Abstract: It is well established that ionizing radiation induces a variety of damage in DNA by direct effects that are mediated by one-electron oxidation and indirect effects that are mediated by the reaction of water radiolysis products, e.g., hydroxyl radicals (•OH). In cellular DNA, direct and indirect effects appear to have about an equal effect toward DNA damage. We have shown that ϒ-(gamma) ray irradiation of aqueous solutions of DNA, during which •OH is the major damaging ROS can lead to the formation several lesions. On the other hand, the methylation and oxidative demethylation of cytosine in CpG dinucleotides plays a critical role in the gene regulation. The C5 position of cytosine in CG dinucleotides is frequently methylated by DNA methyl transferees (DNMTs) and constitutes 4-5% of the total cytosine. Here, my PhD research work focuses on the analysis of oxidative base modifications of model compounds of methylated and non methylated oligonucleotides, isolated DNA (calf-thymus DNA) and F98 cultured cell by gamma radiation. In addition, we identified a series of modifications of the 2-deoxyribose moiety of DNA arising from the exposure of isolated and cellular DNA to ionizing radiation. We also studied one electron oxidation of cellular DNA in cultured human HeLa cells initiated by intense nanosecond 266 nm laser pulse irradiation, which produces cross-links between guanine and thymine bases (G*-T*). To achieve these goals, we developed several methods based on mass spectrometry to analyze base modifications in isolated DNA and cellular DNA. / Résumé : Les radiations ionisantes induisent une variété de dommages à l'ADN selon des effets directs, correspondant à une oxydation suite à l’éjection d’un électron, et indirecte, médiés par une réaction avec les produits issus de la radiolyse de l’eau environnante, tels que les radicaux hydroxyles (•OH). Au sein d’une cellule, l’importance relative des effets directs et indirects semble être quantitativement similaire en ce qui concerne les dommages induits à l'ADN cellulaire. Nous avons démontré que l'irradiation par rayons Υ-(gamma) de solutions aqueuses d'ADN, dont l’action délétère est principalement véhiculé e par l’intermédiaire des radicaux hydroxyles, peut induire sur l’ADN la formation de toute une palette de modifications. D'autre part, la méthylation et la déméthylation oxydative de la cytosine au sein de couples de dinucléotides CpG jouent un rôle essentiel dans la régulation des gènes. La position C5 de cette cytosine se retrouve fréquemment méthylée par les méthyltransférases (DNMTs) et constitue alors 4-5% de l’ensemble de la cytosine présente au sein de l’ADN. Mon projet de recherche est centralisé autour de l'analyse de la modification des bases de l’ADN suite à leur oxydation dans des composés modèles constitués d'oligonucléotides méthylés et non-méthylés, puis dans l'ADN isolé (extrait de cellules de thymus de veau) et enfin au sein de cultures cellulaires F98 ayant subies une irradiation par rayons Υ-(gamma). De plus, nous avons identifié une série de modifications spécifiques au groupement fonctionnel 2-désoxyribose de l'ADN résultant de l'exposition de l'ADN isolé et cellulaire aux rayonnements ionisants. Nous avons également étudié les conséquences de l’irradiation par des impulsions lasers nanoseconde à 266 nm de cultures cellulaires de lignée humaine (HeLa). Responsable d’une réaction d’oxydation suite à l’éjection d’un électron, l’identification des modifications induites à l’ADN cellulaire suite à l’irradiation laser a permis de mettre en évidence des pontages ADN-ADN caractéristiques entre les bases guanine et thymine (G*-T*). Pour atteindre ces objectifs, nous avons développé plusieurs méthodes d’analyse des modifications de bases au sein de l’ADN isolé et de l'ADN cellulaire basées sur la spectroscopie de masse.

