The role of highly conserved amino acids within the carboxy-terminal hydrophobic domain of herpes simplex virus I (HSV-I) glycoprotein gB was studied by introducing point mutations using the method of site directed mutagenesis. A segment of this hydrophobic domain of glycoprotein gB contains a nuclear envelope (NE) targeting signal and the effect of these point mutations on targeting to the nuclear envelope was determined. A complementation assay was employed to determine the effect these mutations have on HSV-I infectivity .The point mutations created within the transmembrane domain of glycoprotein gB had no effect on nuclear envelope targeting and localization. However, single point mutations introduced into the first and second hydrophobic domains of glycoprotein gB, G₇₄₃R and F₇₇₀S, affected the targeting and localization of full-length glycoprotein gB at the nuclear envelope. When the transmembrane domain ofHSV-I glycoprotein gB containing the following point mutations A₇₉₀Q, A₇₉₁S, A₇₈₆S, A₇₈₆Y and A₇₉₀S, was introduced into a chimeric protein consisting of the cytoplasmic domain and ectodomain of a plasma membrane protein, vesicular stomatitis virus glycoprotein G, NE targeting and localization were affected. These point mutations may affect the targeting of glycoprotein gB by altering the structure of the targeting signal within the protein. It can be hypothesized that the presence of the cytoplasmic domain. ectodomain domain, and the first and second transmembrane domains within full-length glycoprotein gB can compensate for the effect these point mutations have on nuclear envelope targeting. since the same point mutations had no effect on the targeting · and localization of full-length glycoprotein gB. Complementation assays showed that the glycoprotein gB mutants, A₇₈₆S, A₇₈₆Y, A₇₈₆N, A₇₉₀Q, A₇₉₁S, F₇₇₀S, or G₇₄₃R, were unable to complement a gB-null virus even though these mutant proteins are localized at the nuclear envelope. These proteins may not have been incorporated into the viral capsid due to misfolding or due to the fact that sequences required for interaction with other viral proteins were lost. Another possibility is that the mutant proteins were incorporated into the HSV virion but were not biologically active. / Thesis / Master of Science (MS)

The glycoprotein gB of HSV-1 is involved in viral entry and membrane fusion functions. It is glycosylated, forms homodimers, and is transported to both the inner nuclear membrane and plasma membrane in infected cells. The gB glycoprotein contains a potential membrane anchoring hydrophobic sequence of 69 amino acids, located near the carboxy terminus of the glycoprotein. This sequence is predicted to span the membrane bilayer three times, and can thus be divided into three distinct segments, each of which could span the bilayer. To define both the membrane anchoring sequence and the role of this 69 residue hydrophobic domain, a series of deletion mutants were constructed. These mutants have one, two or all three of the predicted membrane spanning segments deleted, in every combination possible, thereby creating a total of 7 deletion mutants. These mutant constructs were expressed in COS-1 cells using transient expression systems. All the mutant constructs were expressed and glycosylated in a manner similar to the wild type gB glycoprotein. Mutant glycoproteins lacking the third (or most carboxy terminal) predicted spanning segment of this 69 residue hydrophobic domain were found to be secreted from the cells, indicating that this segment may specify the membrane anchoring domain of the gB glycoprotein. Further, the mutant glycoproteins containing this third segment were localized in the nuclear envelope, while mutants lacking this segment were not. All the deletion mutants, except for one, were however defective in intracellular transport and processing of the N-linked oligosaccharides. The only mutant that showed any intracellular transport and processing had only the third segment deleted, but even this mutant was transported and processed much slower than the wild type glycoprotein. The mutant glycoproteins also failed to complement a gB-null virus. These results suggest that the carboxy terminus hydrophobic domain contains essential structural determinants of the gB glycoprotein. / Thesis / Master of Science (MS)

