Herpes simplex virus (HSV) causes recurrent orofacial and genital infections and establishes latent infection in sensory neurons. During latency all virus genes are supressed except the latency associated transcripts which are transcribed from latency associated gene (LAT). It is established that HSV LAT promoter mutants have lower levels of spontaneous reactivation rates in small animal models compared to wild virus. However, the variation in the LAT promoter has not been studied in viruses from clinical samples in humans. The aim of the sudy was to evaluate the sequence variation in herpes simplex virus latency associated gene promoter from clinical samples by developing and applying molecular methods and correlate with herpes infection clinical features. In this study a new PCR method specific for HSV LAT promoter was developed and HSV LAT promoter DNA sequences from Lithuanian and Swedish mucocutaneous and cerebrospinal fluid clinical samples were analyzed. HSV type 2 was found to be the main cause of genital herpes in the population of the Lithuanian patients. All cases of orofacial herpes simplex infection were caused by HSV type 1. The structure of the LAT promoter region was studied in 145 HSV clinical samples. HSV LAT promoter was found to be G+C rich and contained variable homopolimer tracts. An inter- and intrastrain variability of homopolimer tracts in the promoter region was detected, potentially giving rise to a large variation at the protein level, leading to... [to full text] / Herpes simplex virusas sukelia recidyvuojančią burnos-veido ir lytinių organų infekciją. Latentinėje būklėje šis virusas glūdi sensoriniuose ganglijuose. Latencijos metu visi HSV genai yra supresuoti, išskyrus su latencija susijusį geną (LAT). Tyrimais nustatyta, kad tarp HSV LAT promotoriaus mutantų reaktyvacijos dažnis laboratorinių gyvūnėlių modeliuose yra mažesnis nei laukinių virusų. Nėra atlikta tyrimų, kurie nagrinėtų LAT promotoriaus sekų variaciją herpes simplex virusuose, išskirtuose iš žmonių klinikinių mėginių. Šio tyrimo tikslas buvo įvertinti herpes simplex viruso LAT promotoriaus sekų įvairovę molekulinės diagnostikos metodais bei palyginti su infekcijos klinikiniais požymiais. Tuo tikslu buvo sukurtas PGR metodas HSV LAT promotoriaus analizei atlikti. Buvo ištirta Lietuvos ir Švedijos klinikiniuose odos-gleivinių bei cerebrospinalinio skysčio mėginiuose rasto herpes simplex viruso promotoriaus DNR sekų įvairovė. Tyrimo metu rasta, kad 2 tipo herpes simplex virusas buvo pagrindinė lytinių organų HSV infekcijos priežastis tarp Lietuvos pacientų. Visuose veido srities bėrimuose rasta 1 tipo HSV. HSV LAT promotoriaus sekos ištirtos 145 klinikiniuose mėginiuose. Nustatyta, kad HSV LAT promotoriaus sekos yra gausios GC ir turi variabilias homopolimerinių nukleotidų sritis, kurios varijuoja tarp viruso padermių ir pačių padermių viduje. Ši variacija gali turėti įtakos baltymų sintezei, o drauge ir fenotipo pokyčiams. Nenustatytas ryšys tarp HSV LAT promotoriaus... [toliau žr. visą tekstą]

A substituição dos conservantes sintéticos em cosméticos por produtos naturais é uma tendência, direcionando as pesquisas para a busca de novas alternativas de compostos antimicrobianos naturais. Extratos de bambus asiáticos têm ampla aplicação na indústria cosmética, sendo muito utilizados em diversas formulações. No Brasil, a triagem química de espécies nativas de bambus para futuras aplicações na indústria ainda é incipiente. O objetivo do trabalho foi avaliar as atividades antioxidante e antimicrobiana de extratos e frações de folhas e colmos de A. simplex McClure & Smith. Extratos brutos de folhas e colmos foram obtidos por percolação com etanol 60%, liofilizados, e fracionados com solventes de polaridade crescente. A capacidade antioxidante foi quantificada pelo método de redução do radical DPPH, sendo a fração clorofórmica