Orientador: Marcelo Alexandre Prado / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-21T09:52:53Z (GMT). No. of bitstreams: 1 MirandaeSilva_LigiaMaria_M.pdf: 148256 bytes, checksum: 789cac2002bb2e8dcb1bf70832d395b6 (MD5) Previous issue date: 2012 / Resumo: O crescente aumento populacional em escala mundial, tornou necessário um grande esforço por parte da agricultura para aumentar, a cada ano, a produção de alimentos para atender as necessidades do mercado externo e interno do Brasil. Recursos técnicos e científicos passaram então, a serem aplicados em busca da melhoria na produção dos cultivos,principalmente mediante o uso de fertilizantes e praguicidas. Com isso, a sociedade se deparou com problemas de ordem de equilíbrio ambiental e saúde pública, pois devido à contínua diversificação dos fitoparasitas, surgem, a todo momento, reduções do período de tempo entre aplicações consecutivas, e mais importante talvez, usos de doses mais altas e emprego simultâneo de diferentes pesticidas, por parte dos agricultores, objetivando complementar ações específicas ou alcançar efeitos sinérgicos para maiores rendimentos na produção. Tal situação traz como conseqüência óbvia e direta, o aumento, inaceitável, dos riscos de contaminação do meio ambiente com resíduos químicos de defensívos da área agropecuarista prejudiciais à saúde, o que leva a inúmeros problemas relativos à segurança alimentar dos produtos consumidos, e à uma preocupação de âmbito nacional evidenciada pela criação do Programa de Análise de Resíduos de Agrotóxicos em alimentos (PARA) da ANVISA. O aumento na necessidade de detecção e quantificação destes compostos, acarretou o desenvolvimento de pesquisas no setor, a fim de atingir uma melhoria na eficiência,qualidade e rapidez de resposta nas análises. A possibilidade do estudo de não apenas um de cada vez, mas de até 300 compostos sendo extraídos, detectados e quantificados simultâneamente se tornou a saída mais viável, tanto qualitativa quanto economicamente, facilitando o monitoramento contínuo do fornecimento de produtos do setor alimentício pelos chamados métodos multiresíduos. O presentre trabalho teve como princípio a validação de um método multiresíduo para análise de 14 analitos usando uma técnica de alto poder de concentração e limpeza do extrato como o GPC (Gel Permeation Chromatography) e detecção e quantificação por GC-MS/MS e LC-MS/MS. Os pesticidas investigados englobam classes como: acaricidas, inseticidas, fungicidas, nematicidas e formicidas de aplicação foliar, em sementes ou em solo, sendo que o acefato, metamidofós, acetamiprido e o thiamethoxan foram extraídos de amostras de batata e feijão e analisados por LC-MS/MS e a azoxistrobina, bifentrina, carbofuran, chlorotalonil, clorpirifós, clorfenapir, etofenprox, famoxadone,metalaxil, procimidone e o tebuconazole em amostras de batata e tomate e analisados por GCMS/MS. Os limites de detecção (LD) encontrados variaram de 0,06 a 2,89µg/L, e os coeficientes de variação (CV), de 0,036 a 2,036%. As recuperações foram determinadas em cada tipo de amostras, e os valores encontrados estavam entre 93,34% e 109,67%. Nenhuma das matrizes utilizadas apresentaram resultados insatisfatórios e o método utilizado mostrouse robusto e de fácil aplicação para todos os analitos testados / Abstract: The growing population worldwide, has required a great effort on the part of agriculture to increase each year, the production of food to meet the needs of external and internal market of Brazil. Technical and scientific resources spent then, to be applied in pursuit of improved crop production, mainly through the use of fertilizers and pesticides.With this, the company encountered problems in the balance of environmental and public health, since due to the continuous diversification of plant parasites, arise at any moment,reductions in the time period between consecutive applications, and perhaps most important,uses more doses high and simultaneous use of different pesticides by farmers, aiming to complete specific actions or to achieve synergistic effects in producing higher yields. This situation brings obvious and direct consequence, the increase unacceptable risk of environmental contamination with chemical residues from pesticides in farms are harmful to health, which leads to numerous problems relating to food safety of the products consumed, and to a concern nationwide evidenced by the creation of the Program Analysis of Pesticide Residues in Food (TO) of ANVISA. The increase in the necessity for detection and quantification of these compounds, led the development of research in the sector in order to achieve an improvement in efficiency, quality and responsiveness in the analyzes. The possibility of studying not just one at a time, but up to 300 compounds being extracted,detected and quantified