Herpes simplex Virus Type 1 expresses three different classes of genes, immediate early, early, and late, during a lytic infection. Immediate early genes are the first class of genes expressed and they are the only genes expressed independently of de novo viral protein synthesis. This unique characteristic is thought to be the result of the activation of immediate early genes by Vmw65, a protein brought into the cell as a component of the infecting virion. Vmw65 transactivates through the target sequence TAATGARAT (R= purine) which is present at least once in all immediate early transcription regulatory regions. By inserting minimal synthetic promoters, containing the TAATGARAT sequence, into the thymidine kinase locus of the herpes simplex virus type 1 genome I determined that transactivation by Vmw65 is not sufficient to confer on the linked sequences the complete immediate early pattern of gene expression. Furthermore through a transient expression assay I determined that the TAATGARAT sequence element, by itself, when linked to a TATA box is sufficient to act as a target for Vmw65 transactivation. / Thesis / Master of Science (MS)

Infection of a host cell by the Herpes Simplex Virus Type 1 leads to the efficient reprogramming of the cells' synthetic machinery to replicate the viral genome ultimately producing progeny virions. Two proteins introduced upon viral fusion are thought to initiate this effect. The potent transactivator of immediate early genes (VP16) and the mRNA destabilizing virion host shutoff protein (vhs), work in concert with one another to invoke the cascade of viral gene expression, and to destroy pre-existing cellular mRNA. Due to the non-specific nature of vhs induced mRNA degradation, its activity is downregulated at later times during infection to spare virally encoded mRNA. Recent evidence has shown that VP16 is responsible for this vhs downregulation, a process thought to occur by mutual interactions between the two proteins and a potential compartmentalization of vhs within the nucleus (Lam 𝘦𝘵 𝘢𝘭., 1996; Smibert 𝘦𝘵 𝘢𝘭., 1994). Furthermore, such an event is also thought to position vhs so it can be efficiently packaged, a supposition supported by the observation that vhs lacking the ability to bind VP16 is not incorporated into new virions (Read 𝘦𝘵 𝘢𝘭. , 1993). To ascertain if VP16 was indeed capable of relocalizing vhs to the nucleus of a cell in the absence of any other viral factors, we created multiple constructs consisting of various portions of vhs fused in frame to the fluorescent marker protein EGFP. In addition, various truncated forms of VP16 were also fused to EGFP for the purpose of delineating the region of VP16 that is responsible for VP16 and possibly vhs nuclear localization. Co-transfection experiments utilizing EGFP-vhs fusions demonstrated that vhs relocalizes to perinuclear regions in the presence of VP16, an effect absolutely dependent upon its ability to interact with VP16. In addition, deletion mapping of VP16 implicated the region spanning amino acids 335 to 355 as being necessary for this localization, with a stretch of 15 amino acids (330 to 344) appearing to constitute a putative bipartite nuclear localization signal. Interestingly, our observation that the vhs/VP16 complex localizes to a region of the cell thought to ultimately encompass the tegument of new virions gives credence to the notion that this interaction and subsequent localization may indeed function to package vhs into new virions. Furthermore, it is also suggested that vhs may in fact be downregulated at intermediate times during infection through VP16 mediated compartmentalization within the nucleus. For these reasons we propose that the disruption of the vhs/VP16 interaction could severely abrogate the infectivity of HSV and as such could present a novel target for antiviral intervention. / Thesis / Master of Science (MS)

Thèse ou mémoire avec insertion d'articles / Le virus de l'herpès simplex de type 1 (VHS-1) est un virus enveloppé hautement contagieux et répandu dans le monde entier, où 67 % de la population mondiale est infectée. Ce virus cause des maladies incurables bénignes, dont la plus commune est l'herpès labial (ou boutons de fièvre), soit des lésions autour de la bouche. Toutefois, des infections bien plus graves, telles que l'encéphalite, peuvent aussi survenir. Peu d'études ont exploré le VHS-1 dans un contexte agroalimentaire. Bien que le VHS-1 ne soit pas considéré comme un virus d'origine alimentaire, certains travaux ont démontré qu'il pouvait survivre dans l'environnement alimentaire. En plus de persister sur des surfaces couramment rencontrées en industrie alimentaire et sur des aliments fréquemment consommés, la majorité des personnes infectées sont des porteurs asymptomatiques du VHS-1, ce qui contribue grandement à la propagation de celui-ci. D'ailleurs, la main-d'œuvre pratiquant de mauvaises habitudes d'hygiène et le concept de partage entre les individus (partage d'aliments, de boissons, d'ustensiles, etc.) sont des facteurs qui augmentent le risque de transmission du VHS-1. Ce mémoire avait comme objectif d'étudier la persistance du VHS-1 sur des surfaces en contact avec les aliments et sur des aliments prêts-à-manger qui peuvent se partager ; en plus d'évaluer également le potentiel de transfert du VHS-1 d'une surface non poreuse vers un gant. Ce projet de maîtrise a démontré que les surfaces et les aliments pouvaient être des vecteurs potentiels de transmission du VHS-1, puisqu'une récupération du virus infectieux était possible selon les conditions environnementales. En effet, la température, le pH des aliments et potentiellement des composés avec des propriétés antivirales ont affecté la persistance du VHS-1. En conclusion, les résultats de ce mémoire ont pour but de sensibiliser et d'éduquer la population en ce qui a trait aux risques de transmission du VHS-1 entre les personnes. / Herpes simplex virus type 1 (HSV-1) is an enveloped virus that is highly contagious and widespread throughout the world, where 67 % of the world's population is infected. This virus causes mild lifelong diseases, which herpes labialis (or cold sores) is the most common one. However, much more serious infections, such as encephalitis, can also occur. Few studies have explored HSV-1 in an agri-food context. Although HSV-1 is not considered a foodborne virus, some studies have shown that it can survive in the food environment. In addition to persisting on surfaces commonly encountered in the food industry and on frequently consumed foods, most infected people are asymptomatic carriers of HSV-1, which greatly contributes to the spread of this virus. Moreover, the food handlers that have poor hygiene practices and the concept of sharing (sharing food, drinks, utensils, etc.) are factors that may increase HSV-1 transmission. The objective of this thesis was to study the persistence of HSV-1 on surfaces in contact with food and ready-to-eat (RTE) foods that can be shared; and assess the potential for transmission of HSV-1 from a non-porous surface to a glove. This master's project demonstrated that surfaces and food could be potential vectors of HSV-1 since recovery of infectious virus was possible depending on environmental conditions. Indeed, the persistence of HSV-1 was affected by the temperature, the food pH and potentially compounds with antiviral properties. In conclusion, the results of this thesis are intended to raise awareness and educate the population regarding the risks of transmission of HSV-1 between people.

In CBA/NJ mice, splenic natural killer (NK) cell activity varies with stages of estrous. Susceptibility of ICR mice to intravaginal inoculation of herpes simplex virus type 2 (HSV-2) decreases with age. Susceptibility of female ICR and CBA/NJ mice to HSV-2 inoculated intravaginally and intraperitoneally was examined during the estrous cycle. In cycling ICR mice, greatest susceptibility to intravaginal inoculation was observed during diestrous and the least during metestrous. CBA/NJ mice were most susceptible to intravaginal inoculation of HSV-2 during diestrous. ICR mice were ovariectomized to mimic diestrous and found to be highly susceptible to intravaginal inoculation at all virus doses. No difference in susceptibility among phases of the estrous cycle was seen following intraperitoneal inoculation.

Métodos tipo simplex são a base dos principais softwares utilizados na resolução de problemas de otimização linear. A implementação computacional direta destes métodos, assim como são descritos na teoria, leva a resultados indesejáveis na resolução de problemas reais de grande porte. Assim, a utilização de técnicas computacionais adequadas é fundamental para uma implementação eficiente e estável. Neste trabalho, as principais técnicas são discutidas, com enfoque naquelas que buscam proporcionar a estabilidade numérica do método: utilização de tolerâncias, estabilização do teste da razão, mudança de escala e representação da matriz básica. Para este último tópico, são apresentadas duas técnicas, a Forma Produto da Inversa e a Decomposição LU. A análise das abordagens é feita baseando-se na resolução dos problemas da biblioteca Netlib / Simplex-type methods are the basis of the main linear optimization solvers. The straightforward implementation of these methods as they are presented in theory yield unexpected results in solving reallife large-scale problems. Hence, it is essencial to use suitable computational techniques for an efficient and stable implementation. In this thesis, we address the main techniques focusing on those which aim for numerical stability of the method: use of tolerances, stable ratio test, scaling and representation of the basis matrix. For the latter topic, we present two techniques, the Product Form of Inverse and the LU decomposition. The Netlib problems are solved using the approaches addressed and the results are analyzed

Este projeto teve por objetivo a implementação de software para formulação de ração de frangos de corte utilizando Algoritmo Genético (AG). A geração da população inicial foi direcionada, impedindo a geração de indivíduos que possuíam características restritivas. Realizou-se três experimentos, sendo o primeiro para definição do tamanho da população, número de gerações e método de seleção de pais, o segundo para comparar a formulação de ração do AG com a do Simplex e o terceiro para verificar a variabilidade de resultados do AG. O experimento 1 foi realizado em delineamento inteiramente ao acaso, com tratamentos arranjados em esquema fatorial 2 x 5 x 19, sendo os fatores: métodos de seleção de pais (roleta e torneio de três), tamanho de população (200, 360, 500, 1.000 e 1.500 indivíduos) e número de geração (10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 e 1.000), totalizando 190 tratamentos, com 20 repetições resultando em 3.800 observações. A cada observação registrou-se o fitness que foi submetido a análise de variância e quando significativa (P<0,05) aplicou-se o teste de Scott-Knott (5%). No experimento 2 foram formuladas três rações, sendo uma ração pelo método Simplex e duas pelo AG. As rações formuladas com AG utilizaram os parâmetros de tamanho de população, método de seleção de pais e número de gerações definidos no experimento 1. Os resultados obtidos pelo AG proporcionaram rações que apresentam uma diferença média no atendimento das necessidades nutricionais de 0,34% para a ração formulada pelo método roleta e de 0,16% pelo método torneio de três, sendo essas diferenças pequenas e que provavelmente não impactam sobre o desempenho animal e sobre as características de carcaça. A variação de resultados existente no AG, devido a sua característica heurística, foi testada no experimento 3 por intermédio de 100 execuções para cada método de seleção de pais, roleta e torneio de três, utilizando os mesmos parâmetros de tamanho de população e número de gerações das rações formuladas no experimento 2. Os resultados obtidos demonstram baixa dispersão nos dados. Conclui-se que o AG é uma estratégia de otimização eficiente para formulação de rações para frangos de corte, pois aproxima-se do atendimento exato da exigência nutricional, com variação pequena, e com mínimo custo. / The objective of the present project was to implement software for the formulation of broiler chicken feed using a Genetic Algorithm (GA). The generation of the initial population was directed, preventing the production of individuals with restrictive characteristics. A total of three experiments were carried out: the first one to define the population size, number of generations, and the method of parent selection; the second to compare ration formulation using the GA with that of the Simplex method, and the third to verify result variability using the GA. Experiment 1 was performed in a completely randomized design, with arranged treatments in a 2 x 5 x 19 factorial scheme, assessing the following factors: parent selection methods (roulette-wheel selection and tournament selection of three), population size (200, 360, 500, 1 000 and 1 500 individuals), and number of generations (10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 and 1 000 ), totaling 190 treatments, with 20 repetitions each, resulting in 3 800 recordings. At each observation, the registered fitness was submitted to variance analysis, and if significant (P < 0.05), the Scott-Knott test (5%) was applied. In the second experiment, three rations were formulated: one by the Simplex method, and two employing the GA. The feeds formulated with the GA used the parameters of population size, parent selection method, and number of generations, defined in experiment 1. The results obtained by the GA provided feeds that exhibited a mean difference in nutritional requirements of 0.34% for the ration formulated by the roulette-wheel method and 0.16% for the tournament selection of three technique. These differences are considered small and may not impact on animal performance and carcass characteristics. The variation regarding the GA results, given its heuristic attribute, was tested in experiment 3 using 100 repetitions of each method of parent selection, employing the same parameters regarding population size and number of generations of the rations formulated in experiment 2. The obtained results demonstrate low data dispersion. In conclusion, the GA is an efficient optimization strategy for the formulation of broiler chicken feeds, since it approximates the exact fulfillment of the nutritional requirement, with small variation, and with minimum cost.

Made available in DSpace on 2017-07-21T14:32:37Z (GMT). No. of bitstreams: 1 Guilherme Arielo.pdf: 1339457 bytes, checksum: 8bc042278a333bce7c569d21d7195e80 (MD5) Previous issue date: 2013-02-22 / Brazil is one of the most advanced countries, in the technological point of view, regarding the production and use of ethanol as fuel. It is used mostly in the gasoline blend, and is applied to exclusive use of direct fuel. In order to obtain it, different types of biomass can be explored, and sugar cane is both the main source of fuel generation. In the process of ethanol production, many factors may cause interference in expected yields. The aim of this study was to determine the best process conditions for fermentation of aqueous jerivá pulp extract using the response surface methodology and optimization simplex. Design was incomplete factorial 33 with yeast extract, yeast and NH4H2PO4 as independent variables and ethanol yield, the response. Regression analysis showed that the model is predictive and the simplex optimization indicated a formulation containing 0.35 g L-1 yeast extract, 6.33 g L-1 yeast and 0.30 g L-1 and NH4H2PO4, for an optimum yield of 85.40% ethanol. To validate the predictive equation, experiment was repeated in triplicate under optimum conditions, yielding, and an average yield of 87.15% was obtained. According to application of the t test, it was found that there was no significant difference, at 5% level, between the mean value obtained and the value obtained by simplex optimization. / O Brasil é um dos países mais avançados, do ponto de vista tecnológico, na produção e no uso do etanol como combustível. O etanol é usado na sua maior parte na mistura da gasolina, e aplicado como uso exclusivo de combustível direto, no Brasil. Para sua obtenção se pode utilizar de diferentes formas de biomassa, sendo a cana de açúcar no a principal fonte de geração do combustível. No processo de produção de etanol vários fatores interferem no rendimento. O objetivo deste trabalho foi determinar as melhores condições do processo de fermentação alcoólica do extrato aquoso da polpa de jerivá, utilizando a metodologia de superfície de resposta e otimização simplex. Foi empregado o delineamento fatorial incompleto 33 tendo o extrato de levedura, NH4H2PO4 e levedura, como variáveis independentes e o rendimento da produção de álcool como resposta. A análise da regressão mostrou que o modelo é preditivo e a otimização simplex indicou uma formulação contendo 0,35 g L-1 de extrato de levedura, 6,33 g L-1 de levedura e 0,30 g L-1 de NH4H2PO4 para um rendimento ótimo de produção de etanol de 85,40%. Para a validação da equação preditiva o experimento foi repetido em triplicata, nas condições ótimas estabelecidas, obtendo-se um valor médio de 87,15% de rendimento. Aplicando-se o teste t, verificou-se que não houve diferença significativa, em nível de 5%, entre o valor médio obtido e aquele indicado na otimização simplex.

Title: Properties of Poulsen simplices Author: Zdeněk Jaroň Department: Department of Mathematical Analysis Supervisor: Doc. RNDr. Jiří Spurný, Ph.D. Abstract: In the present thesis, we study a generalisation of concept of the Poulsen simplex in general, non-metrizable case. First, for any given simplex F we con- struct a new one S, containing F as a face, having dense set of extreme points and preserving some important properties of F. In the next part, we employ this con- struction to build up, for any given infinite cardinal κ, two simplices S1, S2 with dense extreme boundary, with density character equal to κ and with spaces of affine functions Ac (S1) and Ac (S2) having the same density character, but which are not affinely homeomorphic. Keywords: Poulsen simplex, projective limit, Helly space