Hepatitis C virus (HCV) is remarkably capable of efficiently hijacking host cell pathways including lipid metabolism in the liver in order to create pro-viral environments for pathogenesis. It is becoming increasingly clear that identifying small molecule inhibitors that target host factors exploited by the virus will expand available HCV treatment options. As such, a thorough understanding of host-virus interactions is critical to the development of alternative therapeutic strategies. Hepatic lipid droplets (LDs) are recruited by HCV to play essential roles in the viral lifecycle. The intracellular location of LDs is modified upon interacting with viral structural core protein. This enables formation of platforms that support viral particle assembly. Because these interactions are non-static, capturing its dynamic processes in order to better understand viral assembly can be achieved with label-free molecular imaging enhanced with live-cell capabilities. Chemical biology approaches that includes CARS microscopy employed in a multi-modal imaging system was used to probe interactions between HCV and host LDs. By successfully tracking LD trajectories, we identified core protein’s ability to alter LD speed and control for LD directionality. Using protein expression model systems that allowed for simultaneous tracking of core protein and LDs, our data revealed that mutations in the core protein region that vary in hydrophobicity and LD binding strengths, are factors that control for differential modulation of LD kinetics. Furthermore, we measured bidirectional LD travels runs and velocities, and observed critical properties by which core protein induces LD migration towards regions of viral particle assembly. Given that many steps in the HCV lifecycle are directly linked to host lipid metabolism, it is not surprising that disrupting lipid biosynthetic pathways would negatively affect viral replication. From this outlook, we explored small molecule inhibitors that targeted several lipid metabolic pathways to study its antiviral properties. Using fluorescent probes covalently labeled to viral RNA, we captured the visualization of disrupted replication complexes upon antagonizing nuclear hormone receptors that are linked to regulating lipid homeostasis. Correspondingly, biochemistry and molecular imaging techniques were also employed to identify novel antiviral mechanisms of small molecule inhibitors that target additional HCV-dependent lipid metabolic pathways.

Vesicles are simple structures, but display complex, non-linear dynamics in fluid flow. I investigate the deformation of nanometer-sized vesicles, both fully-inflated and those with excess area, as they travel in tightly confined capillaries. By varying both channel size and flow strength, I simulate vesicles as they transition from steady-state to unstable shapes, and then rupture in strong flow fields. By employing a molecular dynamics model of the vesicle, fluid, and capillary system one is able to rupture the lipid bilayer of these vesicles. This is unique in that most other numerical methods for modelling vesicles are unable to show rupture. The rupture of fully-inflated vesicles is applicable to drug delivery in which the release of the encapsulated medicine needs to be controlled. The deformation and rupture of vesicles with excess area could be applicable to red blood cells which have similar rheological properties.

Mass spectrometry has increasingly become the method of choice for the analysis of complex biological samples, including proteins and lipids. This thesis describes the development of MS-based analytical methodologies for the analysis of complex proteomic and lipidomic samples. Chapter 3 describes the development of microfluidic proteomic reactors, in the formats of SCX reactor, SCX 96-well plate reactor, and SAX reactor, for the enzymatic digestion of complex proteomic samples for subsequent LC-MS/MS analysis. These microfluidic proteomic reactors greatly simplified the enzymatic digestion of complex proteomic samples by combining multiple processing steps, such as rapid extraction and enrichment of proteins. Furthermore, chemical and enzymatic treatments of proteins were all performed in a few nanoliters effective volume, resulting in an increased protein digestion efficacy. After the protein digestion process, the resulting peptides were eluted in buffers that were compatible with HPLC-MS/MS analysis. In chapter 4, a methodology based on nano-HPLC-ESI-MS/MS for the analysis of PAF and