simultaneously output became more viable, both qualitatively and economically, facilitating continuous monitoring of the supply of products by the food industry called methods multiresidue. The principle presentre work was the validation of a multiresidue method for analysis of 14 analytes using a technique of high power concentration and cleanup of the extract as GPC (Gel Permeation Chromatography) and detection and quantification by GC-MS/MS and LC- MS / MS. The pesticides investigated include classes such as acaricides, insecticides, fungicides, insecticides and nematicides foliar, seed or soil,and acephate, methamidophos, and Acetamiprid thiamethoxan were extracted from samples of potatoes and beans and analyzed by LC-MS / MS and azoxystrobin, bifenthrin, carbofuran,chlorothalonil, chlorpyrifos, chlorfenapyr, etofenprox, famoxadone, metalaxyl, procymidone and tebuconazole in samples of potato and tomato and analyzed by GC-MS/MS. The limits of detection (LOD) ranged from 0.06 to 2.89 mg / L, and the coefficients of variation (CV), 0.036 to 2.036%. The recoveries were determined for each type of samples, and the values were between 93.34% and 109.67%. None of the arrays used had unsatisfactory results and method proved to be robust and easy to apply for all analytes tested / Mestrado / Ciência de Alimentos / Mestra em Ciência de Alimentos

Ce projet de recherche comporte deux parties principales qui possèdent comme lien unificateur l’amélioration des méthodes et techniques utilisées actuellement pour évaluer aussi bien l’activité que l’expression des cytochromes P450 (CYP450s) et menant par la suite à leur application en clinique. Le premier volet de ce projet de recherche porte sur le développement de méthodes LC-MS/MS pour un cocktail de 7 substrats marqueurs des CYP450s. Notre objectif est de développer et valider une méthode LC-MS/MS spécifique et sensible permettant l’évaluation des activités des CYP1A2, 2B6, 2C9, 2C19, 2D6, 3A4/5 et 2E1 suivant l’administration orale et à faible dose d’un cocktail de substrats marqueurs chez des patients et sujets sains. Les méthodes développées peuvent être utilisées pour évaluer les mécanismes de variabilité interindividuelle comme l’impact de polymorphismes génétiques, de facteurs environnementaux et de maladies dans le processus de métabolisme et d’élimination des médicaments, mais également pour investiguer les interactions médicamenteuses. Ce cocktail a été appliqué avec succès, dans un projet clinique portant sur l’évaluation des effets du diabète sur la capacité métabolique par les CYP450s. Le deuxième volet de ce projet de recherche vise à développer des méthodes analytiques par LC-HRMS afin de caractériser et quantifier les CYP1A1, 1A2, 1B1, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2J2, 3A4, 3A5, 3A7 et 4F2 dans l’intestin grêle humain. Notre hypothèse suggère que les CYP450s retrouvés le long de l’intestin grêle peuvent affecter significativement l’effet de premier passage de certains médicaments administrés par voie orale et influencer leurs concentrations plasmatiques et conséquemment, leurs effets pharmacologiques et/ou toxiques. Ma participation à ce projet a permis d’identifier des peptides protéotypiques par digestion in silico et in vitro et de développer des méthodes de quantification absolue par LC-HRMS. Ce projet est une première étape dans la caractérisation des CYP450s majeurs le long de l'intestin grêle. Il permettra de mieux comprendre les mécanismes de variabilité interindividuelle dans la réponse aux médicaments associés au processus d'absorption intestinal et de mieux prédire la variabilité dans la biodisponibilité des médicaments et de développer des modèles pharmacocinétiques plus complexes. / This research project is divided into two sections, both aiming at the development of sensitive and specific LC-MS methods to evaluate activity and expression of CYP450 and finally, looking at their clinical application. The first section of this research project focuses on the development of analytical methods by LC-MS/MS for a seven CYP450 probe-drug cocktail. Although these cocktails have shown value they also suffer from many limitations. Our objective was to develop and validate highly sensitive and selective LC-MS/MS assays allowing the determination of CYP1A2, 2B6, 2C9, 2C19, 2D6, 3A4/5 and 2E1 activities following administration of low oral doses of a modified CYP450 probe-drug cocktail in patients. These methods can be used to phenotype CYP450 activities, evaluate inter-individual variabilities, study the impact of pathological conditions on drug metabolism and elimination, and evaluate drug-drug interactions. Our CYP450 cocktail assays have been successfully applied to phenotype CYP450 activities in type 2 diabetic patients. The second section of this project aims at the development of a LC-HRMS method for the characterization and absolute quantification of CYP1A1, 1A2, 1B1, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2J2, 3A4, 3A5, 3A7 and 4F2 in the human small intestine. Our hypothesis suggests that CYP450 isoenzymes found along the small intestine can significantly affect the first-pass effect of certain drugs administered orally and thus influence their pharmacological and/or toxic effects. My participation in this project allowed to identify proteotypic peptides by in silico and in vitro digestion and to develop LC-HRMS methods allowing the absolute quantification of CYP450. This project is a first step in the characterization of the main CYP450 along the small intestine. This project will allow a better understanding of inter-individual variability in drug response associated with intestinal absorption of drugs, a better prediction of variability in drug bioavailability and to develop more complex pharmacokinetic models.

Plasmodium falciparum is a protozoan parasite responsible for causing the most severe form of malaria in humans. This species is responsible for over 90% of malaria mortalities which occur predominantly in Africa. An increase in drug resistant parasites in recent years is threatening the progress made against malaria and thus new antimalarial drugs and vaccines are needed to combat this disease. During the intraerythrocytic phase, merozoites egress from mature schizonts to invade new uninfected erythrocytes. Glycophosphatidylinositol (GPI) -anchored proteins cover most of the exterior surface of the merozoite prior to invasion, while other GPI-anchored proteins are released onto the merozoite surface through apical organelle secretions. These proteins are involved in interactions with erythrocytes and are thought to be vital to erythrocyte invasion. GPI-anchored proteins have also been implicated as a cause of pathogenic symptoms and activation of immune components. These proteins are then released or cleaved to enable merozoite entry into the erythrocyte. Several enzymes are thought to be involved in their cleavage including the serine proteases subtilisin-like proteases (SUB) 1 and 2, and phosphatidylinositol-phospholipase C (PIPLC); GPI-anchored proteins are also generally sensitive to phospholipase A2 (PLA2). Cleaved proteins are released into the host blood system, while uncleaved proteins are carried into the erythrocyte during invasion. Merozoites have a limited period in which they retain invasive capacity. A previous lack of available techniques that are specifically adapted to merozoite analysis has resulted in an incomplete understanding of invasion and GPI-anchored protein involvement in invasion. This study aimed to determine how GPI-anchored proteins on the merozoite surface are altered in the invasive phase, and explore the possibility of using merozoite GPI-anchored proteins as potential drug targets to block erythrocyte invasion. Optimised methods of in vitro parasite culturing which produce highly synchronised merozoites was essential to this study. Parasite culturing techniques were optimised by utilising low haematocrit cultures with frequent culture splitting and optimised synchronisation. The “Malarwheel” is a tool that was developed for this research to provide a means for scheduling sorbitol treatments and MACs isolations. This tool and optimised culturing methods enabled large volumes of highly synchronised invasive merozoites to be harvested. Four compounds (vanadate, edelfosine, dioctyl sodium sulfosuccinate (DSS), and gentamicin) suspected to interfere with GPIanchored cleavage or processes were screened on intraerythrocytic stages and merozoites. Antimalarial and anti-invasive properties of these compounds were screened by modified malaria SYBR Green I-based fluorescence (MSF) assay and merozoite invasion assays (MIA) respectively. DSS and gentamicin showed limited potential as antimalarials or as anti-invasive agents. Vanadate and edelfosine both showed antimalarial and anti-invasive activity, while edelfosine was the most potent anti-invasive agent at physiological concentrations. The merozoite GPI-anchored proteome was analysed by sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) followed by complete gel lane analyses conducted by liquid chromatography-tandem mass spectrometry (LC-MS/MS) on soluble and pelleted merozoite proteins in samples from either invasive or non-invasive merozoites. Thirteen known or predicted GPI-anchored proteins were identified in samples. Several changes were identified in merozoite GPI-anchored proteins between the invasive phase and after its completion, and minor differences were observed following treatment with edelfosine. Edelfosine showed partial inhibition of erythrocyte invasion, however, the primary cause of inhibition cannot be directly related to interferences with GPI-anchored proteins. These results suggest that GPIanchored proteins are controlled by various complex processes, and are cleaved or processed by diverse mechanisms during the invasive phase. These mechanisms may be controlled by multiple signals which effect proteins or groups of proteins in specific ways. These signals may be influenced by “checkpoints” during invasion processes including the time period after egress from schizonts, and possibly the recognition of erythrocyte targets. These methods and results provide a foundation for future research to enable culturing of P. falciparum parasites specifically for merozoite research, and to identify merozoite proteins active during the invasive phase. These results confirm and challenge previous ideas reported in literature on the GPI-anchored processes of merozoites and further characterise less studied GPIanchored proteins. The results suggest that the processes controlling GPI-anchored proteins may be more complex than previously thought. These results form a basis to further identify and characterise GPI-anchored proteins in the aim to develop antimalarial medications and vaccines that target merozoites and their GPI-anchored processes. / Dissertation (MSc)--University of Pretoria, 2017. / Pharmacology / MSc / Unrestricted

La cholestase intrahépatique de la grossesse (CIG) est la maladie du foie la plus répandue au cours de la grossesse. Elle est caractérisée par un prurit et est associée à une augmentation de la concentration des acides biliaires dans le sang, ce qui peut mener à un risque accru de conséquences périnatales indésirables, y compris un accouchement prématuré spontané et une augmentation des risques de mort de l’enfant à l’accouchement, entre autres. Le traitement médical de cette maladie repose actuellement sur l’acide ursodésoxycholique (UDCA) qui diminue le prurit et les anomalies biochimiques maternelles dans certains cas. Actuellement, le diagnostic de la CIG est posé suite à un test de quantification des acides biliaires sériques totaux par une méthode enzymatique. Nous émettons l'hypothèse que certains profils d’acides biliaires permettraient d’évaluer le risque de complications chez les femmes atteintes de CIG. En analysant les profilages, il pourrait être possible de déterminer la ou les espèces responsables de ces complications et ainsi déterminer des sous-groupes de patientes plus à risque de complications ou qui répondraient mieux au traitement. De plus, nous pensons que le traitement à l’UDCA, étant lui-même un acide biliaire, pourrait interférer lors de la quantification des acides biliaires totaux sériques, particulièrement dans les cas les plus problématiques de CIG où de fortes doses de ce composé sont administrées. Si c’était le cas, cela ferait en sorte que les valeurs de référence pourraient être modifiées en fonction du traitement administré. Le projet de recherche présenté vise au développement d’une méthode de quantification des acides biliaires sériques par la chromatographie liquide couplée à un spectromètre de masse en tandem (LC-MS/MS), qui permettrait un profilage des acides biliaires sériques chez les femmes enceintes atteintes de la CIG et qui permettrait également d’évaluer l’effet du traitement à l’UDCA sur ce profilage. Une méthode de quantification des acides biliaires par chromatographie liquide couplée à un spectromètre de masse en tandem a été développée et validée. Les surnageants obtenus par précipitation de protéines avec le méthanol ont été injectés sur le LC-MS/MS. La séparation est réalisée par chromatographie en phase inverse sur une colonne C18 de type interactions hydrophobes. Les transitions ioniques sur le spectromètre de masse ont été déterminées pour toutes les espèces d’acides biliaires au préalable et l’acide cholique deutéré, l’acide chénodésoxycholique deutéré ainsi que l’acide désoxycholique deutéré ont été utilisés comme standards internes. Quinze acides biliaires, y compris les acides biliaires conjugués et libres, ont été séparés et quantifiés par LC–MS/MS en utilisant l’ionisation par électro nébulisation (ESI) en mode ion négatif. La quantification a été réalisée en mode de surveillance de réactions multiples (MRM) avec des méthodes de courbes d'étalonnage externes. Les coefficients de corrélation des courbes standards pour tous les acides biliaires étaient supérieurs à 0,9966. La méthode développée a démontré une précision acceptable, avec une imprécision intra analyse inférieure à 3,2% pour toutes les espèces d’acide biliaire étudiées (pour des échantillons à 0,8 et 5 μg/mL) et une imprécision inter analyse inférieure à 15%. Une suppression d’ion moyenne de 8,2% a été observée, qui a été jugée acceptable. Une bonne corrélation a été obtenue entre la méthode LC-MS/MS et une méthode enzymatique (r=0,964). En