Candida albicans is an opportunistic fungal pathogen that may cause mucutaneous infection and/or disseminated candidasis if the host defense system is impaired (such as those in HIV patients). Cell surface of C. albicans is the frontier where initial interplay between host-pathogen takes place and therefore is of great importance in understanding the mechanism of hostpathogen interaction. MALDI-TOF-MS analysis of intact fungal cells yielded mass signatures for rapid species differentiation, strain grouping and yeast morphogenesis monitoring. Cell surface biotinylations at low temperature (4°C), enzymatic digestion of the intact fungal cell surface proteins ("whole cell shaving"), biotin-avidin affinity enrichment of biotinylated peptides, liquid chromatography mass spectrometry (LC-MS) based proteomic approach were employed for unambiguous identification of cell wall/cell wall associated proteins and the exposed peptide segments of these proteins. SILAC (Stable Isotope Labeling by Amino acids in Cell Culture) based CWP quantification analyses were performed to monitor CWP accumulation level change in response to hyphae induction. Information on surface exposed peptide segments and regulation of cell wall/surface protein during morphogenesis provided new candidates to the pool of potential peptide targets for protective vaccine development. A New type of "fluorous" (fluorinated alkane) affinity gained popularity due to its low level nonspecific protein/peptide binding. Fluorous labeling reagents that target primary amine groups in proteins/peptides were synthesized and characterized. The acid labile linker in the labeling reagents allows cleavage of the bulky fluorous tag moiety and the long oligo ethylene glycol (OEG) spacer after fluorous affinity purification. Upon collision induced decomposition, the labeled peptide ion yielded a characteristic fragment that could be retrieved from the residual portion of fluorous affinity tag, and serve as a marker to indicate that the relevant peptide had been successfully labeled. Results showed that both the protein/peptide labeling and affinity enrichment/separation process were highly efficient.

Humans are exposed to electrophilically reactive compounds, both formed endogenously and from exogenous exposure. Such compounds could react and form stable reaction products, adducts, at nucleophilic sites in proteins and DNA. The formation of adducts constitutes a risk for effects, such as cancer and contact allergy, and plays a role in ageing processes. Adducts to proteins offer a possibility to measure electrophilic compounds in vivo. Adductomic approaches aim to study the totality of adducts, to specific biomolecules, by mass spectrometric screening. This thesis describes the development and application of an adductomic approach for the screening of unknown adducts to N-terminal valine (Val) in hemoglobin (Hb) by liquid chromatography tandem mass spectrometry (LC/MS/MS). The adductomic approach is based on the FIRE procedure, a modified Edman procedure for the analysis of adducts to N-terminal Val in Hb by LC/MS/MS. The adduct screening was performed by stepwise scanning of precursor ions in small mass increments and monitoring four fragments common for derivatives of detached Val adducts, in the multiple reaction monitoring mode. Samples from 12 smokers/nonsmokers were screened with the adductomic approach, and seven previously identified adducts and 19 unknown adducts were detected. A semiquantitative approach was applied for approximate quantification of adduct levels. A strategy for identifying unknown Hb adducts using adductome LC/MS/MS data was formulated and applied for the identification of unknown adducts. Identifications were based on the observed m/z of precursor ions and retention times combined with databases and Log P calculations. Hypothesized adducts were generated in vitro for comparison and matching with the corresponding unknown adducts. Five identified adducts correspond to the precursor electrophiles ethyl vinyl ketone (EVK), glyoxal, methylglyoxal, acrylic acid, and 1-octen-3-one. These adducts, except the adducts corresponding to glyoxal and methylglyoxal, have not been observed as protein adducts before.  