dos colmos a que apresentou melhor atividade (IC50= 24,02 µg/mL). A atividade antimicrobiana foi determinada pelo método de microdiluição frente Escherichia coli (ATCC 8739), Staphylococcus aureus (ATCC 6538), Pseudomonas aeruginosa (ATCC 9027), Candida albicans (ATCC 10231) e Apergillus brasilensis (ATCC 16404). Os extratos brutos e frações de folhas e colmos de A. simplex apresentaram baixa atividade antimicrobiana com concentração inibitória mínima (CIM) > 1mg/mL para todos os micro-organismos. O efeito sinérgico dos extratos com parabenos foi realizado utilizando o delineamento experimental centroide simplex para uma mistura de metilparabeno, propilparabeno e extrato. As misturas foram testadas frente aos micro-organismos utilizados no ensaio de atividade antimicrobiana. O sinergismo foi observado em maior intensidade em S. aureus e C. albicans. Adicionalmente, os resultados da quantificação de flavonoides e compostos fenólicos totais sugeriram que o sinergismo entre esses compostos e os parabenos poderia ser responsável pela atividade antimicrobiana. / The replacing synthetic preservatives in cosmetics with natural products, is a tendency, research to investigate new natural antimicrobial compounds. Although Asian bamboo extracts have wide application in the cosmetics industry and are widely used in various formulations, Brazilian native species were not yet been investigated for future industrial applications. Thus, the purpose of this study was to evaluate the antioxidant and antimicrobial activities of extracts and fractions from A. simplex McClure & Smith leaves and culms. Crude leaf and culm extracts were obtained by percolation with 60% ethanol, lyophilized and fractionated with increasing polarity solvents. Antioxidant capacity was measured by the DPPH radical scavenging method in which the culm chloroform fraction presented the highest activity (IC50=24.02 µg/mL). Antimicrobial activity was determined by the microdilution method against Escherichia coli (ATCC 8739), Staphylococcus aureus (ATCC 6538), Pseudomonas aeruginosa (ATCC 9027), Candida albicans (ATCC 10231) and Apergillus brasiliensis (ATCC 16404). All the extracts and fractions from A. simplex showed low antimicrobial activity with Minimal Inhibitory Concentration (MIC) > 1 mg/mL for all microorganisms. The synergistic effect of the extracts mixed with methyl and propyl parabens was tested using a simplex centroid design. The mixtures were tested against the same microorganisms used for extracts evaluation. A stronger synergic effect was observed for S. aureus and C. albicans. Additionaly, the quantification results of flavonoids and total phenolic compounds suggested that the synergism among these compounds and the parabenos could be responsible for the antimicrobial activity.

Made available in DSpace on 2018-06-27T18:55:57Z (GMT). No. of bitstreams: 3 Danieli Ferrari da Rosa.pdf: 3718059 bytes, checksum: bb8ef19a5af8a3faa7687e991e0d5c3d (MD5) Danieli Ferrari da Rosa.pdf.txt: 158457 bytes, checksum: b6b83ac5211b5e9ee8b9dfba9feba1d9 (MD5) Danieli Ferrari da Rosa.pdf.jpg: 3270 bytes, checksum: 1f0862e05ecb2e7b1da2056322eeeaab (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The sialic acid is the generic name of carboxylated monosaccharides family with nine carbon glycoconjugated at terminal portion. These molecule family are involved in several biological processes such cell recognition processes, platelet adhesion, migration, invasion and metastatic potential, it also work as a receptor for bacteria and viruses. High concentrations of total sialic acid in the blood have been reported in different groups of patients with brain tumors, leukemia, melanoma, carcinoma and other kinds of cancers. The cleavage of sialic acid is a crucial step in virus infection influenzae, since this acid is part of the cellular receptor that the