HUMAN were down-regulated. Protein named TITIN_HUMAN, DESP_HUMAN, PTN13_HUMAN were up-regulated. High dose of N-acetylcysteine (NAC) was recently reported for a therapy of H1N1 influenza pneumonia. NAC was used as a small-molecule organic probe to investigate the protein expression of human lung carcinoma cell line (A549) infected by influenza virus H1N1. The obtained results showed that NAC kept cells away from apoptosis. Virus-infected cells were arrested in G0/G1 phase. The lowest cell population of G0/G1 phase was detected when the cells were treated by 10 mM NAC for one day. Software analysis showed that 4 proteins had close relationship. The results indicated that NAC as a small-molecule probe might effect the proteins expression of A549 cells infected by the H1N1 virus

Orientador: Clarice Weis Arns / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T15:29:47Z (GMT). No. of bitstreams: 1 Rosa_GislaineNonino_M.pdf: 911936 bytes, checksum: 7194af83e7f30e4bcab42d6488202031 (MD5) Previous issue date: 2007 / Resumo: A cinomose canina tem sido relatada como uma das mais importantes doenças infecto contagiosas dos canídeos selvagens e domésticos. É causada por um agente viral denominado vírus da cinomose canina (CDV). Os vírus pertencem à família Paramyxoviridae , gênero Morbillivirus. São vírus envelopados, com genoma composto por uma fita simples de RNA, polaridade negativa, não segmentado. Alto grau de variabilidade genética no gene da hemaglutinina ( H) tem sido encontrada entre estirpes virais recentes e vacinais, e estas variações podem estar relacionadas ao aumento da ocorrência global da cinomose. No presente estudo, amostras biológicas de cães com sintomatologia sugestiva da cinomose foram analisadas geneticamente. Para a detecção de um fragmento de 882 pb do gene H do CDV padronizou-se uma RT-PCR que mostrou-se capaz de amplificar o material genético viral em amostras de urina, líquido céfalo-raquidiano, sistema nervoso central (SNC), baço , pulmão , fígado , rins, linfonodos, bexiga e timo. As amostras positivas de acordo com a RT-PCR foram encaminhadas para o sequenciamento e análise filogenética. Os resultados encontrados sugerem que estas amostras assemelham-se geneticamente aos vírus agrupados nas linhagens Européia e Ásia-1. A estirpe vacinal Lederle, utilizada como padrão, foi agrupada junto a linhagem América-1 onde encontram-se todas as estirpes vacinais , também conhecidas como "Old CDVs" . Os achados deste trabalho apontam para a ocorrência de estirpes geneticamente distintas daquelas utilizadas na produção de vacinas e mais estudos são necessários para a avaliação da eficácia das vacinas disponíveis, propiciando um melhor controle da doença / Abstract: Canine distemper has been reported as one of the most important infectious diseases of wild and domestic canids. It is caused by a viral agent called canine distemper virus (CDV). Viruses belonging to the family Paramyxoviridae, genus Morbillivirus. They are enveloped viruses with genome composed of a single strand of RNA, negative polarity, not segmented. High degree of genetic variability in the gene for hemagglutinin (H) has been found between recent viral strains and vaccination strains, and these variations may be related to increased overall occurrence distemper. In this study, biological samples from dogs with symptoms suggestive of distemper were analyzed genetically. For the detection of a 882 base pairs (bp) fragment of the gene of CDV H was standardized a RT-PCR wich proved to be capable of amplifying the viral genetic material in urine, cerebrospinal fluid, central nervous system (CNS), spleen , lungs, liver, kidneys, lymph nodes, bladder and thymus. Positive samples according to RT-PCR were sent for sequencing and phylogenetic analysis. The results suggest that these samples are similar to viruses genetically clustered lineages in European and Asia-1. The Lederle vaccine strain, used as standard, was grouped with Latin-1 lineages which are all vaccine strains, also known as "old CDVs." The findings of this study point to the occurrence of genetically distinct strains from those used in vaccine production and more studies are needed to assess the efficacy of vaccines available, providing better control of the disease / Mestrado / Microbiologia / Mestra em Genética e Biologia Molecular

Orientador: Valdemiro Carlos Sgarbieri / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Agricola e de Alimentos / Made available in DSpace on 2018-07-17T05:11:04Z (GMT). No. of bitstreams: 1 Junqueira_RobertoGoncalves_M.pdf: 5051016 bytes, checksum: b32d877ce7ee71afa402a015ea108c53 (MD5) Previous issue date: 1979 / Resumo: Foi realizado um estudo inicial de extração, fracionamento e recuperação das proteínas e da atividade hemaglutinante de sementes de feijão Rosinha G2 (Phaseolus vulgaris). O extrato bruto foi obtido em NaCl O,5M, e as proteínas foram fracionadas por diferença de solubilidade em meios de baixa força iônica, após diálise por 36 horas contra água destilada. A atividade mostrou maior tendência em concentrar-se na fração solúvel (ALB), contudo, uma atividade específica hemaglutinante quatro vezes menor foi detectada na fração insolúvel (GLB). Eletroforeticamente, estas frações não se mostraram completamente distintas e quando foram fracionadas pelo tamanho molecular de seus constituintes, por cromatografia em SephadexG-200, a atividade hemaglutinante apresentou-se, aproximadamente, em uma mesma faixa de peso molecular.A hemaglutinina do feijão Rosinha G2 foi purificada por uma sequência de etapas envolvendo cromatografia de afinidade em ConA-Sepharose e cromatografia em gel de Sephadex G-200. A análise eletroforética, em géis de poliacrilamida, a pH 8,4, da preparação obtida na primeira etapa da purificação, componente CII-Af, indicou que a atividade hemaglutinante é devida à presença de, pelo menos três grupos de glicoproteínas. A preparação obtida na última etapa de purificação, componente CII-S, apresentou-se corno urna banda larga e densa, sendo o perfil interpretado corno um fenômeno de agregação entre os constituintes da preparação hemaglutinante final. Quando este material foi analisado por eletrofocalização em gel de polia crilamida revelou que a atividade hemaglutinante é devida ao componente de ponto isoelétrico na faixa de pH de 5,5 - 5,7, sendo contaminado, principalmente, por um componente de ponto isoelétrico em pH = 4,7, que não apresentou atividade hemaglutinante. Foi observado que os eritr5citos bovinos, tripsinizados, quando tratados com soluções de hemaglutinina, mostravam afinidade por superfícies de acetato de celulose. Esta afinidade foi interpretada como interações inespecíficas, devidas a forças de natureza polar, que foram também responsabilizadas pela agregação observada no componente CII-S. A hemaglutinina purificada, CII-S, teve sua composição de aminoácidos determinada, revelando-se deficiente em aminoácidos sulfurados (traços) mas rica em ácido aspártico (11,64%) e serina (7,66%). Revelou-se uma glicoproteína, contendo 8,30% de açúcares neutros e 2,21% de glicosamina. Por cromatografia em gel, a hemaglutinina apresentou um raio molecular de Stokes de 43 A e um peso molecular de 136.000, O raio molecular foi utilizado para o cálculo de sua constante de difusão, 5,0 cm2.s-1. Por ultracentrifugação em gradiente de densidade, foi determinado seu coeficiente de sedimentação, 6,9S e calculou-se seu volume específico parcial, 0,75 ml.g-l, pela equação de Svedberg, para uma diluição infinita. Estes dados foram utilizados para estimar-se o comportamento hidrodinâmico da hemaglutinina, pela determinação de seu quociente friccional, f/f= 1,3. Este valor indica que a proteína pode assumir combinações de hidratação e as simetria dentro dos seguintes limites: 0,9g de água por grama de proteína e a/b = 6, se o modelo de um elips5ide prolato for escolhido. As propriedades químicas e físico-químicas, determinadas para a hemaglutinina do feijão Rosinha G2, mostraram que é semelhante em muitos aspectos fundamentais a outras hemaglutininas purificadas, de diferentes variedades de Phaseolus vulgaris / Abstract: The present is a preliminary study on some physicochemical properties of the bean (Phaseolus vulgaris, variety "Rosinha G2")proteins such as solubility, electrophoretic mobility,molecular weight distributions and hemagglutinating activity. The crude extract was prepared by extracting the flour with a O.5M NaCl solution. The proteins were fractionated using their differences in solubility as the ionic strength was lowered by means of dialysis against distilled water for 36 hours. Most of the hemagglutinating activity was found in the soluble fraction (albumins). Some activity was detected in the insoluble fraction (globulins), which was four times lower than that found in the albumins. However, it was shown electrophoretically that the hemagglutinating components were similar in both fractions. When the protein constituents were separated on the bases of their molecular size, the hemagglutinating activity was found approximately on the same range of molecular weights. The hemagglutinin prepared from this bean was obtained by a sequence of steps involving affinity chromatography on ConA-Sepharose and gel chromatography on Sephadex G-200. The agglutinating preparation obtained by affinity chromatography, component CII-Af, was shown by polyacrylamied-gel electrophoresis to be composed of, at least, three groups of active glycoproteins. Under the same conditions, the preparation obtained at the last step of purification, component CII-B, gave a broad band, which was interpreted to be the result of aggregation. The gel-electrofocusing pattern of this material showed that the hemagglutinating activity was associated with the component separated in the pH range of 5.5-5.7. Other in active components were also present, particularly at pH4.7. It was noted during the hemagglutinating activity tests that the erythrocytes after treatment with hemagglutinin solutions adhered to the cellulose acetate surfaces. This affinity was probably due to nonspecific interactions occuring between groups of polar nature, which could be responsible for the aggregation of the constituents of CII-S.The purified hemagglutinin was characterized as a glycoprotein, containing 8.30% of neutral sugars (as mannose) and 2.12% of hexosamine (asglucosamine). It was shown to be deficient in sulfur ¿ containing amino acids (traces) but rich in aspartic acid(11.64%) and serine (7.66%). The hemagglutinin revealed a Stokes radius of 43R and a molecular weight of 136,000, as obtained by gel chromatography. The molecular radius was used to calculate the approximate value of the diffusion constant, D20,w = 5.0 x 10-7cm2.s-1. The sedimentation coefficient which was determined in a linear sucrose density was 6.9S. These parameters were substituted in the Svedberg equation to determine its partial specific volume, v = 0.75 ml.g-l. The hydrodynamic behavior of the hemagglutinin was estimated by the calculation of its frictional ratio, f/fo = 1.3. This value indicated that the protein had a maximum hydration of 0.9 g of water per gram of protein and a maximum axial ratio, a/b = 6 if a prolate ellipsoid of revolution was chosen as the model. The chemical and physicochemical properties of the hemagglutinin' prepared from the bean "Rosinha G2" were similar in many fundamental aspects to other hemagglutinins obtained from the seeds of different varieties of PhaseoZus vuZgaris / Mestrado / Mestre em Ciência de Alimentos

