The lipid bilayer membrane of enveloped viruses, such as human immunodeficiency virus type 1 (HIV-1), plays an important role in key steps of the infection, including cell binding and uptake. Phosphatidylserine (PS) and monosialotetrahexosylganglioside (GM1) are examples of two host-derived lipids in the membrane of enveloped virus particles that are known to contribute to virus attachment, uptake, and ultimately dissemination. A quantitative characterization of their contribution to the functionality of the virus requires information about their relative concentrations in the viral membrane. In this dissertation, a gold nanoparticle (NP) binding assay for probing relative PS and GM1 lipid concentrations in the outer leaflet of different virus-like particles (VLPs) using small sample sizes is introduced. The assay evaluates both scattering intensity and resonance wavelength and determines relative NP densities through plasmon coupling as a measure for the target lipid concentrations in the NP-labeled VLP membrane. The performed studies reveal significant differences in the membrane of HIV-1 and Ebola VLPs that assemble at different intracellular sites and pave the way to an optical quantification of lipid concentration in virus particles at physiological titers. In addition, this technique was used in another application to improve the understanding of the relationship between the membrane PS lipid and the infectivity of HIV-2 and murine leukemia virus (MLV). The composition of the membrane, in particular the cholesterol (chol) content, determines its fluidity. As differences in the membrane composition of individual virus particles can lead to different intracellular fates, biophysical tools capable of probing the membrane fluidity on the single-virus level are required. In this dissertation, we demonstrate that fluctuations in the polarization of light scattered off gold or silver nanoparticle (NP)-labeled virus-like-particles (VLPs) encode information about the membrane fluidity of individual VLPs. We developed a plasmonic polarization fluctuation tracking microscopy (PFTM) which facilitated, for the first time, the investigation of the effect of chol content on the membrane fluidity and its dependence on temperature on the single-VLP level. Chol extraction studies with different methyl-β-cyclodextrin (MβCD) concentrations yielded a gradual decrease in polarization fluctuations as function of time. The PFTM revealed chol content and fluidity heterogeneities of an HIV-1 VLP population.