virus uses during the process of cellular internalization. The neuraminidase, an enzyme produced by the virus, cleaves the bond between sialic acid and the viral glycoproteins, allowing the entry of viruses into cells.The aim of this study was the analysis of serum sialic acid levels in murine melanoma and Herpes Simplex virus-1 (HSV-1) infection model. In the tumor model were used C57BL/6 and in the viral model BALB/c mice. Mice were injected with 2x105 B16F10 cells subcutaneously in the thigh and the tumor progression was followed each day till it became visible. The HSV-1 infection was conducted by intraperitoneally injection of with 102 PFU of virus. The sialic acid in serum samples was quantified by thiobarbituric method in spectrophotometer at 549 nm. A standard curve with commercial sialic acid was used as parameter for quantification. The results showed that in tumor model the sialic acid was increased compared with control group and have significant difference (p <0.05) in the first day after administration of cells. For the viral infection the concentration of sialic acid showed a significant difference (p <0,05) in the first day after infection when compared infected with control group. The histological analysis in thigh of mice performed 24 hours after administration of B16F10 cells were found compact groups of round or polygonal melanocytes with clear and large cytoplasm, irregular chromatin, hyperchromatic and vacuolated nuclei, eosinophilic nucleoli and atypical mitosis. / O ácido siálico é o nome genérico dado a família de monossacarídeos carboxilados com nove átomos de carbono que aparece na porção terminal de glicoconjugados. Estas moléculas estão envolvidas em vários processos biológicos, tais como, processos de reconhecimento celular, adesão plaquetária, migração, invasão, potencial metastático, sendo também um receptor para bactérias e vírus. O aumento das concentrações séricas de ácido siálico total tem sido descrito em vários grupos de pacientes que sofrem de tumores cerebrais, leucemia, melanoma, carcinoma e outros tipos de cânceres. A clivagem do ácido siálico é um passo crucial para a infecção do vírus Influenza, uma vez que este ácido é parte do receptor celular usado pelo vírus durante o processo de internalização celular. A neuraminidase, enzima produzida pelo vírus, cliva a ligação entre o ácido siálico e as glicoproteínas virais, permitindo a entrada dos vírus nas células. O objetivo desse estudo foi analisar os níveis séricos de ácido siálico em modelo de melanoma murino e modelo de infecção herpética (HSV-1). No modelo tumoral foram utilizados camundongos C57BL/6 e no modelo viral camundongos BALB/c. Os camundongos receberam 2x105 células B16F10 através da administração subcutânea na coxa e a progressão do tumor foi acompanhada todos os dias até o tumor se tornar visível. A infecção com HSV-1 foi realizada através da administração intraperitoneal de 102 PFU de vírus. O ácido siálico das amostras de soro foram quantificadas pelo método tiobarbitúrico em espectrofotômetro à 549 nm. Uma curva padrão com ácido siálico comercial foi usada como parâmetro para a quantificação. Os resultados mostraram que as concentrações de ácido siálico no modelo tumoral foram aumentadas nos animais com tumor quando comparadas ao grupo controle e houve diferença significativa (p< 0,05) no primeiro dia após a administração das células. Para o modelo de infecção viral houve diferença significativa (p< 0,05) no primeiro dia após a infecção quando comparado o grupo infectado com o controle. Na análise histológica da coxa dos camundongos realizada após 24 horas da administração de células B16F10 foram encontrados grupos compactos de melanócitos arredondados ou poligonais, com citoplasma amplo e claro, cromatina irregular, núcleos hipercromáticos e vacuolizados, nucléolos eosinofílicos e mitoses atípicas.