conclusion, une méthode fonctionnelle, efficace et rapide a été développée pour quantifier les acides biliaires sériques individuels et différents profils d’acides biliaires représentant une large gamme de concentrations ont été comparés. La comparaison des profilages d’acides biliaires suggère que les acides biliaires principaux responsables de l’augmentation de la concentration des acides biliaires totaux dans le sang pour des échantillons à une concentration de plus de 10 μmol/L sont l’acide cholique glyco-conjugué (GCA), l’acide cholique tauro-conjugué (TCA) ainsi que l’acide ursodésoxycholique glyco- conjugué (GUDCA). Cette nouvelle méthode validée, et les données préliminaires sur les profils d’acides biliaires dans les échantillons cliniques, permettront de lancer des analyses cliniques prospectives pour évaluer l’effet du traitement par l’UDCA sur les concentrations totales d’acides biliaires sériques et sur les profils d’acides biliaires individuels chez les patientes atteintes de la CIG. / Intrahepatic cholestasis of pregnancy (ICP) is the most common liver disease during pregnancy. It is characterized by pruritus and is associated with an increased concentration of bile acids in blood, which may lead to an increased risk of perinatal consequences, including spontaneous preterm delivery and an increased risk of death at birth, among others. The medical treatment of this disease currently relies on ursodeoxycholic acid (UDCA) which reduces pruritus and maternal biochemical abnormalities in some cases. Currently, the diagnosis of ICP is made using an enzymatic assay to measure total serum bile acids. We hypothesize that profiling of the individual bile acids would make it possible to assess the risk of complications in women with ICP. By analyzing the bile acid profiles, it could be possible to determine which specie(s) is responsible for these complications and thus to distinguish subgroups of patients at higher risk of complications or who would respond better to treatment. In addition, we believe that UDCA treatment, being a bile acid itself, could interfere with the quantification of total serum bile acids, particularly in the most problematic cases of CIG where high doses of this compound are administered. If this was the case, it would mean that the reference values would need to be changed depending on the administered treatment. The research project aims to develop and validate a method for quantifying bile acids in serum by liquid chromatography coupled to a tandem mass spectrometer (LC-MS/MS), which would allow profiling of serum bile acids in affected women and which would also make it possible later to evaluate the effects of UDCA treatment on this profiling. A method for the quantification of bile acids by liquid chromatography coupled to tandem mass spectrometry has been developed and validated. The supernatants obtained by precipitation of proteins with methanol were injected onto the LC-MS/MS. The separation was carried out using reversed-phase chromatography on a C18 hydrophobic interactions type column. Ionic transitions on the mass spectrometer were determined for all bile acids species beforehand and deuterated cholic acid, deuterated chenodeoxycholic acid and deuterated deoxycholic acid were used as internal standards. Fifteen bile acids, including conjugated and free bile acids, were separated and quantified by LC–MS/MS using electrospray ionization (ESI) in negative ion mode. Quantification was performed in multiple reaction monitoring (MRM) mode with external calibration curve methods. Correlation coefficients for standard curves for all bile acids were greater than 0.9966. The method developed showed acceptable precision, with intra-assay imprecision of less than 3.2% for all the bile acid species studied (for samples at 0.8 and 5 μg/mL) and inter-assay imprecision under 15%. An average ion suppression of 8.2% was observed, which was judged acceptable. Finally, a good correlation was obtained between the LC-MS/MS method and an enzymatic method (r = 0.964). In conclusion, a functional, efficient and rapid method was developed to quantify the individual serum bile acids and different bile acids profiles representing a wide range of concentrations were compared. The comparison of the bile acid profiles suggests that the main bile acids responsible for the increase in total bile acids concentration in blood for samples at a concentration of more than 10 μmol/L are glycocholic acid (GCA), taurocholic acid (TCA), glycoursodeoxycholic acid (GUDCA). This new validated method, and the preliminary data on bile acid profiles in clinical samples, will allow us to initiate prospective clinical analyses to assess the effect of UDCA treatment on total bile acid concentrations and profiles in patients with intrahepatic cholestasis of pregnancy.

As in many other fields, biology is faced with enormous amounts ofdata that contains valuable information that is yet to be extracted. The field of proteomics, the study of proteins, has the luxury of having large repositories containing data from tandem mass-spectrometry experiments, readily accessible for everyone who is interested. At the same time, there is still a lot to discover about proteins as the main actors in cell processes and cell signaling. In this thesis, we explore several methods to extract more information from the available data using methods from statistics and machine learning. In particular, we introduce MaRaCluster, a new method for clustering mass spectra on large-scale datasets. This method uses statistical methods to assess similarity between mass spectra, followed by the conservative complete-linkage clustering algorithm.The combination of these two resulted in up to 40% more peptide identifications on its consensus spectra compared to the state of the art method. Second, we attempt to clarify and promote protein-level false discovery rates (FDRs). Frequently, studies fail to report protein-level FDRs even though the proteins are actually the entities of interest. We provided a framework in which to discuss protein-level FDRs in a systematic manner to open up the discussion and take away potential hesitance. We also benchmarked some scalable protein inference methods and included the best one in the Percolator package. Furthermore, we added functionality to the Percolator package to accommodate the analysis of studies in which many runs are aggregated. This reduced the run time for a recent study regarding a draft human proteome from almost a full day to just 10 minutes on a commodity computer, resulting in a list of proteins together with their corresponding protein-level FDRs. / <p>QC 20160412</p>

Summary: Many primary human tumors and tumor cell lines highly express human L-type amino acid transporter 1 (hLAT1); cancerous cells in vivo are strongly linked to LAT1 expression. Synthetic chemistry and in vitro screening efforts have afforded a variety of novel and highly hLAT1 selective compounds, such as JPH203 1. In a recent report, we demonstrated that 1 has potent in vitro and in vivo activity. JPH203 was intravenously administered to produce significant growth inhibition against HT-29 tumors transplanted in nude mice. The current work develops a robust LC/MS-MS method to monitor 1 and its major Phase II metabolite N-acetyl-JPH203 2 from biological samples. We have conducted in vitro and in vivo experiments and the major scientific findings are: i) the major route of biotransformation of 1 is Phase II metabolism to produce 2; ii) metabolite 2 is formed in various organs/tissues (i.e. blood, liver, kidney); and iii) as dogs, which are deficient in NAT genes, do not produce 2, the dog will not be an appropriate toxicological model to evaluate 1.

No description available.

Protein carbonylation, a marker of excessive oxidative stress, has been studied in the context of multiple human diseases related to oxidative stress. The variety of post-translational carbonyl modifications (carbonyl PTMs) and their low concentrations in plasma challenge their reproducible identification and quantitation. However, carbonyl-specific biotinylated derivatization tags (e.g., aldehyde reactive probe, ARP) allow for targeting carbonyl PTMs by enriching proteins and peptides carrying these modifications. In this study, an oxidized human serum albumin protein model (OxHSA) and plasma from a healthy donor were derivatized with ARP, digested with trypsin, and enriched using biotin-avidin affinity chromatography prior to nano reversed-phase chromatography coupled online to electrospray ionization tandem mass spectrometry with travelling wave ion mobility spectrometry (nRPC-ESI-MS/MS-TWIMS). The presented workflow addresses several analytical challenges by using ARP-specific fragment ions to reliably identify ARP peptides. Furthermore, the reproducible recovery and relative quantitation of ARP peptides were validated. Human serum albumin (HSA) in plasma was heavily modified by a variety of direct amino acid oxidation products and adducts from reactive carbonyl species (RCS), with most RCS modifications being detected in six hotspots, i.e., Lys10, Lys190, Lys199, Lys281, Lys432, and Lys525 of mature HSA.

Venezuelan equine encephalitis virus (VEEV) is an alphavirus that causes disease in humans and equines eliciting both an agricultural and public health threat. In humans, the disease typically presents as a febrile illness with common signs of fever and malaise. Four to fourteen percent of Venezuelan equine encephalitis (VEE) cases are associated with severe neurological complications due to encephalitis caused by VEEV's propensity to infect the brain. Public health concerns are exacerbated by VEEV's aerosolization capabilities, low infectious dose and affordability to mass produce. These qualities drove interest in the pathogen as a bioweapon by the US and the former Soviet Union during the cold war. As a precautionary response to VEEV's notoriety as a biothreat, the National Institute of Allergies and Infectious Diseases has classified VEEV as a category B priority pathogen, and the Human Health Services and United States Department of Agriculture list live virulent strains of VEEV as a select agent and require the pathogen to be manipulated in highly regulated biosafety level 3 (BSL3) facilities. There are currently no FDA approved vaccines or antivirals to target VEEV or other closely related alphaviruses associated with clinical disease in humans. The research performed in this dissertation aimed to elucidate new antiviral targets and treatments to help bridge gaps in current understanding of alphaviruses. The current market lacks available antibodies for E1 specific isolation. In response, a recombinant VEEV TC-83 was produced with a V5 tag at the C-terminal of the E1 sequence to enable VEEV E1 detection. Sequencing was used to verify V5 insertion in the plasmid and immunoprecipitation was used to verify V5 insertion within the E1 glycoprotein. Replication kinetics experiments verified the virus replicated similarly to the parental VEEV TC-83 strain, while passaging experiments verified the tag was highly stable for up to 10 passages. This research produced a cost-effective and highly efficient means to probe and isolate the E1 glycoprotein without modifying the viability of the virus. Knowledge of host protein interactions with VEEV E1 glycoprotein has been limited, with most E1 research focusing on its fusion capabilities. Utilizing 293-T cells infected with E1-V5 TC-83, co-immunoprecipitation was performed to isolate E1 and associated interactors. A total of 486 host and 5 viral protein interactors of E1 were discovered after normalization to the negative control. The top peptide spectrum matches (PSMs) revealed a number of chaperone proteins and ubiquitin proteins as top interactors of VEEV E1. These results effectively revealed a number of previously unknown alphavirus interactions that can be targeted by antivirals and explored further for implications in viral replication. LC-MS/MS results showed that protein disulfide isomerase family A member 6 (PDIA6) interacted with E1. High PSMs, presence in all 3 replicates, similar cellular localization to E1 and known associations between other viruses and protein disulfide isomerase (PDI) family members made this protein an optimum target for further analysis. Co-immunoprecipitation and co-localization experiments were used to validate the LC-MS/MS results. Involvement of PDIs in VEEV replication were explored utilizing two known PDI inhibitors, LOC14 and Nitazoxanide. LOC14, a non-FDA approved broad-spectrum PDI inhibitor, showed broad-spectrum alphavirus antiviral potential, decreasing titers of VEEV TC-83, VEEV Trinidad Donkey strain, eastern equine encephalitis virus (EEEV), chikungunya virus (CHIKV) and Sindbis (SINV) virus in a dose dependent manner. Nitazoxanide, an FDA approved drug known to inhibit PDIA3, was shown to have minimal toxicity and effectively reduced VEEV TC-83 and EEEV titers at concentrations with 100% cell viability. Time of addition assays, E1 expression time course studies, and early event assays showed PDI inhibition with these drugs effects early viral production events. RNA quantification, confocal microscopy and biotin switch assay experiments show that the drugs also prevented proper folding of the E1 glycoprotein and decreased expression of E1 on the peripheral membrane. With no current treatments for alphaviruses, these data provide an effective broad-spectrum target that affects viral replication at multiple stages in-vitro. Nitazoxanide also presents as a promising, non-toxic drug that could be repurposed to combat a number of clinically relevant alphaviruses. Valosin containing protein (VCP) was also shown to interact with the E1 glycoprotein. Exploration of VCP's interaction with alphavirus E1 has never been explored, yet it was previously shown to be involved in alphavirus replication. Co-localization and co-immunoprecipitation experiments were performed validating the interaction between VCP and E1. siRNA knockdown of VCP in 293-T cells and U87-MG cells showed a significant reduction in VEEV TC-83 titers. The allosteric VCP inhibitor, NMS-873, also reduced VEEV TC-83 titers, but was shown to be less effective against CHIKV, SINV and EEEV, suggesting the NMS-873 mechanism is more selective for VEEV. Mechanism experiments showed that reduction of VCP with NMS-873 inhibits early events of VEEV replication. These results elucidate VCP's association with E1 and show that VCP can be targeted to decrease VEEV viral replication. / Doctor of Philosophy / Venezuelan equine encephalitis virus (VEEV) causes disease in humans, as well as horses, donkeys and other closely related animals. In humans, the virus causes a flu-like disease and sometimes swelling of the brain. This can be associated with symptoms such as light sensitivity, confusion and sometimes coma. Prior to the Cold War, VEEV was researched by the US and previous Soviet Union's militaries in hopes to deploy the virus as a bioweapon. Current treaties prevent active production of such weapons, yet allows for defensive research to continue in preparation for a worst-case scenario. Currently no FDA approved medications or vaccines exist to combat the virus further exacerbating concerns. In order to protect laboratorians and prevent unintentional or intentional introduction of the virus into the community, the virus is only manipulated in highly secure facilities with barriers that separate the virus from personnel and the outside environment. A component of the virus called E1, allows for the virus to be released from a structure, called an endosome, that transports the virus into the cell. Currently, E1 is mostly known for this function, yet our research found that E1 interacts with 486 protein components of the host cell, suggesting a more elaborate role of E1 than previously understood. This list of interactors provides numerous new targets for potential medications to combat VEEV and other closely related viruses. Discovered E1 interactors, protein disulfide isomerase family A member 6 (PDIA6) and valosin containing protein (VCP), were validated through extensive experimentation and their function in viral replication was further explored. Protein disulfide isomerases (PDI), such as PDIA6, play an important role in folding proteins, which are cellular components made of organic building blocks called amino acids. PDIs do so by creating organic pillars, called disulfide bonds, between two cysteine amino acid residues. These disulfide bonds contribute to the 3D shape of the proteins they fold which are essential for the protein's function. E1 of VEEV has a total of eight disulfide bonds within its structure, highlighting that disulfide bonds are likely essential for the protein's structure, and therefore, function. We verified that E1 could not properly fold without PDI function by using two compounds that prevented PDI from forming or breaking disulfide bonds, specifically LOC14 and FDA approved drug nitazoxanide. Cells treated with one of either compound before and after infection with VEEV, were found to produce E1 protein with significantly less disulfide bonds therefore producing less viable virus. Further experiments also showed that the compounds also affected early stages in the virus production cycle. These two mechanisms explain the significant reduction in production of VEEV and related viruses when PDI is inhibited. These results provide a new VEEV drug target, PDIs, as well as two compounds that can potentially be used to combat VEEV and other related viruses that have no current treatment options. Another host interactor, VCP, functions throughout the cell and is known for unfolding of numerous substrates, including proteins. It is involved in numerous cellular functions thus making this interactor a promising target for drug treatment. Cells with reduced VCP function were shown to produce less progeny VEEV. Cells treated with NMS-873, a compound that reduces VCP function was also shown to reduce VEEV production. NMS-863 inhibition of VCP was shown to effect early events in VEEV replication. These results further emphasize the E1 interactors discovered are invaluable novel targets for VEEV drug treatment.

TRPC6, the sixth member of the family of canonical transient receptor potential (TRP) channels, contributes to a variety of physiological processes and human pathologies. This study extends the knowledge on the newly developed TRPC6 blocker SH045 with respect to its main target organs beyond the description of plasma kinetics. According to the plasma concentration-time course in mice, SH045 is measurable up to 24 h after administration of 20 mg/kg BW (i.v.) and up to 6 h orally. The short plasma half-life and rather low oral bioavailability are contrasted by its reported high potency. Dosage limits were not worked out, but absence of safety concerns for 20 mg/kg BW supports further dose exploration. The disposition of SH045 is described. In particular, a high extravascular distribution, most prominent in lung, and a considerable renal elimination of SH045 were observed. SH045 is a substrate of CYP3A4 and CYP2A6. Hydroxylated and glucuronidated metabolites were identified under optimized LC-MS/MS conditions. The results guide a reasonable selection of dose and application route of SH045 for target-directed preclinical studies in vivo with one of the rare high potent and subtype-selective TRPC6 inhibitors available