O Vírus Respiratório Sincicial Humano (HRSV) é um dos patógenos mais importantes do trato respiratório. Analisamos as interações das proteínas virais nucleoproteína (N), fosfoproteína (P) e matriz (M) em células HEK-293T. N interage com as proteínas celulares Hsp70, PRMT5 e WDR77; P com Hsp70 e Tropomiosina; e M com Nucleofosmina e Tropomiosina. Cada gene celular foi co-expresso em bactérias com um gene viral possibilitando a co-precipitação das proteínas. Analisamos a interação entre Hsp70 e N ou P, confirmando sua ocorrência em bactérias. Com um conjunto de proteínas mutantes, definimos que as interações são através dos amino terminais de N e P, ou do carboxi terminal de P, e do domínio amino terminal de Hsp70. Superexpressão de Hsp70 por transfecção provocou efeito de estímulo sobre a replicação de HRSV. Imunizações em camundongos com vacinas de DNA para N e P mostraram a indução de resposta celular e humoral. Ensaios de desafio resultaram em redução da carga viral após imunização com N, indicando potencial para sua aplicação em formulação vacinal. / Human respiratory syncytial virus (HRSV) is one of the most important pathogens of the respiratory tract. We analyzed the interactions of viral nucleoprotein (N), phosphoprotein (P) and matrix (M) in HEK-293T. N interacts with the cellular proteins Hsp70, PRMT5 and WDR77; P interacts with Hsp70 and Tropomyosin; and M with Nucleophosmin and Tropomyosin. Each cellular gene was co-expressed with a viral gene in bacteria allowing co-precipitation of proteins. Hsp70 co-expression with N or P proteins confirmed that these interactions also occur in bacteria. Using a set of mutants we found that the N and P amino terminus, P carboxy terminus, and Hsp70 amino terminal domain participate in the interactions. The overexpression of Hsp70 by transfection resulted in stimulation of HRSV replication. Mice immunization with N and P showed that DNA vaccines were capable of inducing humoral and cellular response. In challenge assays it was possible to detect significant virus titer reduction in animals immunized with N, indicating its potential for a vaccine formulation.