Le criblage par siRNA de 49 protéines de l'hôte qui sont incorporées dans les particules matures du virus herpès simplex de type 1 (VHS-1) a révélé l'importance d'au moins 15 d’entre elle pour infectivité du virus (Stegen, C et al. 2013). Parmi celle-ci figure la protéine humaine DDX3X, qui est une ARN hélicase ATP-dépendante. Cette protéine multifonctionnelle participe à différents stages de l'expression génique, tels que la transcription, la maturation et le transport d'ARNm ainsi que la traduction. DDX3X est impliquée dans la réplication de plusieurs virus tels que le Virus de l’immunodéficience humaine de type 1 (VIH-1), l'hépatite B (VHB), le virus de la vaccine (VACV) et le virus de l'hépatite C (VHC). Le rôle exact de DDX3X dans le cycle de réplication du VHS-1 est toutefois inconnu. Ce mémoire consiste en l’étude détaillée de l'interaction de DDX3X avec le virus. De manière surprenante, tant l’inhibition que la surexpression de DDX3X réduit de manière significative l'infectivité du VHS-1. Fait intéressant, lorsque nous avons restauré la déplétion de DDX3X par une construction résistante aux ARNi utilisés, le virus pouvait de nouveau infecter les cellules efficacement, indiquant que le virus est sensible aux quantités de cette protéine de son hôte. Nos résultats indiquent de plus que le virus modifie la localisation de DDX3X et cause son agrégation tôt dès les premiers temps de l'infection. Cependant, le virus ne modifie pas les niveaux cellulaires de DDX3X dans deux des trois lignées cellulaires examinées. Nous avons également pu établir que cette protéine n'a pas d'effet sur l'entrée du VHS-1, suggérant qu’elle agit à un stade ultérieure de l’infection. En examinant cette relation plus en détail, nos résultats ont démontré que l’inhibition ou la surexpression de DDX3X inhibent toutes deux la production de nouvelles particules virales en réduisant l'expression des diverses classes cinétiques des protéines virales et ce au niveau de leur transcription. Malgré le rôle connu DDX3X dans la stimulation de la réponse immunitaire innée et la production d’interférons de type I, l’impact de DDX3X sur la réplication du VHS-1 est ici indépendante de cette fonction. Ces travaux démontrent donc une nouvelle voie d’action de DDX3X sur les virus en agissant directement sur la transcription de gènes viraux et la réplication du génome d’un virus à ADN. En comprenant mieux cette interactions hôtepathogène, il est maintenant envisageable de concevoir des nouvelles approches thérapeutiques contre ce virus. / siRNA screening of 49 host proteins that are known to be incorporated in the mature virions of herpes simplex virus type 1 (HSV-1) revealed the importance of at least 15 cellular proteins for viral infectivity (Stegen, C et al. 2013). Among these, was the human protein DDX3X, a DEAD-box ATP-dependent RNA helicase. This multifunctional protein participates in different stages of gene expression such as mRNA transcription, maturation, mRNA export and translation. DDX3X has been shown to be involved in the replication of several viruses such as human immunodeficiency virus type 1 (HIV-1), hepatitis B virus (HBV) vaccinia virus (VACV) and hepatitis C virus (HCV). The exact role of DDX3X in HSV-1 replication cycle is not known. Here we sought to find the detailed interaction between DDX3X with HSV-1. Surprisingly, the down-regulation as well as overexpression of DDX3X, significantly reduced the infectivity of HSV-1, indicating that the virus is sensitive to the precise levels of DDX3X. Accordingly, when we rescued DDX3X back to its normal cellular levels by sequential transfection of DDX3X siRNA and siRNA resistant DDX3X plasmid, the virus was able to infect cells efficiently compare to wild-type conditions. Furthermore, the virus changes the localization of DDX3X and causes its aggregation at early times in the infection. However, the virus does not change the cellular levels of DDX3X in at least two of three different cell lines tested. Using a luciferase assay we were able to establish that this protein has no effect on the entry of HSV-1. In fact, depleting or overexpressing DDX3X impaired the production on newly assembled viral particles by blocking the expression of all classes of viral proteins at the transcription level. Despite the known role of DDX3X in the stimulation of innate immune response and interferon type I production, DDX3X appears to act on HSV-1 replication independently of this pathway. This highlights a novel route of action of DDX3X by acting at the transcription level and the consequent genome replication of a DNA virus. By better understanding such pathogen interactions, it might now be possible to design novel therapeutic approaches.

Le virus de l’Herpès simplex de type 1 (HSV-1) est le pathogène humain responsable des lésions herpétiques labiales, plus communément appelé « feux sauvages ». Annuellement, il est responsable de plusieurs cas d’encéphalites et d’infections de l’appareil visuel qui sont la principale cause de cécité en Amérique du Nord. Bien qu’il existe quelques traitements antiviraux, aucun vaccin ou médicament ne permet de prévenir ou de guérir les infections causées par ce virus. Aujourd’hui, les infections produites par l’HSV-1 sont présentes partout sur la planète. Récemment, une étude en protéomique effectuée sur les virus matures extracellulaires a permis d’identifier la présence d’ubiquitines libres et d’enzymes reliées à la machinerie d’ubiquitination dans le virus. De plus, le virus exploite cette machinerie au cours de l’infection. Il est connu que certaines protéines virales sont ubiquitinées durant une infection et que le virus imite même certaines enzymes d’ubiquitination. Nous avons donc entrepris des recherches afin d’identifier des protéines virales ubiquitinées qui pourraient être présentes dans les virus matures ainsi que leurs rôles potentiels. La protéine majeure de la capside, VP5, un constituant très important du virus, a été identifiée. Nos recherches nous ont permis de caractériser le type d’ubiquitination, une monoubiquitination sur les lysines K810 et/ou K1275 de VP5. Le rôle que pourrait jouer l’ubiquitination de VP5 dans le cycle de réplication virale et dans les virus matures n’est toutefois pas encore connu. / Herpes simplex virus type 1 (HSV-1) is the human pathogen responsible for herpetic lesion such as cold sores. On a yearly basis, it is responsible for many cases of encephalitis and infections of the eye that are the most common cause of blindness in North America. Antiviral treatments exist, but no vaccines or drugs are able to prevent or cure the diseases caused by this virus. Today, infections caused by HSV-1 are present all around the world. Recently a proteomics approach was used to study mature extracellular viruses. This study highlighted the presence in the virus of free ubiquitin and ubiquitin related enzymes. Furthermore, the virus exploits this machinery during the course of infection. Also, it is known that certain virally encoded proteins are ubiquitinated and that the virus mimics some ubiquitin related enzymes. Our researches focused on identifying ubiquitinated viral proteins that could be present in mature extracellular viruses and their potential roles. The major capsid protein, VP5, an important virus component, was identified. We characterised the type of ubiquitination, a monoubiquitination of lysine K810 and/or K1275 of VP5. The role that could play the ubiquitination of VP5 in the viral cell cycle and in mature virions has yet to be identified.

Les virus exploitent la machinerie cellulaire de l’hôte de façon très variée et plusieurs types vont même jusqu’à incorporer certaines protéines cellulaires. Nous avons récemment effectué la première analyse protéomique du virion mature de l’Herpès simplex de type 1 (HSV-1), ce qui nous a permis de déterminer que jusqu’à 49 protéines cellulaires différentes se retrouvaient dans ce virus (Loret, S. et al. (2008). "Comprehensive characterization of extracellular herpes simplex virus type 1 virions." J Virol 82(17): 8605-18.). Afin de déterminer leur importance dans le cycle de réplication d’HSV-1, nous avons mis au point un système de criblage nous permettant de quantifier le virus produit et relâché dans le milieu extracellulaire en utilisant un virus marqué à la GFP ainsi que des petits ARN interférents (pARNi) ciblant spécifiquement ces protéines cellulaires. Cette approche nous a permis de démontrer que 17 des protéines identifiées précédemment jouaient un rôle critique dans la réplication d’HSV-1, suggérant ainsi que leur incorporation dans le virus n’est pas aléatoire. Nous avons ensuite examiné le rôle d’une de ces protéines, DDX3X (DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, X-linked), une protéine multifonctionnelle connue pour son implication dans les cycles de réplication de plusieurs virus humains. À l’aide de pARNi ainsi que de différentes lignées cellulaires, dont une lignée DDX3X thermosensible, nous avons démontré que l’inhibition de DDX3X résultait en une diminution du nombre de capsides intracellulaires et induisait une importante diminution de l’expression des gènes viraux. Nous avons aussi démontré que la fraction de DDX3X incorporée dans le virion contribuait activement au cycle infectieux d’HSV-1. Ces résultats confirment l’intérêt de notre approche afin d’étudier les interactions hôte-pathogène en plus de démontrer la contribution des protéines cellulaires incorporées à HSV-1 dans l’infection virale. / Viruses exploit the cellular machineries in many ways and several viruses specifically incorporate host proteins. To understand their biological relevance, we recently performed the first comprehensive characterization of the mature herpes simplex virus type 1 (HSV-1) in which up to 49 distinct cellular proteins were identified by mass spectrometry. In the present study, we sought to identify which of these cellular factors are critical for the HSV-1 life cycle. To this end, we performed a functional screen using small interfering RNA (siRNA) and a GFP-tagged virus, which indicated that at least 17 of the virion-incorporated host proteins alter HSV-1 proliferation in cell culture. Interestingly, these include several Rab GTPases and other intracellular transport components as well as proteins involved in signal transduction, gene regulation and immunity. Among them, the DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, X-linked protein (DDX3X) is a multifunctional molecule previously linked to several other viruses. Its relevance for HSV-1 was further confirmed with different siRNA reagents and cell lines, including a DDX3X thermosensitive cell line. We found that DDX3X inactivation reduced intracellular capsid abundance via a strong inhibition of viral gene expression. We also report evidence that the pool of DDX3X present in the mature virions actively contributes to HSV-1 life cycle. Altogether, this highlights a powerful and biologically relevant approach to characterize host-pathogen interactions and points to the important contribution host proteins within mature viral particles.

Le virus Herpes Simplex de type 1 (HSV-1) possède un mode d’infection particulier dit bimodal. Il peut soit se répliquer de manière active lors d’une phase dite lytique soit migrer dans les neurones et rester en latence. Il peut réactiver pour rétablir une infection lytique. Une protéine virale majeure dans la réactivation de HSV-1 est ICP0. C’est une protéine nucléaire à activité E3 ubiquitine ligase, qui possède la particularité d’induire la dégradation par le protéasome de plusieurs protéines centromériques constitutives, ce qui provoque une déstabilisation du centromère interphasique. Mon équipe a découvert une réponse cellulaire à l’instabilité centromérique, induite par la protéine ICP0, et nommée iCDR (pour interphase Centromere Damage Response.). L’objectif général de ma thèse est de déterminer les modifications structurales que subissent les centromères endommagés par ICP0 à l’origine de l’iCDR et probablement de la réactivation. J’ai pu démontrer qu’ICP0 affectait toute la structure protéique étroitement associée aux centromères durant l’interphase. Suite à ces résultats, j’ai pu démontrer, par des analyses de digestion de chromatine à la nucléase microccocale (MNAse), que l’occupation nucléosomique de la chromatine centromérique suite à l’activité d’ICP0 était affectée de façon significative. Une étude in vivo effectuée à partir de tissus nerveux provenant de souris infectées de manière latente, a permis de démontrer une co-localisation entre les génomes HSV-1 latents et les centromères. Cette co-localisation est associée à une répression transcriptionnelle du virus. Les résultats de ma thèse montrent donc que les effets d’ICP0 sur la déstabilisation des centromères sont en relation avec un rôle de ces centromères durant la latence. Ceci suggère fortement une implication de la déstabilisation des centromères dans le processus de réactivation contrôlé par ICP0. / The Herpes Simplex type 1 (HSV-1) virus possesses a bimodal mode of infection. It can either replicates in an active way during the lytic cycle, or it can infect neurons and stay in latency. HSV-1 reactivates from latently infected neurons for re-establishing a lytic infection. A major viral protein implicated in reactivation is ICP0. It is a nuclear E3 ubiquitin-ligase, which has the particularity to induce the proteasome-mediated degradation of several constitutive centromeric proteins. This activity severely destabilizes the interphase centromere. My team has discovered a novel cellular response triggered by the estabilization of centromeres by ICP0, called iCDR (interphase Centromere Damage Response). The general aim of my thesis is to determine the centromere structural modifications induced by ICP0 that can trigger the iCDR and probably the reactivation. I was able to demonstrate that ICP0 affected the entire proteinacious structure of interphase centromeres. Following this, I showed by micrococcal nuclease (MNase) digestion approach that the nucleosomal organization of centromeric chromatin was significantly affected by ICP0. An in vivo study in nervous tissues coming from latently infected mice enabled to show a co-localization between latent HSV-1 genomes and centromeres. This co-localisation is linked to a transcriptional repression of the virus. The results of my thesis show that the destabilization of centromere by ICP0 correlates with a role of the centromeres during latency. This strongly suggests an implication of centromere destabilization in the ICP0-controlled reactivation process.