Probable exposure sources to these electrophiles are diet and/or endogenous formation. The observation of these adducts motivate further studies to evaluate possible contributions to health risks, as well as their potential as biomarkers of exposure. The adduct from EVK was quantitatively assessed through different experiments to estimate the daily internal dose (area under the concentration-time-curve, AUC). EVK is about 2 × 103 more reactive than the reference compound acrylamide. The EVK adduct was shown to be unstable, with a relatively short half-life. The daily AUC in humans of EVK was estimated to be about 20 times lower than the corresponding AUC of acrylamide from intake via food. To confirm the observation of the detected unknown adducts and obtain a statistical foundation, analysis of unknown adducts were performed in large sets of blood samples (n = 50–120) from human cohorts. The majority of the previously detected unknown adducts were found in all analyzed samples, and the levels of many adducts showed large variations between individuals. The cause and significance of these observed variations are not yet clarified, but are of importance for the directions of future studies. In conclusion, a new approach for identification of unknown human exposure to electrophiles was developed and successfully applied. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Submitted.</p>

Alors que le protéome de l'ovocyte isolé, aux stades VG et MII a déjà été étudié, celui du follicule en croissance n'a jamais été décrit.Dans cette étude, nous avons cherché à identifier, comparer et caractériser les profils protéiques de follicules ovariens de souris à trois stades de leur développement in vitro distincts morphologiquement : follicules secondaires en début de culture - stade initial (IS), follicules avec une rupture complète de la membrane de Slavjanski (RMS) et follicules avec une cavité similaire à l'antrum (FA).Après un préfractionnement par IEF et une analyse LC-MS/MS en deux configurations (1D et 2D), 1403 protéines ont pu être identifiées dans le follicule ovarien de souris. 43,4 % (609) des protéines identifiées étaient communes aux trois stades et d'autres ont été identifiées uniquement à un stade : 71 au stade IS, 182 au stade RMS et 193 au stade FA. De plus, on a identifié 365 protéines qui n'avaient pas été décrites antérieurement dans le protéome de l'ovocyte ce qui pourrais indiquer qu'elles sont exprimées dans les cellules somatiques du follicule. Des analyses qualitatives et quantitatives complémentaires ont démontré une surreprésentation pour 44 fonctions biologiques par rapport aux fonctions biologiques des gènes constituant le génome de Mus musculus et mis en évidence des différences d'expression et d'abondance des protéines liées au cycle cellulaire, à la fixation des ions de calcium et à la glycolyse selon le stade de développement. Ces résultats représentent un point de départ pour beaucoup d'autres études de caractérisation moléculaire du développement folliculaire. / Until now only the proteome of isolated oocyte from fully grown follicle were described with the aim of oocyte maturation characterization. However the ability of oocyte to mature is acquired during its development within the follicle. Thus in this study we proposed a protein identification and characterization of whole mice ovarian follicle at three morphological stages during in vitro development: early secondary stage, described as initial stage (IS), follicles with a complete Slavjanski membrane rupture (RMS) and follicles with an antrum like cavity formation (FA). Using an IEF pre fractionation before protein digestion and two configurations of LC-MS/MS analysis (1D and 2D), 1403 proteins were successfully identified in the murine ovarian follicle. From those, 43.4 % (609) were commonly identified in the three stages and some were identified only at one single stage: 71 at IS stage, 182 at RMS stage and 193 at FA stage. Compared to the proteomes of isolated oocyte previously described, 365 proteins were only identified in our samples indicating that those ones were probably expressed in the somatic cells of the follicle. Additional qualitative and quantitative analysis highlighted 44 biological processes over represented in our samples when compared to Mus musculus gene database and different expressions and protein abundance implicated in cell cycle, calcium ion binding and glycolysis, throughout in vitro follicle development. This report represents so far the most complete catalogue of follicle proteins and could be an important milestone in the proteomic study of the follicle metabolism throughout in vitro development.