O benefício do consumo de compostos bioativos, como os carotenóides, tem sido amplamente demonstrado pela literatura científica. No entanto, alguns destes bioativos (como os carotenos), devido à sua hidrofobicidade, apresentam dificuldades para serem incorporados em formulações alimentícias aquosas, além de serem, dependendo da matriz alimentícia na qual estão inseridos, dificilmente absorvidos no tratogastrointestinal - ou seja, possuem limitada biodisponibilidade. Tais problemas podem ser contornados através da micro e da nanoencapsulação. O presente trabalho de Mestrado teve como objetivo utilizar a triestearina e o ácido esteárico para a encapsulação do beta-caroteno em micro e nanopartículas lipídicas sólidas, a caracterização físico-química das estruturas formadas e a avaliação da estabilidade química e microestrutural das mesmas. Nos sistemas microparticulados de ácido esteárico (AE) foi utilizado como tensoativo o polisorbato 80 e foram produzidos com 4 e 6% de lipídio, na ausência e na presença de alfa-tocoferol, e todos se mostram extremamente estáveis em relação à distribuição do tamanho médio das partículas, mas somente as partículas que continham alfa-tocoferol conseguiram preservar o beta-caroteno ao longo do período de 7 meses de armazenagem. No caso das micropartículas de triestearina também foram produzidos sistema com 4 e 6% de lipídio total, e a presença do hidrocolóide goma xantana foi essencial para evitar a floculação e permitir a estabilidade do sistema, e foram testadas formulações contendo misturas de tensoativos fosfatidilcolina de soja e polisorbato 60 e fosfatidilcolina de soja e polisorbato 20. Dentre tais sistemas, somente as micropartículas sólidas estabilizadas com polisorbato 60 se mostraram estáveis em relação ao tamanho médio das partículas, e o sistema com menor quantidade de lipídio manteve-se resistente à floculação até o 4º mês de estocagem. Sistemas nanoparticulados foram produzidos com 6% de lipídio total, testando-se uma e duas passagens no homogeneizador à alta pressão. Os dados obtidos indicaram que as nanopartículas lipídicas de AE não diferiram em relação à distribuição de tamanho, mas apresentaram aumento do diâmetro de partícula ao longo do tempo de estocagem. Por sua vez, para as nanopartículas de triestearina os sistemas (tanto com uma quanto com duas passagens no homogeneizador a alta pressão) se mostraram estáveis até cerca de dois meses de armazenagem, em termos de diâmetro médio de partícula, sendo que a distribuição de tamanho se mostrou mais homogênea para o sistema com duas passagens. A microestrutura de todos os sistemas foi avaliada por difratometria de raio-X (DRX) e calorimetria diferencial de varredura (DSC), e a quantidade de beta-caroteno preservada ao longo do tempo foi monitorada espectofometricamente e por colorimetria instrumental. De maneira geral, os sistemas microparticulados se mostraram melhores do que os nanoparticulados, tanto do ponto de vista de estabilidade da estrutura quanto da preservação do beta-caroteno. / The benefits from the consumption of bioactive compounds, like carotenoids, have been widely demonstrated for scientific literature. However, some of this compounds (like carotenes), due totheir hydrophobicity, are difficult to be incorporated in aqueous food formulations, and, depending on the food matrices where they are introduced, are hardly absorbed in the gastrointestinal tract - in order words, they present limited bioavailability. These problems can be overcome by micro and nanoencapsulation. In this context, the objective of this study was to investigate the temporal stability of beta-carotene encapsulated in solid lipid micro and nano particles produced with a mixture of stearic acid or tristearin and sunflower oil, monitoring the microstructure of the systems by X-ray diffractometry, differential scanning calorimetry, zeta potential and particle size measurements, and try to link the preservation of beta-carotene with microstructural considerations. The surfactant used for the stearic acid microparticulate systems was polysorbate 80 and formulations with 4 and 6% of total lipid were produced, in the absence and presence of alpha-tocopherol, and all systems showed high stability in terms of average particle diameter and size distribution, but only the particles containing alpha-tocopherol preserved the content of beta-carotene during the storage period of 7 months In the case of the tristearin microparticles the presence of a hydrocolloid (xanthan gum) was essential for avoid flocculation and improves the system stability, and formulations containing mixtures of surfactants (soybean phosphatidylcholine and polysorbate 60 and phosphatidylcholine and polysorbate 20) were tested. Among such systems, only the solidmicroparticles stabilized with phosphatidylcholine and polysorbate 60 showed stability in terms of average particle diameter and size distribution, and the system with less concentration of solid lipid did not show significant destabilization until the 4th month of storage. As for the nanoparticulated systems, formulations with 6% of total lipid were produced, testing one and two passages in high pressure homogenizer. Our results indicated the stearic acid solid nanoparticles did not exhibitalterations of size distribution, but average particle diameter increased along the time. On the other hand, the triestearin nanoparticles (both with one and two passage in high pressure homogeneizer) showed stability until two months of storage, in terms of average particle diameter, and the size distribution demonstrated to be more homogeneous for the systems submitted to two passages. As an overall conclusion, the microparticulated systems seemed to be more stable than the nanoparticulated ones, from the point of view of structure stability as well as in terms of beta-carotene preservation of beta-carotene.

Freshwater zooplankton crustacean Daphnia frequently face strong temperature fluctuations in its natural environment, which necessitates adaptive plastic responses. This study focuses on changes in lipid peroxidation and total oxidative capacity in Daphnia tissues in response to long-term and short-term temperature changes. Long-term acclimation to 28ºC helped Daphnia survive longer at lethally high temperatures. This difference, however, was not accompanied by changes in lipid peroxidation, indicating that it isn’t a good measure of damage or predictor of temperature tolerance. On the other hand, total oxidation capacity was lower 28ºC- than in 18ºC-acclimated Daphnia, suggesting that acclimation resulted in higher amounts of antioxidants in Daphnia tissues. Exposure to hypoxia, known to up-regulate antioxidant pathways in Daphnia, further elevated heat tolerance in 28ºC- acclimated individuals. Yet, manipulations of glutathione, an important antioxidant, while predictably affecting oxidative capacity, didn’t influence heat tolerance in Daphnia, suggesting that other antioxidants may play a significant role in it.

Orientador: Antônio Fernando Monteiro Camargo / Banca: Rogério Falleiros Carvalho / Banca: Irineu Bianchini Junior / Resumo: O estresse oxidativo causado pela produção de espécies reativas de oxigênio (EROs) é uma das respostas que as plantas apresentam frente a um estresse ambiental. Nosso objetivo foi avaliar o estresse oxidativo das macrófitas aquáticas Crinum americanum e Spartina alterniflora em duas condições: salinidade e nutrientes que ocorrem no estuário do rio Itanhaém (SP). Raízes e folhas (5 plantas) de C. americanum foram coletadas em cada área (5 amostras/área). O material vegetal para análises de clorofila (a+b), carotenóides, peróxido de hidrogênio (H2O2), malonaldeído (MDA) e enzimas antioxidantes SOD e CAT foram acondicionado em N2 líquido no campo e posteriormente armazenado em freezer a -80 °C. Também foram coletados em cada réplica material vegetal para avaliar o teor de N e P totais da planta e do sedimento Os dados obtidos foram submetidos à Análise de Variância (ANOVA) e comparados pelo teste de Tukey (p < 0,05). Os resultados obtidos mostraram que: C. americanum e S. alterniflora sofreram maior estresse oxidativo em alto e médio estuário, respectivamente; Os indivíduos de S. alterniflora sofreram maior estresse na área de lançamento de esgoto. / Abstract: The oxidative stress caused by the production of reactive oxygen species (ROS) is one of the responses that plants exhibit facing environmental stress. Our objective was to evaluate the oxidative stress of the aquatic macrophytes Crinum americanum and Spartina alterniflora under two conditions: salinity and nutrients that occur in the estuary of the Itanhaém river (SP). Roots and leaves (5 plants) of C. americanum were collected in each area (5 samples / area). The plant material for analysis of chlorophyll (a + b), carotenoids, H2O2, MDA and antioxidant enzymes SOD and CAT with platform in N2 without field and liquid in freezer at -80 ° C. Vegetable material was also collected in each replicate to evaluate the total N and P content of the plant and of the sediment. The data were submitted to Analysis of Variance (ANOVA) and compared by the Tukey test (p <0.05). The results showed that: C. americanum and S. alterniflora suffered higher oxidative stress in upper and middle estuary, respectively; The State of São Paulo changes have suffered greater stress in the area of sewage / Mestre

Interleukin-1-beta (IL-1β) is a potent proinflammatory cytokine. A primary outcome of IL-1β signaling is the activation of NFκB, a transcription factor that induces a large number of immune molecules, apoptotic factors, anti-apoptotic factors, and other transcription factors. Recent work has demonstrated that the activation of NFκB involves a multistep redox-signaling cascade that requires endocytosis of the interleukin receptor (IL-1R1)/ligand pair and superoxide production by NADPH oxidase 2 (Nox2) within the resulting newly formed early endosome. Hydrogen peroxide produced by the rapid dismutation of superoxide is necessary for the subsequent downstream recruitment of IL-1R1 effectors (TRAF6, IKK kinases) and ultimately the activation of NFκB. In this thesis, I have further dissected the spatial and temporal events that coordinate signaling processes of the IL-1β pathway. Using a combination of biophotonic imaging, immunofluorescence imaging, and lipid raft density gradient isolation, I demonstrate that both Nox2 and IL-1R1 are constitutively present in lipid raft microdomains on the plasma membrane. Stimulation by IL-1β induces endocytosis of Nox2 and IL-1R1 from the plasma membrane into caveolin-1, lipid raft positive early endosomes. Further, inhibition of lipid raft mediated endocytosis or deletion of caveolin-1 inhibits activation of NFκB, by IL-1β. We have also identified Vav1 as the Rac1 guanine exchange factor that is recruited to caveolin-1 positive lipid rafts following IL-1β stimulation, and demonstrated that dominant negative Vav1 inhibits NFκB activation by IL-1β. Following this work, I utilized assays for redox sensitivity and mass spectrometry to demonstrate that C70, C73, and C105 are hydrogen peroxide sensitive cysteines within the RING domain of TRAF6. I further demonstrate that hydrogen peroxide does not alter the E3 ubiquitin ligase activity associated with the TRAF6 RING domain. My findings suggest that the redox sensitivity of the RING domain mediates TRAF6 recruitment to the receptor complex. This is supported by the observation that hydrogen peroxide treatment of TRAF6, but not early signaling effectors (IL-1R1, IRAK1, IRAK4, MyD88) mediates TRAF6 recruitment to the IL-1 receptor complex. Further, mutation of the identified redox sensitive cysteines inhibits IL-1β signaling and NFκB activation. This research has helped to refine the understanding of the IL-1β signaling pathway, and may ultimately lead to new therapeutic targets for controlling inflammation.