[pt] Este trabalho busca desenvolver o método Simplex para Redes na solução de problemas de Fluxo de Custo Mínimo. Este método consiste em uma adaptação do método Simplex primal em que são exploradas as características específicas da rede subjacente ao problema ao se buscar a solução ótima em um número finito de árvores geradoras. A árvore geradora ótima será obtida iterativamente através de sucessivas melhorias na estrutura de cada árvore formada. A maior eficiência do Simplex para Redes se dá tanto no menor número de iterações necessárias para se atingir o ótimo, quanto na maior velocidade destas iterações, trata-se, portanto, de um método bastante poderoso na resolução de problemas de Fluxo de Custo Mínimo. Serão, também, abordados aspectos práticos da implementação do algoritmo além da aplicação deste algoritmo implementado em VBA (Visual Basic for Applications) em um problema prático a título de exemplificação. / [en] The current work intends to develop a Network Simplex Method for solving Minimum Cost Flow problems. Such method consists of a primal Simplex Method adaptation in which specific characteristics of the network underlying the problem are investigated by searching for the optimal solution within a finite number of spanning trees. The optimal spanning tree is iteratively obtained through successive structure improvements in each formed tree. The higher efficiency of Network Simplex lies both in fewer iterations necessary to achieve the optimum and in the higher speed of these iterations. Therefore, it is a powerful method for solving Minimum Cost Flow Problems. Practical aspects of implementing the algorithm will be discussed, as well as the algorithm´s implementation in VBA (Visual Basic for Applications) through a practical instance.

The Beta distribution is a bounded domain distribution which has dominated the modeling the distribution of random variable that assume value between 0 and 1. Bounded domain distributions arising in various situations such as rates, proportions and index. Motivated by an analysis of electoral votes percentages (where a distribution with support on the positive real numbers was used, although a distribution with limited support could be more suitable) we focus on alternative distributions to Beta distribution with emphasis in regression models. In this work, initially we present the Simplex mixture model as a flexible model to modeling the distribution of bounded random variable then we extend the model to the context of regression models with the inclusion of covariates. The parameters estimation is discussed for both models considering Bayesian inference. We apply these models to simulated data sets in order to investigate the performance of the estimators. The results obtained were satisfactory for all the cases investigated. Finally, we introduce a parameterization of the L-Logistic distribution to be used in the context of regression models and we extend it to a mixture of mixed models. / A distribuição beta é uma distribuição com suporte limitado que tem dominado a modelagem de variáveis aleatórias que assumem valores entre 0 e 1. Distribuições com suporte limitado surgem em várias situações como em taxas, proporções e índices. Motivados por uma análise de porcentagens de votos eleitorais, em que foi assumida uma distribuição com suporte nos números reais positivos quando uma distribuição com suporte limitado seira mais apropriada, focamos em modelos alternativos a distribuição beta com enfase em modelos de regressão. Neste trabalho, apresentamos, inicialmente, um modelo de mistura de distribuições Simplex como um modelo flexível para modelar a distribuição de variáveis aleatórias que assumem valores em um intervalo limitado, em seguida estendemos o modelo para o contexto de modelos de regressão com a inclusão de covariáveis. A estimação dos parâmetros foi discutida para ambos os modelos, considerando o método bayesiano. Aplicamos os dois modelos a dados simulados para investigarmos a performance dos estimadores usados. Os resultados obtidos foram satisfatórios para todos os casos investigados. Finalmente, introduzimos a distribuição L-Logistica no contexto de modelos de regressão e posteriormente estendemos este modelo para o contexto de misturas de modelos de regressão mista.