Influenza is an important respiratory pathogen that infects several million people each year. Currently available flu vaccines have to be updated regularly in order to be effective as the virus changes its composition by antigenic drift and shift. Most of the antibody response generated by these vaccines is strain specific as it is directed against the head domain (HA1) of HA. The HA2 subunit of hemagglutinin is highly conserved and immunogens designed from this subunit are likely to provide protection against multiple strains of the virus. However, expression of HA2 alone in the absence of HA1 resulted in a protein that took up the low pH conformation of HA. Our goal was to design immunogens from HA2 that would fold into the neutral pH form. Sequence analysis of a large number of HA protein sequences was carried out to identify conserved and exposed regions on HA. Several peptide and protein constructs were designed from the stem region of HA. These proteins were expressed in bacteria and purified proteins were used to immunize mice. Immunized mice were challenged with a lethal dose of virus to test for efficacy of the immunogen. Using this approach, stem domain constructs of HA were successfully designed and shown to take up the neutral pH form. These immunogens were also shown to be capable of providing broad range protection. Residues involved in the low pH induced conformational change of HA were identified from studies on HA2 derived peptides.

Bibliography: leaves 160-205. / 205, [128] leaves, [27] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Aims to characterise the region of the chromosome associated with the gene encoding the MFRHA (Mannose-Fucose Resistant Haemagglutinin) / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1994

No description available.

Influenza A and B viruses are major causes of seasonal flu epidemics each year. Hemagglutinin (HA) mediates the binding of virus to host cell and the fusion with host membrane. The crystal of HA in complex with antibody that reveals the mechanism by which antibody recognizes HA may not diffract to high resolution, thereby preventing the accurate interpretation of the structural model. The application of normal mode refinement that aims for improving the structure quality at the low resolution is tested. These studies provide some guidelines for future refinement of HA-antibody complex structures. By comparing the residues constituting the base of the receptor binding site of influenza A and B virus HAs, it is found that they share some similarities, except for a Phe at position 95 of influenza B virus hemagglutinin (BHA) versus Tyr in of influenza A virus hemagglutinin (AHA). The recombinant protein BHA containing the F95Y mutation exhibits the increased receptor binding affinity and specificity. However, recombinant viruses with the Phe95Tyr mutation show lower erythrocyte agglutination titer and decreased binding abilities with different cell lines. The replication of the Phe95Tyr mutant virus in mice is also attenuated. These data suggest that the increased receptor binding ability of HA alone is not advantageous to the pathogenesis of the viruses. The structure of BHA2 (a portion of BHA near the C-terminus) at the post-fusion state has been determined to 2.45 Å resolution. This protein forms a hairpin-like conformation rich in -helices. About 70 residues from the N-terminus is a three-stranded coiled coil, and the remaining of the protein packs in anti-parallel against the groove formed by the central helices. In the post-fusion state of BHA2, the helix converted from the B-loop in pre-fusion state contacts the C-terminal fragment of this protein with more hydrophobic interactions as compared to AHA2. This structure illustrates the distinct stabilization strategy employed by BHA2 to form a post-fusion state that resembles that for AHA2. These studies will further the understanding of BHA with respect to its role in receptor binding ability and fusion.

The optimisation of ligand-macromolecule interactions is fundamental to the design of therapeutic agents. The GRID method is a procedure for determining energetically favourable ligand binding sites on molecules of known structure using an empirical energy potential. In this thesis, it has been extended, tested, and then applied to the design of anti-influenza agents. In the GRID method, the energy of a hydrogen-bond is determined by a function which is dependent on the length of the hydrogen-bond, its orientation at the hydrogen-bond donor and acceptor atoms, and the chemical nature of these atoms. This function has been formulated in order to reproduce experimental observations of hydrogen-bond geometries. The reorientation of hydrogen atoms and lone-pair orbitals on the formation of hydrogen-bonds is calculated analytically. The experimentally observed water structures of crystals of four biological molecules have been used as model systems for testing the GRID method. It has been shown that the location of well-ordered waters can be predicted accurately. The ability of the GRID method to assist in the assignment of water sites during crystallographic refinement has been demonstrated. It has also been shown that waters in the active site of an enzyme may be both stabilized and displaced by a bound substrate. Ligands have been designed to block the highly conserved host cell receptor site of the influenza virus haemagglutinin in order to prevent the attachment of the virus to the host cells. The protein was mapped energetically by program GRID and specific ligand binding sites were identified. Ligands, which exploited these binding sites, were then designed using computer graphics and energy minimization techniques. Some of the designed ligands were peptides and these were synthesised and assayed. Preliminary results indicate that they may possess anti-influenza activity.