Orientador: Maria de Fatima Sonati / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-04T14:49:11Z (GMT). No. of bitstreams: 1 Zaccariotto_TaniaRegina_M.pdf: 3102786 bytes, checksum: 18d48dc5af4798d0abf2b5c3df1208d8 (MD5) Previous issue date: 2005 / Resumo: A haptoglobina (Hp) humana é uma glicoproteína plasmática tetramérica (a2b2), sintetizada no fígado, com propriedades antioxidativas e imunomodulatórias. É codificada por dois alelos codominantes, HP1 e HP2, de cuja combinação resultam três principais genótipos/fenótipos (Hp1-1, Hp2-1 e Hp2-2), os quais apresentam distintas eficiências em suas atividades. Vários autores têm investigado a influência do polimorfismo da Hp na suscetibilidade e evolução clínica de diferentes doenças humanas, incluindo a infecção pelo HIV. Os resultados, entretanto, são controversos, dependendo do aspecto investigado e da origem da população estudada. Foram aqui analisados 387 pacientes brasileiros HIV-1+ (classificados clinicamente em A=177, B=51 e C=159, segundo classificação do Center for Disease Control-CDC-USA) e 142 controles normais, soronegativos, quanto à distribuição dos genótipos de Hp e à influência desse polimorfismo no status de ferro (ferro sérico, ferritina, transferrina e saturação da transferrina) e nas concentrações séricas das proteínas de fase aguda - Hp, proteína C reativa, fibrinogênio e albumina. Entre os pacientes, as contagens de linfócitos T-CD4+ e as determinações de carga viral também foram comparadas. Os resultados não revelaram diferenças significativas na distribuição dos genótipos, nem entre pacientes e controles, nem entre os subgrupos clínicos da doença, embora, entre os pacientes, as proporções genotípicas não tenham correspondido ao equilíbrio de Hardy-Weinberg (p<0,05). Com relação aos parâmetros bioquímicos analisados, a única diferença estatisticamente significante foi quanto as concentrações séricas de Hp nos indivíduos com genótipo Hp2-2, inferiores aos demais, tanto nos pacientes quanto nos controles (p<0,001), resultado também encontrado em estudos prévios em outras populações. Nenhuma outra diferença ou correlação relevante pode ser detectada, sugerindo que, na população aqui investigada, o polimorfismo da Hp não exerce influência importante. Um quarto fenótipo, denominado ¿HP0¿, correspondente à Hp reduzida ou ausente no plasma, foi encontrado em 28% dos pacientes e 12% dos controles normais, mas as bases moleculares não foram esclarecidas, sendo que em parte dos pacientes essa ¿hipohaptoglobinemia¿ pode ser secundária a episódios de hemólise ou disfunção hepática / Abstract: Haptoglobin (Hp) is a plasma protein with antioxidant and immunomodulatory properties. Three main genotypes/phenotypes (Hp1-1, Hp2-1, Hp2-2) show distinctive efficiencies in their activities and have been related to susceptibility and/or outcome in different diseases, including HIV-infection. We compared the Hp genotype distribution between 387 Brazilian HIV-1-seropositive patients, sub-classified in A, B and C according to the Center for Disease Control-USA, and 142 healthy controls. We also investigated the influence of this polymorphism on the iron status (serum iron, ferritin, transferrin, transferrin saturation), the acute-phase proteins Hp, C-reative protein, fibrinogen and albumin, the viral loads and the CD4+ T-lymphocyte counts. There was no significant difference in the Hp genotypes distribution, neither between patients and controls nor among the clinical subgroups, although in the patient group the genotype distribution did not follow the Hardy-Weinberg equilibrium (p<0,05). Apart the lowest Hp concentrations showed by Hp2-2 individuals (patients and controls), no other significant difference or relevant correlation was observed, neither in the genotype frequency distribution nor in the biochemical parameters analyzed here, suggesting that the Hp polymorphism does not exert an important influence on these variables in the population investigated. The HP0 phenotype, correspondent to absent or reduced Hp in the plasma, was detected in 28% patients and 12% controls, but the molecular basis was not determined and, in part of the patients, this hypohaptoglobinemia can be secondary to hemolysis or liver dysfunction / Mestrado / Ciencias Biomedicas / Mestre em Ciências Médicas

Trabajo de Investigación Requisito para optar al Título de Cirujano Dentista / Introducción: Lesiones bucomáxilofaciales conocidas se asocian con estados de inmunosupresión en pacientes VIH/SIDA. La presencia de estas lesiones puede ser el primer signo clínico de la infección. Determinar la asociación entre las lesiones orales, diferentes niveles de inmunosupresión y carga viral es de gran ayuda para el diagnóstico prematuro, control de la enfermedad y evaluación de la terapia. Materiales y Método: Estudio observacional analítico de corte transversal en adultos diagnosticados con VIH/SIDA atendidos en el Hospital San Juan de Dios, entre Julio 2012 y Julio 2015. Se realizó examen intraoral y diagnóstico de las lesiones orales de acuerdo al criterio clínico de EC-Clearinghouse. El grado de inmunosupresión se basó en el recuento de linfocitos TCD4+ y carga viral más cercana a la fecha del ingreso al estudio. Se categorizó a los pacientes de acuerdo al grado de inmunosupresión en: Ausente (>500 cél/mm 3 ), moderada (201-499 cél/mm 3 ) y severa (<200 cél/mm 3 ) y carga viral (CV) en: indetectable (<500 copias/ml), moderada (501-9.999 copias/ml) y alta (>10.000 copias/ml). El análisis de asociación de variables se realizó mediante el test de Chi 2 de Pearson y exacta de Fisher y luego se determinó la magnitud del efecto de las variables mediante razones de prevalencia (RP) de Poisson Robusta. Resultados: 79 pacientes cumplieron con los criterios de inclusión. El 29,11% de los pacientes presentó lesiones bucomáxilofaciales asociadas a VIH con un recuento promedio de linfocitos TCD4+ de 333,91 cél/mm 3 y el 70,89% restante presentó un recuento promedio de 383,67 cél/mm 3 . En cuanto a la carga viral, el recuento promedio fue de 38.746 copias/ml en pacientes sin lesión y 21.372 copias/ml con lesión. Se observó que es 1.77 veces más probable presentar una lesión asociada a VIH/SIDA con recuentos de TCD4+ menores a 200 cél/mm (IC 95% 0,77-4,11). En pacientes con una carga viral alta no se observó una mayor prevalencia de lesiones estadísticamente significativa (p= 0,749). Conclusiones: Existe una relación estadísticamente significativa entre el recuento de Linfocitos TCD4+ y la presencia de manifestaciones bucomáxilofaciales fuertemente asociadas a la infección por VIH/SIDA., siendo más prevalentes en pacientes con inmunosupresión severa. En relación a la carga viral y la presencia de lesiones no existe significancia estadística. / Adscrito a proyecto PRIO-ODO 14/003