Linear optimization tools are used to solve many problems that arise in our day-to-day lives. The linear optimization models and methodologies help to find, for example, the best amount of ingredients in our food, the most suitable routes and timetables for the buses and trains we take, and the right way to invest our savings. We would cite many other situations that involves linear optimization, since a large number of companies around the world base their decisions in solutions which are provided by the linear optimization methodologies. In this thesis, we propose theoretical and computational developments to improve the performance of important linear optimization methods. Namely, we address simplex type methods, interior point methods, the column generation technique and the branch-and-price method. In simplex-type methods, we investigate a variant which exploits special features of problems which are formulated in the general form. We present a novel theoretical description of the method and propose how to efficiently implement this method in practice. Furthermore, we propose how to use the primal-dual interior point method to improve the column generation technique. This results in the primal-dual column generation method, which is more stable in practice and has a better overall performance in relation to other column generation strategies. The primal-dual interior point method also oers advantageous features which can be exploited in the context of the branch-and-price method. We show that these features improves the branching operation and the generation of columns and valid inequalities. For all the strategies which are proposed in this thesis, we present the results of computational experiments which involves publicly available, well-known instances from the literature. The results indicate that these strategies help to improve the performance of the linear optimization methodologies. In particular for a class of problems, namely the vehicle routing problem with time windows, the interior point branch-and-price method proposed in this study was up to 33 times faster than a state-of-the-art implementation available in the literature / Ferramentas de otimização linear são usadas para resolver diversos problemas do nosso dia-a- dia. Os modelos e as metodologias de otimização linear ajudam a obter, por exemplo, a melhor quantidade de ingredientes na nossa alimentação, os horários e as rotas de ônibus e trens que tomamos, e a maneira certa para investir nossas economias. Muitas outras situações que envolvem otimização linear poderiam ser aqui citadas, já que um grande número de empresas em todo o mundo baseia suas decisões em soluções obtidas pelos métodos de otimização linear. Nesta tese, são propostos desenvolvimentos teóricos e computacionais para melhorar o desempenho de métodos de otimização linear. Em particular, serão abordados métodos tipo simplex, métodos de pontos interiores, a técnica de geração de colunas e o método branch-and-price. Em métodos tipo simplex, é investigada uma variante que explora as características especiais de problemas formulados na forma geral. Uma nova descrição teórica do método é apresentada e, também, são propostas técnicas computacionais para a implementação eciente do método. Além disso, propõe-se como utilizar o método primal-dual de pontos interiores para melhorar a técnica de geração de colunas. Isto resulta no método primal-dual de geração de colunas, que é mais estável na prática e tem melhor desempenho geral em relação a outras estratégias de geração de colunas. O método primal-dual de pontos interiores também oferece características vantajosas que podem ser exploradas em conjunto com o método branch-and-price. De acordo com a investigação realizada, estas características melhoram a operação de ramificação e a geração de colunas e de desigualdades válidas. Para todas as estratégias propostas neste trabalho, são apresentados os resultados de experimentos computacionais envolvendo problemas de teste bem conhecidos e disponíveis publicamente. Os resultados indicam que as estratégias propostas ajudam a melhorar o desempenho das metodologias de otimização linear. Em particular para uma classe de problemas, o problema de roteamento de veículos com janelas de tempo, o método branch-and-price de pontos interiores proposto neste estudo foi até 33 vezes mais rápido que uma implementação estado-da-arte disponível na literatura