Preterm birth is defined as delivery prior to 37 weeks’ completed gestation. It affects an average of 11% of pregnancies worldwide and is the leading cause of death in children under age 5. Many studies have identified associations between pregnancy lipid levels and increased risk for preterm birth. This thesis investigates the role of genetic variability associated with lipids and its relationship with preterm birth, and the relationship between pre-pregnancy dyslipidemia and risk for preterm birth. Genetic variability in the form of single-nucleotide polymorphisms, previously identified by genome-wide association studies for association with lipid levels, was analyzed for association with risk for preterm birth. The study population included 992 women in California with banked 2nd trimester serum samples. Serum lipid levels and DNA were used. Genetic risk scores were constructed for each subject using published SNPs associated with lipid levels as an indicator of genetic burden. These genetic risk scores were then analyzed for association with risk for preterm birth. The GRS were not associated with the overall risk for preterm birth. However, a higher HDL-C GRS was associated with increased risk for spontaneous preterm birth. Higher triglyceride and total cholesterol GRS were associated with decreased risk for spontaneous preterm birth. The relationship between pre-pregnancy dyslipidemia and risk for preterm birth was assessed in a cohort of 2,962,434 women giving birth in the state of California from 2007-2012. Dyslipidemia, as defined by medical diagnostic codes, was associated with a 1.5-fold increase in risk for preterm birth. This association was consistent across race/ethnicity, body mass index, type of dyslipidemia, and type of preterm birth. This thesis identified counter-intuitive associations between lipid GRS and spontaneous preterm birth, while also identifying a strong relationship between pre-pregnancy dyslipidemia and all types of preterm birth including spontaneous. Together, these findings suggest that the previously reported associations between lipids and preterm birth may be reflecting unidentified dyslipidemias. One possible interpretation of the counter-intuitive genetic findings is that while extreme dyslipidemia predisposes to preterm birth a genetic predisposition to low total cholesterol also confers increased risk for spontaneous preterm birth. An alternative explanation is that these results are simply an artefact of the data and additional genetic loci and lifestyle factors confer stronger effects on risk for spontaneous PTB than the effects of the genetic loci included in this thesis.

The detection and analysis of lipids in biological matrices for clinical applications poses many challenges, but rapid and reliable detection will prove invaluable for clinical diagnosis. Herein, we report the application of drop-casted Ag nanoplatelets as surface enhanced Raman scattering (SERS) substrates for qualitative detection of 20-hydroxyeicosatetraenoic acid (20-HETE), which is a potential biomarker for diagnosis of hypertensive disorders. Biomarker peaks of 20-HETE can be reliably detected and differentiated from those of the structurally similar lipids (arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid) commonly found in human blood, even 1 pM concentrations. Additionally, one study mixed 20-HETE with three structurally similar lipids at concentrations several orders of magnitude greater than the target lipid and 20-HETE could still be detected under these conditions. These experiments demonstrate the viability of SERS for the rapid and reliable detection of endogenous bioactive lipids, which has significant clinical impact in enabling point of care diagnostics.

Alcoholic liver disease (ALD) is one of the most common liver diseases worldwide characterized by the accumulation of lipids within the liver, inflammation and the possibility of progressing to cirrhosis and liver failure. More importantly, there are currently no effective treatments for ALD and liver transplantation remains the only therapeutic option for end-stage liver disease. Previous studies have shown that ALD is a result of a combination of endoplasmic reticulum (ER) stress, lipid metabolism dysregulation and inflammation. It has been previously reported that alcohol disrupts gut microbiota homeostasis and causes increased endotoxins that contribute to the pathology of ALD. However, the detailed mechanism(s) underlying ALD and disease progression is poorly understood. We have discovered that sphingosine kinase 2 (SphK2) deficient (SphK2-/-) mice on an alcohol diet exhibit increased steatosis and inflammation compared to wild type mice. Sphingosine 1-phosphate receptor 2 (S1PR2) and SphK2 have been previously shown to play a key role in nutrient metabolism and signaling. However, their roles in alcohol-induced liver injury have not been characterized. The overall objective of this study is to determine the molecular mechanism(s) by which disruption of S1PR2-mediated SphK2 signaling contributes to ALD. The effects of alcohol on mouse primary hepatocytes and cultured RAW264.7 macrophages were examined. The acute on chronic alcohol mouse model from NIAAA that recapitulates the drinking pattern of human ALD patients was used to study the effects of SphK2 deficiency in ALD. In addition, 60-day chronic alcohol mouse model was used to determine whether a more severe form of ALD was present in SphK2-/- mice. The results indicated that SphK2-/- mice on an alcohol diet exhibited an increased amount of hepatic steatosis compared to wild type mice. Genes regulating lipid metabolism were also dysregulated in SphK2-/- mice. SphK2-/- mice also had increased inflammation and liver injury as shown by an upregulation of inflammatory markers and increased levels of liver enzymes. Moreover, SphK2 protein expression levels were downregulated in the human livers of alcoholic cirrhotic and hepatocellular carcinoma (HCC) patients. These findings contribute to a greater understanding of the pathophysiology of ALD and could provide information on the development of novel therapeutics against ALD.