This experiment compares the implications of two methods of measuring viral particles, specifically Influenza particles in human cell lines in vitro. These strategies include plaque assays and xCELLigence screening. Plaque assays, also known as reduction assays, are plates that are overlaid with semi-solid medium that limits the spread of the virus and shows where each particle is located based on the "plaque" or empty space on the plates where cells have died and been removed. xCELLigence screening is a newer program that checks for "impedance", an artificial number that will measure the cells killed by virus as well as cell to cell interaction on a 96 well plate that utilizes gold microelectrodes. Both methods have variables that can make them useful in certain situations, however, the focus is on how reliable the xCELLigence program is in comparison to more traditional methods of quantifying viral particles.

Rubella virus (RV) is a small, enveloped, positive-stranded RNA virus in the family Togaviridae, and bears striking similarities to the prototype alphaviruses Semliki Forest virus (SFV) and Sindbis virus (SV) in terms of genome organization and structural protein expression strategy. However unlike alphaviruses, RV infection of cultured cells is characterized by relatively long latency periods, slow replication kinetics, limited cytopathology, and the ability to establish a persistent infection in virtually every cell line capable of supporting its growth. RV virions contain three structural proteins C, E2, and El which are derived by post-translational processing of a precursor polyprotein pllO (NF₂-C-E2-El-COOH). Processing and intracellular transport of RV structural proteins has been studied by jn vitro and jn vivo expression of RV cDNAs. It was found that targeting of El and E2 into the endoplasmic reticulum was mediated by two independently functioning signal peptides. Coincident with translocation into the ER, both proteins underwent addition of N-linked glycans and proteolytic processing. C protein did not appear to play a role in the processing of pllO. Expression of the RV structural proteins in COS cells revealed that E2 exited the ER, and was transported through the Golgi to the cell surface in an El-independent manner, although coexpression of El seemed to increase the rate of transport. Conversely, El was retained in a Golgi-like region and was not found on the plasma membrane in the absence of E2. Oligonucleotide-directed mutagenesis of El and E2 cDNAs showed that El andE2 both contain three N-linked glycans respectively. Lack of glycosylation did not appear to affect the intracellular localization of the RV glycoproteins in COS cells. A number of significant differences between RV and SFV/SV structural protein expression strategies were discovered, and their possible relationship to RV virion assembly are discussed. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