LPC lipid species is described. In this method, lipid extracts from biological samples were separated by nano-flow HPLC prior to being introduced into a Q-TRAP 2000 mass spectrometer, where the lipid species of interest were detected using a precursor ion scan at m/z 184. Absolute quantitation of PAF family lipid species were performed with standard addition method, where 5 standard solutions containing 0.2-1 ng each of C16:0, C18:0 PAF and C16:0, C18:0 lyso-PAF were used in the experiment. Further, the spiking of identical amount of non-endogenous C13:0 LPC at time of extraction allow the relative comparisons of other LPC lipid species of interest between different samples. The developed methods were employed to analyze the changes of PAF and LPC lipid species in NGFdifferentiated PC12 cells, in the posterior/entorhinal cortex of AD patients and TgCRND8 transgenic mice, and over the course of 24 hour exposure of human hNT neurons to Aβ42 treatment, respectively, in comparison to controls. iii Chapter 5 describes the development of a novel shotgun lipidomic methodology for the determination of stereospecificity of diacyl glycerophospholipids including glycerophosphatidic acids (PA), glycerophosphoserines (PS), glycerophosphoglycerols (PG), glycerophosphoinositols(PI), and glycerophosphoethanolamines (PE), which can be conventionally ionized under negative ion mode. The stereospecificity of diacyl glycerophospholipids was determined based on the relative abundance of the lyso-form fragment ions, attributed to the neutral loss of fatty acyl moieties. The fragmentation patterns of a variety of diacyl glycerophospholipid standards were first fully examined over a wide range of collision energy. We observed that lyso-form fragment ions corresponding to the neutral loss of fatty acyl moieties attached to the sn2 position as free fatty acids ([M-Sn2]-) and as ketenes ([M-(Sn2-H2O)]-) exhibited consistently higher intensity than their counter part ions due to the neutral loss of fatty acyl moieties attached to the sn1 position ([M-Sn1]- and [M-(Sn1-H2O)]-). We then examined the product ion spectra of diacyl glycerophospholipids recorded from lipid extracts of rat hepatoma cells, where the stereospecific information of these lipids was conclusively determined.

This thesis is a study of the effect of lipid injections on complement titers of guinea pigs.

Background: Clinical management of dyslipidemia is challenging, particularly hypertriglyceridemia in patients with HIV-infection. Changing combined anti-retroviral therapy (CART) and the use of lipid-lowering drugs have proven useful in treating dyslipidemia in HIV infected patients Objective: To assess the efficacy of lipid lowering drugs (LLDs) and/or CART switching, in the management of HIV-associated dyslipidemia Design: A retrospective, longitudinal cohort study Setting: Phidisa HIV research project, 6 sites in South Africa, period April 2008 and April 2011 Patients: HIV positive South African National Defence Force (SANDF) members and their dependents; who are on CART and are 18 years or older. Four hundred and forty eight participants with dyslipidemia had non-fasted, total serum cholesterol ≥ 8.0mmol/l, serum triglyceride levels ≥4.52 mmol/l and naïve to lipid lowering drugs at baseline. Measurements: Mean change over time of total serum cholesterol and serum triglyceride in the following treatment strategies were used: exercise and dietary advice, lipid-lowering drugs (statins or fibrates or both), CART switches separately and combined lipid lowering drug with ART switch was measured using panel data with first–order autoregressive-response and xtabond. Results: The mean age for a total of 448 participants was 39.9 years; males were 87%, females were only 13%. The participants contributed to 1861 follow-up visits. CD4 count was normally distributed with the baseline mean value of 402 cells/mm3 (18.5%). Mean change over time for total serum cholesterol and triglycerides increased by 0.099 mmol/l (p=0.007) and 0.248 mmol/l (p=0.018) respectively, with an increase in body mass index while an increase in CD4 cell percent decreased mean over time for total serum cholesterol by 0.045 mmol/l (p=0.002). Our hypothesis was confirmed when lipid lowering drugs and ART switch combined treatment strategy even more decrease in the mean total serum cholesterol and triglycerides levels over time by 0.754 mmol/l (p<0.001) and 2.073 mmol/l (p<0.001) respectively compared to the exercise and dietary advice treatment strategy. Our