No abstract available

Esta tese alia dois enfoques principais dentro da Química Farmacêutica. Por um lado, busca explorar uma nova fonte de insumos naturais, cascas de Malus  domestica, com vistas a obtenção dos triterpenos ácidos ursólico (AU) e betulínico (AB); e por outro lado, estuda a relação dos derivados triterpênicos semissintéticos obtidos com a proteína-alvo (PfATP6) destacada atualmente na literatura da terapia da malária com vistas ao planejamento de novos antimaláricos. O primeiro Capítulo inclui o desenvolvimento de um método eficiente, fácil e extremamente rápido onde são combinadas as técnicas de extração por ultrassom (UAE) e análise por Cromatografia Líquida de Alta Eficiência Acoplada a Detector de Espectroscopia de Massas (LC-MS), para identificação e doseamento dos ácidos ursólico (AU) e betulínico (BA) em extratos de cascas frescas de maçã de cinco clones das cultivares Gala e Fuji (“Baigent”, “Fuji Mishima”, “Fuji Suprema”, “Fuji Select” and “Maxi Gala”) oriundas da Região Sul do Brasil. Os parâmetros cromatográficos do método analítico incluem: ionização por eletro spray em modo positivo (ESI+), fluxo de 1,0 mL/min, em modo de eluição isocrático, consistindo de 80% acetonitrila e 20% acetato de amônio 10 mM em pH 6,0 e temperatura ambiente. O método desenvolvido foi validado e mostrou ser seletivo, sensível (LOD e LOQ de 0,087 e 0,266 μg/mL para BA, e 0,398 e 2,117 μg/ mL para UA), coeficiente de regressão linear (r >0.99), preciso, exato e robusto para os analitos de interesse. A otimização do método combinado de UAE com LC-MS permitiu concluir os procedimentos de extração e análise em tempo inferior a 4 h, uma vez que o método não requer a secagem da amostra, etapa que demanda longos tempos de processamento. Este método foi aplicado e forneceu a primeira caracterização fitoquímica dos cinco clones de maçã estudados. Os resultados demonstraram que o método combinado de UAE-LC-MS é adequado às práticas do controle de qualidade. O segundo Capítulo apresenta o estudo da interação dos ligantes semissintéticos derivados dos AU e AB, sintetizados pelo nosso grupo de pesquisa, com a proteína Ca2+-ATPase do Plasmodium falciparum (PfATP6), através do emprego da técnica de Docking Molecular. A PfATP6 é descrita como um importante alvo para novos antimaláricos como Artemisinina (ART), cujo mecanismo de ação inclui, dentre outros, a modulação da homeostasia do cálcio intracelular. Investigações conduziram à hipótese do extravasamento do cálcio, do interior do retículo sarco-endoplasmático como mecanismo plausível para ação dos derivados triterpênicos do AU e AB. Os escores de energia determinados no Docking, de cada um dos nove ligantes (derivados triterpênicos) e quatro compostos de controle (ácidos ursólico e betulínico, artemisinina (ART) e tapsigargina (TPG) foram determinados (análises realizadas em triplicata) e correlacionados com os valores de IC50, para a atividade antimalárica. Os resultados mostraram excelente correlação entre os escores de energia (com a PfATP6) e os valores de IC50, superior a 80% (r > 0,83360). O estudo fornece fortes evidências de que a PfATP6 pode constituir um alvo para os derivados pentacíclicos do estudo, bem como permitiu identificar o perfil conformacional dos ligantes e os principais resíduos do sítio de ligação (SL) da PfATP6 envolvidos nas interações; bem como, contribuiu para a melhor compreensão das propriedades envolvidas na interação do ligante com o receptor e mecanismo de ação. O terceiro Capítulo faz uso de Métodos Clássicos e Quânticos para descrever as mudanças conformacionais da proteína PfATP6. As simulações de Dinâmica Molecular do receptor na forma isolada e complexada com os ligantes foram realizadas durante 10 ns. As conformações finais obtidas para os receptores foram avaliadas em termos RMSD, efeito da presença dos ligantes no sítio de ligação e tipos de interações estabelecidas entre o ligante e o receptor. Os resultados mostraram que as proteínas PfATP6 e SERCA tendem a manter sua conformação nativa e que o modelo utilizado é adequado aos propósitos do estudo. O monitoramento dos resíduos da região citoplasmática das proteínas permitiu evidenciar o efeito alostérico da presença dos ligantes AU e ART no SL, sobre os domínios A e N, da PfATP6. Esse efeito reproduz as conformações E1 e E2, bem estabelecidas para PfATP6, na presença e ausência de Ca2+. As análises de Dinâmica Molecular corroboram os achados do Capítulo II ao evidenciarem o estabelecimento de interações de hidrogênio com os resíduos importantes do SL da PfATP6. Esses resultados fundamentam os indícios de que as bombas de Ca2+-ATPase (SERCA), possam ser de fato, um alvo para os derivados triterpênicos dos AU e AB. / This thesis combines two main focuses within Pharmaceutical Chemistry. On the one hand, it seeks to explore a new source of natural inputs, bark of Malus domestica, in order to obtain ursolic (UA) and betulinic acid (BA) triterpenes. On the other hand, it studies the relation of the semi-synthetic triterpenic derivatives obtained with the target proteins (PfATP6), currently highlighted in the literature on malaria therapy with a view to planning new antimalarial drugs. The first chapter includes the development of an efficient, easy and extremely fast method where ultrasonic extraction techniques (UAE) and high-performance liquid chromatography coupled to mass spectroscopy (LC-MS) are combined for identification and assay of ursolic acid (UA) and betulinic acid (BA) in fresh apple peel extracts from five clones of the Gala and Fuji cultivars (Baigent, Fuji Mishima, Fuji Suprema, Fuji Select and Maxi Gala ") in the Southern Region of Brazil. Chromatographic parameters of the analytical method include: electrospray ionization (ESI +), flow rate of 1.0 mL/min in isocratic elution mode, consisting of 80% acetonitrile and 20% 10 mM ammonium acetate at pH 6.0 and room temperature. The method was validated and proved to be selective, sensitive (LOD and LOQ of 0.087 and 0.266 μg/mL for BA, and 0.398 and 2.117 μg/mL for UA), linear regression coefficient (r> 0.99), accurate, robust for analytes of interest. The optimization of the combined method of UAE with LC-MS allowed to complete the procedures of extraction and analysis in less than 4 h, since the method does not require drying the sample, a stage that demands long processing times. This method was applied and provided the first phytochemical characterization of the five apple clones studied. The results demonstrated that the combined UAE-LC-MS method is suitable for quality control practices. The second chapter presents the study of the interaction of the semi-synthetic ligands derived from the UA and BA, synthesized by our research group, with the Plasmodium falciparum Ca2+-ATPase protein (PfATP6), using the Molecular Docking technique. PfATP6 is described as an important target for new antimalarials such as Artemisinin (ART), whose action mechanism includes, among others, the modulation of intracellular calcium homeostasis. Investigations led to the hypothesis of extravasation of calcium from the interior of the sarco-endoplasmic reticulum as a plausible mechanism for the action of the triterpenic derivatives of UA and BA. The Docking energy scores (binding energy) of each of the nine ligands (triterpenic derivatives) and four control compounds (ursolic and betulinic acids, artemisinin (ART) and tapsigargine (TPG)) with PfATP6, were calculated (analyses performed in triplicate) and correlated with its antimalarial IC50 value. The results showed an excellent correlation between energy scores (with PfATP6) and IC50 values, higher than 80% (r > 0.83360). The study supplies strong evidence that PfATP6 may be a target for the pentacyclic derivatives of the study, and also allowed identifying the conformational profile of the ligands and the main residues of the PfATP6 binding site (BS) involved in the interactions. It further contributed to a better understanding of the properties involved in the interaction of the ligand with the receptor and its action mechanism. The third chapter uses Classical and Quantum Methods to describe the conformational changes of the PfATP6 protein. The Molecular Dynamics simulations of the receptor in the isolated and complexed form with the ligands were performed for 10 ns. The final conformations obtained for the receptors were evaluated in RMSD terms, effect of the presence of ligands at the binding site and types of interactions established between the ligand and the receptor. The results showed that the PfATP6 and SERCA proteins tend to maintain their native conformations and that the model used is adequate for the purposes of the study. The monitoring of the residues of the cytoplasmic region of the proteins allowed evidencing the allosteric effect of the presence of UA and ART ligands in BS, on the A- and N-domains of PfATP6. This effect reproduces the well-established E1 and E2 conformations for PfATP6, in the presence and absence of Ca2+, respectively. Molecular Dynamics analyses corroborate the findings of Chapter II, by showing the possibility of establishing hydrogen interactions with the important residues of PfATP6 BS. These results support the evidence that Ca2+-ATPase (SERCA) pumps may be a target for the triterpenic derivatives of UA and BA.

De nos jours, il y a une volonté générale de se tourner vers une agriculture plus respectueuse de l’environnement et du consommateur se traduisant notamment par une démarche de réduction des intrants chimiques. Dans un contexte de développement durable, la recherche de produits naturels pour lutter contre les maladies et les ravageurs suscite un regain d’intérêt. Dans cette thèse, des extraits hydro-alcooliques issus de coproduits de la vigne (sarment, cep, racine) et d’essences forestières (écorce d'épicéa, nœud de pin) se sont révélés être une excellente source de polyphénols bioactifs, en particulier en stilbènes complexes. En effet, ces extraits ont démontré un large spectre d’activités contre différentes maladies végétales. En particulier, un potentiel oomycide contre le mildiou de la vigne et une capacité insecticide contre un parasite des Solanacées sont rapportés. En outre, la pertinence de l'utilisation de la « chimie verte » pour extraire les stilbènes comme méthode alternative aux solvants organiques a été mise en évidence. Les présents résultats renforcent une voie de recherche originale pour faire progresser une viticulture et une agriculture plus durables, en utilisant des produits de biocontrôle moins toxiques et biodégradables, constituant ainsi une solution possible et réaliste pour lutter contre les pathogènes des plantes. / Nowadays, is a priority to turn towards a more eco- and consumer friendly agriculture resulting in the reduction of the chemical inputs. In a context of a sustainable development, the investigation of natural products to fight against diseases and pests raised a renewed interest. In this thesis, hydroalcoholic extracts derived from grapevine (cane, wood, root) and forest species (spruce bark, pine knot) by-products have demonstrated to be a great source of bioactive polyphenols, and particularly in complex stilbenes. Indeed, these extracts have proved to confer a broad spectrum of activities against different major plant diseases. In particular, an oomycide potential against downy mildew of the vine and an insecticidal capacity against Solanaceae pest were reported. Furthermore, the relevant use of “green chemistry” to extract stilbenes as an alternative method of organic solvents has been highlighted. The present findings strengthen an original line of research to advance in a more sustainable viticulture and agriculture, using less toxic and biodegradable biocontrol products, being this a possible and realistic solution to combat plant pathogens.

Submitted by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2016-04-07T12:58:36Z No. of bitstreams: 1 HEMERSON SILVA DA ROSA.pdf: 1232470 bytes, checksum: b64c04e3acdc1a67c9bd8559b68fb261 (MD5) / Made available in DSpace on 2016-04-07T12:58:36Z (GMT). No. of bitstreams: 1 HEMERSON SILVA DA ROSA.pdf: 1232470 bytes, checksum: b64c04e3acdc1a67c9bd8559b68fb261 (MD5) Previous issue date: 2013-01-24 / Sida tuberculata (Malvaceae), conhecida popularmente como “guanxuma”, é uma espécie vegetal de porte herbáceo, bem representada na região sul do Brasil. Na cultura popular é utilizada para tratamento de diversas enfermidades, em especial àquelas relacionadas ao diabetes e ao colesterol elevado. Para algumas espécies, existem relatos de eventual potencial antimicrobiano. Considerando a ausência de estudos sobre esta planta, o presente trabalho investigou a composição química dos extratos brutos de S. tuberculata e avaliou seu potencial antifúngico in vitro. Após a coleta e identificação, o material vegetal foi submetido aos processos de secagem e trituração. Submeteu-se a extração a frio por percolação, utilizando-se como solvente solução hidroetanólica a 40% para folhas e 70% para raízes. Para fins de comparação foram feitas extrações aquosas por infusão. Na sequência, foram determinados os teores de fenólicos totais e flavonóides totais. Posteriormente, as amostras foram analisadas através de método por CLAE-UV, com seleção das condições cromatográficas de melhor eficiência de separação. Sendo estas estabelecidas, efetuou-se análise por LC-MS em modo ESI positivo. Para os ensaios da atividade antifúngica foram utilizados os protocolos de microdiluição em ágar para determinação da concentração inibitória mínima (CIM) e concentração fungicida mínima (CFM). Também foi aplicada a metodologia para avaliação do potencial de remoção de biofilme em Cateter Venoso Central (CVC). Os resultados obtidos demonstraram um maior teor de fenólicos e flavonóides totais nos extratos das folhas. As análises por LC-UV-MS permitiram a identificação e proposição de cinco compostos, entre ecdisteróides, flavonóides e alcalóides. Nos ensaios de atividade antifúngica os extratos aquosos apresentaram atividade contra linhagens de Candida krusei, com valores de CIM variando entre 3.9 - 62.5 μg/ml para folhas e 1.95 - 31.25 μg/ml para raízes. No teste de remoção de biofilme, os extratos aquosos das folhas demonstraram um maior potencial de remoção. Os dados de composição química obtidos, nas variantes de diferentes partes da