Dyslipidemia often accompanies metabolic diseases such as obesity and type II diabetes mellitus and represents a risk factor for cardiovascular disease. Clearance of triglycerides from the plasma is mediated by lipoprotein lipase (LPL), which hydrolyzes the triglycerides in chylomicrons and VLDL, liberating fatty acids for tissue uptake. LPL functions in the capillaries of the heart, adipose tissue, and skeletal muscle where LPL is anchored to the capillary wall by its endothelial cell transporter GPIHBP1. LPL activity is regulated by several factors including three members of the angiopoietin-like (ANGPTL) family–ANGPTL3, ANGPTL4, and ANGPTL8. How these proteins interact with LPL, especially in the physiological context of LPL anchored to endothelial cells by GPIHBP1, has not been well characterized. In my studies of ANGPTL4, I found when LPL is bound to GPIHBP1, it is partially, but not completely, protected from inactivation by ANGPTL4. Inactivation of LPL by ANGPTL4 leads to the dissociation of active LPL dimers into inactive monomers and I found that these monomers have a greatly reduced affinity for GPIHBP1. ANGPTL4 can be cleaved in vivo, separating the N-terminal coiled-coil domain from the C-terminal fibrinogen like-domain. I found the N-terminal domain alone is a much more potent LPL inhibitor than the full-length protein, even though both appear to have similar binding affinities for LPL-GPIHBP1 complexes. When I investigated ANGPTL3, I found ANGPTL3 itself is not a potent inhibitor of LPL at physiological concentrations, and unlike ANGPTL4, cleavage of ANGPTL3 does not improve its ability to inhibit LPL. Instead I found that ANGPTL3 forms a complex with ANGPTL8, a complex that only forms efficiently when the two proteins are co-expressed, and that this complex allows ANGPTL3 to bind and inhibit LPL. My data provide new insights into how ANGPTL proteins regulate LPL activity and the delivery of fat to tissues.

The continued increase in the demand for fossil fuels combined with their ever dwindling supply has prompted the search for a suitable alternative fuel. The research contained within this dissertation seeks to increase the lipid content of cellular feedstocks, improve extraction efficiencies of lipids, and understand the pathways involved in the production of fatty alcohols and triacylglycerides from microbial feedstocks. As part of this research the diatom, Cheatoceros gracilis, was grown at small and large scale to determine optimal growing conditions. No apparent nutrient stress trigger was required to initiate the accumulation of the biodiesel precursor triacylglyceride, unlike other documented algal strains. A follow-up to this project demonstrated that the microalga C. gracilis may utilize light intensity as a trigger for lipid production. A major difficulty in the production of biofuels from microorganisms is the expensive process of dewatering, drying, and extracting the lipid compounds from the cells. As part of this research, a process has been developed that allows for lipid extraction to occur in the presence of water at a point as low as 2 percent solids or 98 percent water. This process utilizes a single organic solvent that mixes well with microbial lipids, but poorly with water allowing for efficient extraction of lipids and fast solvent to water separation. This process greatly decreases the cost of the microbial biofuels production associated with the removal of water from cell slurries. Triacylglycerides and fatty alcohols are oleochemicals that are commonly used in industrial, pharmaceutical, and consumable processes. A predicted fatty acyl CoA reductase enzyme was cloned into an E. coli vector, expressed, characterized and shown to be active as a dual reductive enzyme reducing a fatty acyl CoA to its respective fatty alcohol, constituting the first enzyme of this type discovered in a bacterium. The process of triacylglyceride production in microbes is fairly well understood; however, the process that regulates this production has not yet been fully explored. As part of this research, the model yeast organism, Yarrowea lipolytica, is utilized to identify essential genes for citrate transport that if removed could result in increasing triacylglyceride production in vivo.