findings showed that combined treatment strategy maintained a decrease in both the mean total serum cholesterol and triglycerides levels over time of 0.283 mmol/l (p=0.038) and 0.941 mmol/l (p=0.016) respectively, when compared to lipid lowering drugs; the mean serum triglycerides over time were also reduced by 0.486 mmol/l (p=0.048) when the combined treatment strategy was compared to CART switch only. Furthermore combined treatment strategy of lipid lowering drugs with ART switch showed significant virological suppression by decreasing log of viral load, 0.486 (p<0.001) when compared to the exercise and dietary advice group. Conclusions: Combining lipid lowering drugs and ART switching as a treatment strategy in the management of HIV-associated dyslipidemia is effective in lowering the mean over time of both total serum cholesterol and triglycerides when compared to exercise and dietary advice strategy, while maintaining virological suppression. / Dissertation (MSc)--University of Pretoria, 2015. / tm2015 / School of Health Systems and Public Health (SHSPH) / MSc / Unrestricted

Orientador: Nelson Eduardo Durán Caballero / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-18T16:38:17Z (GMT). No. of bitstreams: 1 Ridolfi_DanielaMissiani_M.pdf: 1638152 bytes, checksum: e0dcd5884432b022ba79cd8e69b9512f (MD5) Previous issue date: 2011 / Resumo: Tretinoína (ácido todo-trans-retinóico) é empregado no tratamento tópico de várias doenças de pele, no entanto sua utilização é fortemente limitada pelos efeitos colaterais que apresenta e pela sua alta instabilidade química. Neste trabalho tretinoína foi encapsulada em nanopartículas lipídicas sólidas (NLS-TRE) e em NLS recobertas com quitosana (NLS-Quitosana-TRE). Ambas as partículas apresentaram alta eficiência de encapsulação, alta estabilidade física e morfologia esférica. As NLS-Quitosana-TRE apresentaram menor cristalinidade em relação às NLS sem quitosana. A capacidade de transporte das nanopartículas foi limitada pela baixa taxa de solubilização da tretinoína no lipídio fundido, nas condições de preparação. A adição de etanol na preparação das nanopartículas aumentou a capacidade de transporte, no entanto a estabilidade das dispersões foi alterada (as NLS sem quitosana permaneceram estáveis por apenas um mês e as NLS com quitosana se desestabilizaram logo após a preparação). Ambas as partículas não apresentaram potencial citotóxico em células de fibroblastos e queratinócitos. A encapsulação de tretinoína em NLS reduziu de forma significativa sua fototoxidade, o que evidencia o efeito protetor da matriz lipídica. As NLS-Quitosana-TRE apresentaram alta atividade antibacteriana contra as principais bactérias envolvidas na acne (S. epidermidis e P. acnes) e contra a S. aureus, também envolvida em infecções de pele. Os resultados obtidos neste trabalho permitem concluir que as NLS, com e sem recobrimento com quitosana, possuem um grande potencial para encapsulação de tretinoína em aplicações dérmicas. O recobrimento com quitosana pode melhorar ainda mais as propriedades das NLS como sistema carreador de tretinoína, uma vez que as NLS-Quitosana-TRE apresentaram atividade antibacteriana contra bactérias envolvidas em infecções de pele e desta forma podem aumentar a eficácia terapêutica no tratamento tópico da acne e de outras doenças de pele / Abstract: Tretinoin (all-trans retinoic acid) is employed in the topical treatment of various skin diseases, however, its uses is strongly limited by their side effects and high chemical instability. In this work tretinoin was encapsulated in solid lipid nanoparticles (SLN-TRE) and SLN coated with chitosan (SLN-Chitosan-TRE). Both particles exhibited high entrapment efficiency, high physical stability and spherical morphology. The SLN-chitosan-TRE presented lower crystallinity compared to SLN without chitosan. The loading capacity of nanoparticles was limited by the low solubilization rate of tretinoin in the melted lipid at the preparation's conditions. The addition of ethanol in the nanoparticles preparation increased the loading capacity, however the dispersion stability was altered (the SLN without chitosan remained stable by only one month and the SLN with chitosan destabilized after preparation). Both particles were not cytotoxic to either fibroblasts or keratinocytes cells. The tretinoin encapsulation in SLN decreased significantly its phototoxicity, which shows a protector effect by the lipid matrix. The SLN-Chitosan-TRE exhibited high antibacterial activity against the main bacteria involved in the acne (S. epidermidis and P. acnes) and against the S. aureus which is involved in skin infections. The results obtained in this work allows us to conclude that the SLN, with and without coating with chitosan, have a great potential for encapsulation of tretinoin in dermal application. The coating with chitosan can improve the SLN properties as carrier for tretinoin because the SLN-Chitosan-TRE exhibited antibacterial activity against bacteria involved in skin infections and therefore can improve the therapeutic efficacy in the topical treatment of acne and other skin diseases / Mestrado / Físico-Química / Mestre em Química