planta, bem como a atividade antimicrobiana detectada, geram expectativas quanto a novos estudos de exploração do potencial biológico de S. tuberculata. / Sida tuberculata (Malvaceae), popularly known as "guanxuma", is an herbaceous plant species present in southern Brazil. In popular culture, it is used for the treatment of several diseases, such as those related to diabetes and high cholesterol level. For some species of Sida, there are also reports about the antimicrobial potential. Considering the lack of studies at this species, this study proposed an investigation about the chemical composition of Sida tuberculata extracts and their in vitro antifungal activity. After collection and identification, the plant material was submitted to dryness and powdered. Then, it was submitted to extraction by percolation using hydroethanolic solution at 40% and 70% as solvent, to leaves and roots respectively. For comparison, aqueous extracts were obtained by infusion. The total phenolic and flavonoid contents of extracts were determined. The samples were also evaluated by HPLC-UV, testing the chromatographic conditions that promote the better separation efficiency. After, for the identification procedure, the analysis by LC-ESI-MS in positive mode was conducted using previously established conditions. For the antifungal activity assay, the agar microdilution protocols were used for determination of minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC). In addition, the extracts were tested for potential biofilm removal in Central Venous Catheter (CVC). The results demonstrated a higher concentration of total phenolic and flavonoids compounds in the leaves extracts. LC-MS analysis allowed the identification of five components, between ecdysteroids, flavonoids and alkaloids. In the assay of antifungal activity, the aqueous extract had activity against Candida krusei strains, with the MIC values varying between 3.9 - 62.5 μg/ml for leaves and 1.95 - 31.25 μg/ml for roots. In the CVC biofilm removal testing, the aqueous leaves extracts presented a greater potential. The chemical composition data obtained in this work, considering the different parts of plant, as well as the antimicrobial activity detected, bring perspectives of exploring this species concerning its biological potential.

HIV antiretroviral therapy spans several different drug classes, meant to combat various aspects of viral infection and replication. Many authors have argued the benefits of therapeutic drug monitoring (TDM) for the HIV patient including compliance assurance and assessment of appropriate drug concentrations; however, the array of drug chemistries and combinations makes TDM an arduous task. HPLC-UV and LC-MS/MS are both frequent instruments for the quantification of HIV drugs in biological matrices with investigators striving to balance sensitivity and affordability. Plasma, the dominant matrix for these analyses, is prepared using protein precipitation, liquid-liquid extraction or solid-phase extraction depending on the specific complement of analytes. Despite the range of polarities found in drug classes relevant to HIV therapeutics, most chromatographic separations utilize a hydrophobic column (C18 ). Additionally, as the clinically relevant samples for these assays are infected with HIV, along with possible co-infections, another important aspect of sample preparation concerns viral inactivation. Although not routine in clinical practice, many published analytical methods from the previous two decades have demonstrated the ability to conduct TDM in HIV patients receiving various medicinal combinations. This review summarizes the analytical methods relevant to TDM of HIV drugs, while highlighting respective challenges.

The direct sampling of analytes from surfaces under atmospheric conditions followed by mass spectrometric analysis is an ever expanding area of scientific research. Atmospheric pressure surface sampling and ionization techniques for mass spectrometry (MS) offer the ability to interrogate samples that could not be studied under vacuum conditions required of more traditional MS surface analysis techniques. The geometry and nature of materials or surfaces that can be analyzed has been greatly expanded as a result. This dissertation characterizes and shows applications of liquid microjunction surface sampling probe (LMJ-SSP) electrospray ionization systems. The presented work compares traditional analytical work flows with novel analytical workflows utilizing LMJ-SSP-MS technology. The increase of throughput and/or chemical information without the sacrifice of analytical figures of merit is shown and discussed. The readout of analytical surfaces; surfaces where analyte has ended up on a surface in a traditional work flow and not just placed there, constitutes the focus of what is presented in the preceding work. Finally the prospects for spatial liquid chromatography-mass spectrometry (LC-MS) as a powerful analytical technology „in wait‟ is discussed and supported by the presented data.