The interaction between Murine Cytomegalovirus (MCMV) and the cell cycle has been investigated in synchronized murine cells. Based on the following evidence it was concluded that MCMV replication depends upon the host S phase: (1) the normal latent period of viral growth in exponentially growing 3T3 cells (12 h). was protracted until the host S phase (ca. 20 to 24 h) in synchronized cells infected in early G—l; (2) G-l arrested 3T3 cells failed to support viral replication; (3) entry of the virus was equally efficient in G-l, S and exponential cells; (4) in exponential 3T3 cells viral DNA synthesis began at 10 h post infection, and in synchronized cells it began approximately 16 to 18 h after infection, or early S phase. Therefore, the replication of viral DNA requires host S phase events. Another herpesvirus, Herpes Simplex Virus type-1 (HSV-1) replicated independently of S phase. However, a mutant of HSV-1, deficient in its ability to induce thymidine kinase, demonstrated a dependency upon S phase similar to MCMV. These data indicate a key role of thymidine kinase in the ability of HSV-1 to replicate outside of S phase. However, MCMV induced neither a cellular nor a viral thymidine kinase, and thymidine kinase was not essential for normal viral replication. When G-l arrested 3T3 cells were infected with MCMV, viral DNA synthesis did not initiate and the lytic cycle was reversibly blocked. The non-replicating viral genome remained viable in G-l cells and could be activated at any time by stimulating the cells to enter S phase. The G-l non-permissive system was studied to help ascertain the cell cycle requirements of MCMV. Specifically, two approaches were pursued. In vitro endogenous MCMV DNA synthesis was first studied in G-l, S phase, and exponential 3T3 cells. Under the appropriate conditions, nuclei from infected cells synthesized viral DNA when they had the capacity to dp so in vivo. Nuclei from G—1 phase cells synthesized cell DNA only and not viral DNA. Infected G-l and S phase cells contained a new DNA polymerase which was distinguished from the host enzyme by the high salt requirement for maximal activity. The putative viral DNA polymerase was inhibited by antiserum prepared against infected cell proteins. Therefore, the novel DNA polymerase present in infected G-l and S phase cells was a viral gene product. The second approach involved a comparison of viral transcription in permissive and non-permissive 3T3 cells. The kinetics of hybridization in solution were analyzed by a computer program which evaluated the number of viral RNA classes and the fraction of the viral genome coding for each class. This study revealed the following: (1) in permissive cells by 6 h post infection (early, i.e. before viral DNA synthesis), two classes of viral transcripts were detected, differing by 7 fold in concentration. The abundant class was transcribed from approximately 7% of the viral DNA and the scarce class from approximately 20%. (2) in permissive cells at 24 h post infection (late), abundant and scarce classes (differing by 6 fold in concentration) were transcribed from approximately 10 and 33% of the viral DNA respectively. (3) in non-permissive cells at 6 h, only one class of RNA was present, representing approximately 15% of the viral DNA. (4) in non-permissive cells at 24 h post infection, a single RNA class was observed which was transcribed from approximately 24% of the viral DNA. Summation hybridization experiments indicated that in non-permissive (G-l) cells, only those regions of the DNA which code for early RNA are transcribed. A model has been proposed to describe cell cycle dependent replication of MCMV. It is concluded that MCMV does not replicate in G—l cells due to the absence of specific S phase 'helper-functions' which are required either for the initiation of viral DNA synthesis directly, or for the transcription of viral DNA sequences. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

No description available.

The expression of the adenovirus (Ad) genome is co-ordinately regulated and the various early regions of the genome are known to exert feedback controls on each other. The region E1B is known to encode functions which are required for oncogenic transformation by adenovirus yet its potential regulatory role is poorly understood. In this study the regulatory role(s) of the region E1B was investigated at the level of RNA accumulation. The Northern blot technique using various cloned early regions as probes and RNAs from E1B mutants Ad 12, Ad 2 and Ad 5 were used. The levels of region E1A specific RNAs were found to be aberrant. Thus for Ad 12 cyt 68 and Ad 2 dl 250 the levels were higher than wild type ones at late times whereas for Ad 5 dl 313 hr-6 the levels were consistently lower than Ad 5. This implies that the region E1B normally encodes functions which are involved either directly or indirectly in the efficient accumulation of region E1A specific RNA. Other regions also seemed to be affected in these mutants. In cyt 68, region E1B RNA was higher than wild ones at late time, indicating an auto-regulatory mode of control for this region. The expression of region E3, E4 and LS RNA were all perturbed by the E1B mutation as some of the mutants either accumulated higher or lower than wild type levels of RNA. / Thesis / Master of Science (MS)

Viral marketing allows companies to promote their products and services using very small budgets and the viral ad can be considered the holy grail of digital marketing. In this light, the aim of this study is to discuss the main factors in the existing literature that are considered to boost virality and articulate a summarized Virality Theoretical Model. The empirical study included in this thesis involves the monitoring of an actual ad and an assessment of whether it went viral or not and if it follows the guidelines of the Virality Theoretical Model. The empirical study showed that the ad did not go viral and did not include and display all the attributes proposed by the Model. This further indicates, in regards to theory as well as for marketing executives and advertising practitioners, that virality is a complex phenomenon that depends on several different factors involving both content characteristics and dissemination.

Thesis (Ph. D.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "August 2007" Includes bibliographical references.