Orientadores: Priscilla Efraim, Ana Paula Badan Ribeiro / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-26T16:56:00Z (GMT). No. of bitstreams: 1 Lopes_JuliceDutra_D.pdf: 26424877 bytes, checksum: eced1650a81af1c56e7eb7df9b5ca109 (MD5) Previous issue date: 2015 / Resumo: A cristalização de gorduras é um fenômeno complexo que, se controlada, pode ser aplicada em diversos setores das indústrias químicas e de alimentos. Na produção de chocolates, o uso de agentes ativos de nucleação pode se dar pela incorporação de triacilgliceróis de alto ponto de fusão, fundidos ou na forma sólida, à massa de chocolate ou à manteiga de cacau, resultando em um grande número de núcleos de cristalização bem definidos, que consistem na base para a ordenação estrutural do sistema, podendo melhorar e/ou acelerar a etapa de temperagem de chocolates. Baseando-se nestes fatos, o objetivo deste trabalho foi produzir e caracterizar micropartículas lipídicas sólidas (SLM) de óleos totalmente hidrogenados (hardfats) de palma (FHPO), algodão (FHCO), soja (FHSO) e crambe (FHCrO) utilizando a técnica de spray cooling (SC), a fim de se estudar a influência da adição dessas SLM como agentes de nucleação no processo de temperagem de chocolate amargo. As SLM foram produzidas variando-se os parâmetros de processo: temperatura de manutenção dos hardfats (70 a 85 °C), temperatura de solidificação das SLM (0 a 24 °C), diâmetro do atomizador (0,7 e 1 mm) e pressão do ar comprimido de atomização (1,25 a 1,75 kgf/cm2). As SLM produzidas apresentaram polimorfismo ?, formato esférico e diâmetros médios entre 56,12 e 2514,32 ?m, dependendo dos hardfats e das condições de processo utilizadas. A cinética de transição polimórfica das SLM acondicionadas a 25, 35 e 45 °C foi investigada por 141 dias, avaliando-se a configuração visual, o comportamento térmico de fusão e o polimorfismo das SLM. A transição completa das SLM para o polimorfismo de seus respectivos hardfats ocorreu de forma mais rápida no acondicionamento a 45 °C, com transição completa observada nas SLM do FHSO, FHPO e FHCO após 24 h, sem alteração do aspecto visual. Em 35 °C, a transição das SLM do FHPO e FHCO ocorreu após 4 dias e em 25 °C observou-se transição completa apenas nas SLM do FHCO. Para as SLM do FHCrO o polimorfismo ? foi predominante nos tratamentos a 25 e 35 °C e só houve transição polimórfica no tratamento a 45°C após 15 dias. Observou-se redução do tempo da etapa de temperagem de chocolates adicionados de SLM do FHSO com polimorfismo ?, os quais apresentaram tensão de ruptura e cor similares às de chocolates submetidos à temperagem realizada da forma convencional. Os resultados indicam que a técnica de SC é adequada para produção de SLM dos hardfats estudados. Porém, para o uso destas SLM como agentes de cristalização é necessária a indução da transição polimórfica para o polimorfismo mais estável, preferencialmente de forma não estática, para evitar a formação de aglomerados / Abstract: Fats crystallization is a complex phenomenon which, when controlled, can be applied in several chemical and food industries sectors. In chocolate production, the use of nucleating agents can occur by the incorporation of high melting point triglycerides, melted or in solid form. The seeds can be added directly in the chocolate mass or in the cocoa butter, resulting in a large number of well-defined crystallization nuclei, which are the basis for the structural arrangement of the fat crystal network. In this way, the seeds can improve and/or accelerate the chocolate tempering step. Based on these facts, the objective of this research was to produce and to characterize solid lipid microparticles (SLM) from fully hydrogenated oils (hardfats) of palm (FHPO), cottonseed (FHCO), soybean (FHSO) and crambe (FHCrO), using the spray cooling (SC) technique in order to study the influence of the addition