Made available in DSpace on 2014-06-11T19:22:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-01-27Bitstream added on 2014-06-13T20:29:20Z : No. of bitstreams: 1 cravo_hlp_me_sjrp.pdf: 853824 bytes, checksum: 43cbe13f547f1fdf2dfdfbf56e696a9a (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O Vírus Sincicial Respiratório Humano (hRSV) foi identificado em 1957 e mesmo após vários anos de investigação, nenhuma vacina foi desenvolvida. Acredita-se que a chave de inibição da ação viral são suas glicoproteínas de membrana, em especial a proteína de fusão (F), que com auxílio da proteína de ligação (G), é responsável pela instalação do hRSV na célula hospedeira. Há evidências experimentais de que compostos como flavonóides e glicosaminoglicanos podem diminuir a infecção viral, sendo então a proteína F um bom alvo para a ação destes compostos. O presente estudo utilizou de ferramentas de bioinformática para verificar as possíveis regiões de interação da proteína F com a Heparina Sulfatada e Flavonóides. Os programas de bioinformática foram utilizados para: modelagem dos compostos, caracterização e previsão da estrutura secundária da proteína, modelagem da estrutura terciária e docking molecular entre o modelo da proteína F e as estruturas tridimensionais dos Flavonóides e da Heparina Sulfatada. Modelos válidos foram obtidos para as estruturas tridimensionais dos flavonóides e para o modelo completo da proteína F. As características da proteína incluem um alto nível de conservação na seqüência de aminoácidos e, especialmente, em seus sítios de ligação. O docking da proteína com a Heparina, e o virtual screening da biblioteca de Flavonóides e a estrutura da proteína, resultaram em sítios de interação com grande potencial de inibição, uma vez que concordam com evidências experimentais descritos na literatura. A Heparina liga-se ao sítio de clivagem II, importante região para obtenção da atividade de fusão da proteína. Os Flavonóides podem se ligar a região hidrofóbica que desestabiliza... / Human Respiratory Syncytial Virus (hRSV) was identified in 1957 and even after several years of research, no vaccine has been developed yet. It is believed that the key to the inhibition of viral action is its membrane glycoproteins, including the Fusion Protein (F), responsible for the installation of the hRSV in the host cell. There are evidences that compounds such as flavonoids and glycosaminoglycans can decrease the viral infection, and F protein can be a good target for the action of these compounds. The present study checked the possible sites of interaction between F protein and heparin and flavonoids, using computational tools. Bioinformatics programs were used for: modeling compounds, characterization and prediction of protein secondary structure, tertiary structure modeling and the docking between the protein model and the structures of flavonoids and sulfated heparin. Valid models were obtained for flavonoids structures and the complete model of F protein. The characteristics of the protein include a high level of conservation in amino acid sequence and especially in its binding sites. The heparin docking and virtual screening of flavonoids resulted in interaction sites with great potential for inhibition, since they agree with other studies and experimental evidence of F protein inhibition. This study shows that compounds such as sulfated heparin and flavonoids interact in important sites of F protein. Heparin binds to the cleavage site II and flavonoids can bind to the hydrophobic site that destabilizes the formation of the six-helix-bundle region. Both regions are important for conformational changes that F protein undergoes to get its fusion activity. Docking showed that molecular interactions are likely to occur and selected the best candidates for a possible inhibitor. These evidences... (Complete abstract click electronic access below)

Philosophiae Doctor - PhD / Hepatitis C was first recognized as a transfusion-associated liver disease not caused by hepatitis A or hepatitis B virus after serological tests were developed to screen for their presence in the blood. The infectious agent was finally identified with the cloning of the cDNA of hepatitis C virus (HCV) using random polymerase chain reaction (PCR) screening of nucleic acids extracted from plasma of a large pool of chimpanzee infected with non-A non-B hepatitis. NS5B, a membrane-associated RNA-dependent RNA polymerase essential in the replication of HCV, initiates the synthesis of a complementary negative-strand RNA from the genomic positive-strand RNA so that more positive-strand HCV RNA can then be generated from the newly synthesised negative-strand template. The crystal structure of NS5B presented typical fingers, palm and thumb sub-domains encircling the GDD active site, which is also seen in other RNA-dependent RNA polymerases, and is similar to the structure of reverse transcriptase of HIV-1 and murine Moloney leukaemia virus. The last 21 amino acids in the C-terminus of NS5B anchor the protein to the endoplasmic reticulum (ER)-derived membranous web. NS5B has been shown to interact with the core, NS3/NS4A, NS4B and NS5A proteins, either directly or indirectly. Numerous interactions with cellular proteins have also been reported. These proteins are mainly associated with genome replication, vesicular transport, protein kinase C-related kinase 2, P68 (DDX5), α-actinin, nucleolin, human eukaryotic initiation factor 4AII, and human VAMP-associated protein. Previous studies have confirmed that NS5B binds to full-length DDX5. By constructing deletion mutants of DDX5, we proceeded to characterize this interaction between DDX5 and HCV NS5B. We report here the identification of two exclusive HCV NS5B binding sites in DDX5, one in the N-terminal region of amino acids 1 to 384 and the other in the C-terminal region of amino acids 387 to 614. Proteins spanning different regions of DDX5 were expressed and purified for crystallization trials. The N-terminal region of DDX5 from amino acids 1 to 305 which contains the conserved domain I of the DEAD-box helicase was also cloned and expressed in Escherichia coli. The cloning, expression, purification and crystallization conditions are presented in this work. Subsequently, the crystal structure of DDX5 1-305 was solved and the high resolution three-dimensional structure shows that in front of domain I is the highly variable and disordered N terminal region (NTR) of which amino acids 51-78 is observable, but whose function is unknown. This region forms an extensive loop and supplements the core with an additional α-helix. Co-immunoprecipitation experiments demonstrated that the NTR of DDX5 1-305 auto-inhibit its interaction with NS5B. Interestingly, the α-helix in NTR is essential for this auto-inhibition and seems to mediate the interaction between the highly flexible 1-60 residues in NTR and NS5B binding site in DDX5 1-305, presumably located within residues 79-305. Furthermore, co-immunoprecipitation experiments revealed that DDX5 can also interact with other HCV proteins, besides NS5B.

Liver flukes of the family Fasciolidae are parasites of mammals including human. Fascioloides magna and Fasciola hepatica are considered as a veterinary and medically important species occurring also in the Czech Republic. Fascioloides magna and F. hepatica infect wide spectrum of wild and domestic ruminants and in case of F. hepatica human can be also infected. Both flukes are responsible for damage of liver tissue and/or bile-ducts of their definitive hosts causing weight lose, anemia, reduced productivity and in specific cases the death of the host. Effective diagnosis plays the key role in control of F. hepatica and F. magna infections. Current diagnostics is predominantly based on serodiagnostic methods using specific antigens e.g. from excretory-secretory products (ESPs). Due to heterogenity of ESPs, such diagnostic markers can lack the specificity and also the reproducibility of the method is poor. Particular proteins of ESPs are often used in diagnostics of fasciolid flukes. Such approach requires biological material and laboratory procedures associated with identification, purification and antigenicity testing of selected proteins. Recent development of parallel sequencing technologies results in huge amount of genomic, transcriptomic and proteomic data, which are publicly available. Such...

One of the key components of the innate immune system are natural killer (NK) cells. The task of these cells is to induce apoptosis in target cells (e.g., cancer or virally infected cells). The target cells are identified by their interaction with surface receptors of the NK cells. On the surface of the NK cells, there are activating and inhibiting receptors. One of the activating receptors is the natural cytotoxicity receptor NKp46. Several ligands of this receptor have been identified, one of them being the epithelial adhesin Epa1 of yeast Candida glabrata. The invasive candidiasis caused by this yeast is a feared complication for patients with haematological diseases. The use of the NK cells in immunotherapy includes bispecific fusion proteins which can bind to the NK receptor with one part and to tumour antigen with the other part. This work focuses on recombinant preparation of the NKp46 protein. To facilitate a study of the effects of O-glycosylation on the binding of the ligands, a mutation of the glycosylation site NKp46 T225A was prepared. A stably transfected HEK293S GnTI- and HEK293T cells had been prepared and these proteins were then extracellularly secreted. The Epa1 protein had been produced in E. coli bacterial expression system and purified. The binding ability of the Epa1 protein...

Indiana University-Purdue University Indianapolis (IUPUI) / Animals depend on inheriting non-genetic information early in life to grow and develop naturally. This inherited, non-genetic information was previously thought to be limited to DNA modifications and DNA binding proteins. But recent studies have expanded our understanding of inheritance to include RNA and RNA binding proteins. We currently lack methods to identify and enrich for RNA binding proteins that might be involved in providing non-genetic information from mother to daughter cells. Others have developed a method using modified enzyme tags to pulse-label proteins with small molecule fluorescent ligands and follow these proteins as they are inherited by cells. Here I characterized and tested the application of a fluorescent small molecule targeting antibody to enrich for these labeled proteins. I first tested the ability of this antibody to bind to fluorescent ligand-labeled enzymes. I determined that the antibody can efficiently bind to at least one of the labeled enzymes. Second, I determined crystallization conditions for the ligand binding antibody fragment. This thesis sets the stage for structure determination and to test whether this antibody can work in vivo to enrich for RNA binding proteins involved in the delivery of non-genetic information to cells.

Rotavirus, the major cause of life-threatening infantile gastroenteritis, is a member of the Reoviridae family and is considered to be the single most important cause of virus-based severe diarrheal illness in infants and young children particularly 6 months to 2 years of age in industrialized and developing countries. Infection in infants and young children is often accompanied by severe life threatening diarrhea, most commonly following primary infection. Diarrhea is the major cause of death among children around the world. Responsible for 4 to 6 million deaths per year according to the World Health Organization (WHO), diarrhea is especially dangerous for infants and young children. Globally, it is estimated that 1.4 billion episodes of diarrhea occur in children less than five years of age annually. In the United States alone, rotavirus causes more than 3 million cases of childhood diarrhea each year, leading to an estimated 55,000 to 100,000 hospitalizations and 20 to 100 deaths. And is a major cause of mortality for children in developing countries with approximately one million deaths annually. Rotaviruses belong to the family Reoviridae and are spherical 70-nm particles. The virus genome contains 11 segments of double-stranded RNA, each encoding a viral capsid or nonstructural protein. The identification of a rotavirus nonstructural protein gene (NSP4) encoding a peptide, which functions both as a viral enterotoxin and as a factor involved in the acquisition of host cell membrane during virus budding from cells, provides a new approach for mucosal immunization. Protein expression through chloroplast transformation system offers a number of advantages like high level of transgene expression, transgene containment via maternal inheritance, lack of gene silencing and position effect due to site specific gene integration and also the possibility of multi gene engineering in single transformation event. It is also an environmentally friendly approach due to effective gene containment and lack of transgene expression in pollen. To achieve an enhanced immune response to rotavirus infection, a fusion gene encoding the cholera toxin B subunit linked to rotavirus enterotoxin 90 aa protein (CTB-NSP490) was introduced into transgenic chloroplast and was transformed into chloroplast genome of Nicotiana tabacum by homologous recombination. The chloroplast integration of CTB-NSP4(90) fusion gene was confirmed in transgenic tobacco plants by PCR analysis. Southern blot analysis further confirmed site specific gene integration and homoplasmy. Immunoblot analysis of transformed chloroplast confirmed the expression of CTBNSP490 fusion protein both in monomeric and pentameric forms that retained the binding affinity to the enterocytes GM1 ganglioside receptor. Expression levels of CTB-NSP4 protein was quantified by GM1 ganglioside binding ELISA assay; mature leaves expressed CTB-NSP4 fusion protein to upto 2.45 % in total soluble protein, 100-400 fold higher than nuclear expression which was only 0.006%-0.026%. Antibody titration and virus challenge experiments will be performed in mice at Loma Linda University to evaluate the antigenic and protective properties of the chloroplast derived CTB-NSP4 fusion protein.

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Dioxygenase-cytochrome P450 (DOX-CYP) fusion enzymes are present in diverse human and plant pathogenic fungi. They oxygenate fatty acids to lipid mediators which have regula­tory functions in fungal development and toxin production. These enzymes catalyze the for­mation of fatty acid hy­droperoxides which are subsequently converted by the P450 activities or reduced to the corresponding alcohols. The N-terminal DOX domains show catalytic and structural homology to mammalian cyclooxygenases, which belong to the most thoroughly studied human enzymes. 7,8-Linoleate diol synthase (LDS) of the plant pathogenic fungus Gaeumannomyces graminis was the first characterized member of the DOX-CYP fusion enzyme family. It catalyzes the conversion of linoleic acid to 8R-hydroperoxylinoleic acid (HPODE) and subse­quently to 7S,8S-dihy­droxylinoleic acid by its DOX and P450 domains, respectively. By now, several enzymes with homology to 7,8-LDS have been identified in im­portant fungi, e.g., psi fac­tor-producing oxygenase (ppo)A, ppoB, and ppoC, of Aspergillus nidulans and A. fumigatus. By cloning and recombinant expression, ppoA of A. fumigatus was identi­fied as 5,8-LDS. Partial expression of the 8R-DOX domains of 5,8-LDS of A. fumigatus and 7,8-LDS of G. graminis yielded active protein which demonstrates that the DOX activities of LDS are independent of their P450 domains. The latter domains were shown to contain a conserved motif with catalytically important amide residues. As judged by site-directed mutagene­sis studies, 5,8- and 7,8-LDS seem to facilitate heterolytic cleavage of the oxygen-oxygen bond of 8R-HPODE by aid of a glutamine and an asparagine residue, respectively. Cloning and expression of putative DOX-CYP fusion proteins of A. terreus and Fusarium oxysporum led to the discovery of novel enzyme activities, e.g., linoleate 9S-DOX and two allene oxide synthases (AOS), specific for 9R- and 9S-HPODE, respectively. The fungal AOS are present in the P450 domains of two DOX-CYP fusion enzymes and show higher se­quence homology to LDS than to plant AOS and constitute therefore a novel class of AOS. In summary, this thesis describes the discovery of novel fatty acid oxy­genases of human and plant pathogenic fungi and the characterization of their reaction mechanisms.