of these SLM as nucleating agents in dark chocolate tempering process. The SLM were produced by varying the process parameters: hardfats maintenance temperature (70 to 85 °C), SLM solidification temperature (0 to 24 °C), nozzle diameter (0.7 to 1 mm) and atomiser air pressure (1.25 to 1.75 kgf/cm2). The SLM produced presented ? polymorphism, spherical shape and mean diameter between 56.12 and 2514.32 µm, depending on the hardfats and process conditions used. The polymorphic transition kinetics of SLM stored at 25, 35 and 45 °C was investigated by 141 days evaluating the visual configuration, the thermal melting behavior and the SLM polymorphism. The full transition from SLM to polymorphism of their respective hardfats occurred more quickly at 45 °C, with full transition of SLM observed in the SLM of FHSO, FHPO and FHCO after 24 hours, without altering the visual aspect. At 35 °C, the SLM transition of FHPO and FHCO occurred after 4 days, and at 25 °C the complete transition was observed only in the SLM of FHCO. For SLM of FHCrO, ? polymorphism was predominant in the treatments at 25 and 35 °C and there was only polymorphic transition in the treatment at 45 °C after 15 days. It was observed time reduction on the tempering chocolates steps with the addition of SLM of FHSO with ? polymorphism, which showed similar rupture tension and color to the chocolate subjected to the conventional way of tempering. The results indicate that SC technique is suitable for the SLM production of the studied hardfats. However, for use of these SLM as crystallization agents is required to induce the polymorphic transition for the most stable polymorph, preferably non-static way in order to avoid the agglomerates formation / Doutorado / Tecnologia de Alimentos / Doutora em Tecnologia de Alimentos

Polychlorinated biphenyls (PCBs) are ubiquitous environmental pollutants that have been associated with various adverse health effects in humans and wildlife. Dioxin-like PCBs elicit a broad spectrum of biochemical and toxic changes including cardiovascular disorders. Paraoxonase 1 (PON1), an antioxidant enzyme, prevents oxidative stress and plays key roles in the pathogenesis of atherosclerosis. The overall goal of this dissertation is to investigate the mechanism and role of PON1 in the antioxidant defense to the exposure of 3, 3', 4, 4', 5-pentachlorobiphenyl (PCB 126), the most potent congener in PCB family. My overall hypothesis is that: The up-regulation of PON1 is an antioxidant response to PCB 126 exposure, which involves the Ah receptor (AhR) and results in changes in the PON1 protein level and activity which in turn has an influence on the oxidative stress status. First, the responses of PON1 gene expression and activities in serum and liver upon the PCB126 treatment were evaluated in the rat model. I found that PCB 126 up-regulated PON1, gene expression and activities, in a time and dose dependent manner. Next, I investigated the molecular mechanism of this response. My results show that the up-regulation of PON1 by PCB 126 involves the AhR and the XRE-like sequence in the gene promoter region. The up-regulation of PON1 was tissue specific, and this response probably protected liver and serum from lipid oxidation to some extent. The structure-activity relationship studies with PCB congeners indicate that the up-regulation of PON1 was specific to dioxin-like PCB 126. Other different AhR ligands displayed different PON1 induction capabilities. Also, reduction of PON1 activity was found in male rats dosed with non-dioxin-like PCBs. Finally I investigated the interaction, and possible protection, of other antioxidants (Se and NAC) on the response to PCB 126. I found that these antioxidants reduced the magnitude of the response of PON1 to the PCB 126 exposure in liver. Both the increase of PON1 activities and addition of antioxidants may be the reason for the lack in increase of lipid peroxidation. In total, these findings support my hypothesis and suggest that up-regulation of PON1 by PCB 126 may be an adaptive antioxidant mechanism that is involved in the body's antioxidant system.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21841号 / 農博第2354号 / 新制||農||1069(附属図書館) / 学位論文||H31||N5213(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 栗原 達夫, 教授 植田 充美, 教授 小川 順 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21959号 / 医博第4501号 / 新制||医||1037(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 横出 正之, 教授 小杉 眞司, 教授 長船 健二 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM