Advanced fluorescence techniques were used to explore tree distinct topics concerning biological membrane and their interactions. Following thesis is according to the topic divided into three parts: 1) Ionic effects were studied employing time dependent fluorescence shift experiments and molecular dynamic simulations. Combination of these two approaches are suitable to reveal characteristic like mobility and hydration of particular bilayer segment, lipid packing or ion binding sites. Halide anions were reported to adsorb to the cationic lipid bilayer specifically, altering membrane mobility and organization. Changes in observed parameters follows Hofmeister order. Their effect is mediated either by direct ionic interaction (soft, polarizable ions) as well as via alteration of water structure (hard, non-polarizable ions) in proximity of ion molecule. Further, divalent calcium was shown to bind strongly to neutral and negatively charged lipid bilayers. Several types of binding sites depending on calcium concentration were identified. 2) Two complementary lipopeptides, CPK and CPE, incorporated into distinct lipid bilayers serve as a minimal model inducing membrane fusion. Effectiveness of fusion event might be influenced by lipopeptide-membrane and lipopeptide-lipopeptide interaction. To reveal...

Cancer cell esterases are often overexpressed and can have chiral specificities different from that of the corresponding normal cells and can, therefore, be useful targets for activating chemotherapeutic prodrug esters. Prodrug esters are inactive compounds that can be preferentially activated by esterase enzymes. Moreover, cancer cells often exhibit a high level of intrinsic oxidative stress due to an increased formation of reactive oxygen species (ROS) and a decreased expression of some enzymatic antioxidants. Prodrugs designed to induce additional oxidative stress can selectively induce apoptosis in cancer cells already exhibiting a high level of intrinsic oxidative stress. This study focused on the in vitro evaluation of four novel prodrug esters: the R- and S- chiral esters of 4-[(nitrooxy)methyl]phenyl N-acetylalaninate (R- and S-NPAA) and the R- and S- chiral esters of 4-[(nitrooxy)methyl]naphth-1-yl N-acetylalaninate (R- and S-NQM), which are activated, to varying extents, by oxidized protein hydrolase (OPH, EC 3.4.19.1) yielding a quinone methide (QM) intermediate capable of depleting glutathione (GSH), a key intracellular antioxidant. OPH is a serine esterase/protease that is overexpressed in some human tumors and cancer cell lines.Methods: To evaluate the chiral ester prodrugs, we monitored cellular GSH depletion, cellular protein carbonyl levels (an oxidative stress biomarker) and cell viability in tumorigenic and nontumorigenic prostate cancer cell lines.Results: We found that the prodrugs were activated by OPH and subsequently depleted GSH. The S-chiral ester of NPAA (S-NPAA) was two-fold more effective than the R-chiral ester (R-NPAA) in depleting GSH, increasing oxidative stress, inducing apoptosis, and decreasing cell viability in tumorigenic prostate LNCaP cells but had little effect on non-tumorigenic RWPE-1 cells. In addition, we found that that S-NPAA induced apoptosis and decreased cell viability in tumorigenic DU145 and PC3 prostate cell lines. Similar results were found in a COS-7 model that overexpressed active human OPH (COS-7-OPH).Conclusions: Our results suggest that prostate tumors overexpressing OPH and/or exhibiting a high level of intrinsic oxidative stress may be susceptible to QM generating prodrug esters that are targeted to OPH with little effect on non-tumorigenic prostate cells.

The thesis describes time dependent fluorescence shift method and fluorescence correlation spectroscopy method (FCS) with its extensions FLCS, Z-scan FCS and dual-focus FCS applied on specific problems in DNA and lipid research. Compaction mechanism of a DNA molecule smaller than a resolution of a confocal microscope was elucidated. The process was revealed to be "all or non" for a polycation spermine as a condenser in contrast with the gradual compaction caused by a cationic surfactant. Biophysical properties of a phospholipid bilayer influenced by presence of oxidized phospholipids with truncated sn-2 chain were explored. The dynamics of hydrated functional groups in the headgroup region was proved to get faster while the hydration of the headgroup region increased. These effects are in relation with the reorientation of the short sn-2 chains observed in molecular dynamics simulations. Presence of oxidized species may also influence the lateral diffusion of the lipids - a slight increase of the diffusion coefficient was observed. Decrease of hydration and mobility in the headgroup region was found as an influence of heavy water on the phospholipid membrane. These finding are in line with molecular dynamics simulations which show longer lifetimes of hydrogen bonds between water and lipid molecules in...

Biological membranes are mainly composed of two layers of lipids, various kinds of proteins and organic macromolecules, forming the protective barriers that separate the inner milieu of living cells from the environment. The possibility of penetrating the membrane is of great importance for biomedical applications. Recently, a lot of attention has been given to the mechanisms and the details of the interactions between the membrane and nanoparticles, as well as to the development of effective delivery strategies. A manipulation of the hydrophobicity of nanoparticles can facilitate the translocation through the membrane. Modifying the physical/chemical properties of the membrane through oxidation can also influence the delivery of nanoparticles or macromolecules into the cell. In this work, using coarse-grained molecular dynamics simulations, the passive translocation of nanoparticles with a size of about 1 nm and with tunable degrees of hydrophobicity through lipid membranes is studied. It is shown that a window of nanoparticle translocation with a sharp maximum is located at a certain hydrophobicity in between fully hydrophilic and fully hydrophobic characters. By combining direct simulations with umbrella sampling simulations, the free energy landscapes for nanoparticles covering a wide range of hydrophobicities are obtained. The directly observed translocation rate of the nanoparticles can be mapped to the mean escape rate through the calculated free energy landscapes, and the maximum of translocation can be related with the maximally flat free energy landscape. For nanoparticles with the balanced hydrophobicity, the bilayer forms a remaining barrier of a few kBT and can be spontaneously surmounted. Further investigations are conducted to explore the cooperative effects of a larger number of nanoparticles and their impact on membrane properties such as membrane permeability for solvent, the area per lipid, and the orientation order of lipid tails. By calculating the partition of nanoparticles between water and oil phases, the microscopic parameter, i.e. the hydrophobicity of nanoparticles, can be mapped to an experimentally accessible partition coefficient. The studies reveal a generic mechanism for spherical nanoparticles to overcome biological membrane-barriers without the need of biologically activated processes. Two oxidatively modified lipids are studied on coarse-grained level using molecular dynamics simulations. The findings support the view that lipid oxidation leads to a change of the lipid conformation: lipid tails tend to bend toward the lipid head-tail interface due to the presence of hydrophilic oxidized beads. This change in conformation can further influence structural properties, elasticity and membrane permeability: an increase of the area per lipid, accompanied with decrease of the membrane thickness and order parameter of the lipid tails; a sharp drop of stretching modulus; a significant increase of the membrane permeability for water. Oxidized lipid bilayers interacting with NPs of various degrees of hydrophobicity are further studied. The critical hydrophobicity corresponding to the maximum translocation rate of NPs, shifts towards the hydrophilic region, which coincides with the same decrease in percentage of the average hydrophobicity in the core of the membrane upon oxidation. Around the critical point of NPs' hydrophobicity, a significant increase of the translocation rate of NPs through the oxidized bilayers is observed, when compared to non-oxidized bilayers. This is associated with a deterioration of the free energy barrier for NPs inside the oxidized bilayers, resulting from oxidation effects. These findings are consistent with the studies of the mean escape rate through the free energy landscapes using Kramers theory. Regarding the membrane perturbation induced by NPs of various hydrophobicity, the data obtained with oxidized lipid bilayers present the same general trend as in the case of the non-oxidized lipid bilayer. These findings provide a better understanding of the interaction between NPs and oxidized lipid bilayers, and open a possibility to facilitate drug delivery.:1 Introduction 1 1.1 Lipid Bilayers 1 1.2 Oxidized Lipid Bilayers 2 1.3 Experimental Methodology 4 1.4 Lipid Models 5 1.5 The Lipid Bilayer Interacting with NPs 6 1.6 Thesis Overview 7 2 State of the art 9 2.1 Molecular Dynamics Simulations of Lipid Bilayers 9 2.1.1 Equations of Motion and the Integrations of Equations of Motion 10 2.1.2 Interaction Potentials 12 2.1.3 Periodic Boundary Conditions 14 2.1.4 Barostats and Thermostats 15 2.2 Umbrella Sampling Simulation 19 2.2.1 The Basics of Umbrella Sampling Method 20 2.2.2 Analyzing Umbrella Sampling Results by WHAM 23 2.2.3 The Principle of Choosing Bias Potential 24 3 Lipid Membranes interacting with Nanoparticles of Various Degrees of Hydrophobicity 25 3.1 Introduction 25 3.2 Coarse-grained Model and Simulation Setups 27 3.3 Results and Discussions 31 3.3.1 NPs-membrane Interactions 31 3.3.2 NPs Translocation 33 3.3.3 Concentration Effect of NPs 35 3.3.4 The Effect of Hydrophobicity on Kinetic Pathways 38 3.3.5 Potential of Mean Force 39 3.3.6 Hydrophobicity Scale 41 3.3.7 Solvent Permeation and Membrane Perturbation Induced by NPs 45 3.4 Summary 47 4 Coarse-grained Model of Oxidized Lipids and their Interactions with NPs of Varying Hydrophobicities 51 4.1 Introduction 51 4.2 Coarse-grained Model and Simulation Details 52 4.3 Results and Discussions 54 4.3.1 Characterizing the Oxidized Lipid Membranes 54 4.3.2 Oxidized Lipid Membranes Interacting with NPs of Various Degrees of Hydrophobicity 59 4.4 Summary 65 5 Summary and Outlook 69

Předkládaná disertační práce se zabývá přípravou a charakterizací nových biokompatibilních nanovláken s potenciální aplikací v medicíně. V této práci byl výběr jednotlivých složek pro přípravu nanovlákenného materiálu zvolen tak, aby vyhovoval nárokům tkáňového inženýrství. Literární rešerše shrnuje poznatky o elektrostatickém zvlákňování a o jeho parametrech. Dále se věnuje možnostem elektrostatického zvlákňování proteinů kolagenu a želatiny a jejich směsmi se syntetickými polymery a biopolymery a anorganickými plnivy. Teoretická část řeší také různé postupy síťování nanovláken vedoucí ke zlepšení jejich hydrolytické stability a mechanických vlastností. Poslední část je zaměřena na anorganické nanotrubky halloysitu (HNT), které získaly svou pozornost díky svým vynikajícím fyzikálním a biologickým vlastnostem. V experimentální části byly zpracovány dvě případové studie, z nichž každá se zabývá přípravou nanovlákenných biomateriálů s potenciální aplikací v medicíně. První studie je zaměřena na přípravu a charakterizaci nových hydrolyticky stabilních antibakteriálních želatinových nanovláken modifikovaných pomocí oxidované celulózy. Unikátní inhibiční účinky nanovláken byly testovány na kmenu bakterie Escherichia coli pomocí metody chemické bioluminiscence. Kultivované buňky lidského papilárního adenokacinomu plic prokázaly dobrou adhezi a proliferaci k povrchu nanovláken. Druhá část popisuje vliv zdroje a množství anorganických halloysitových nanotrubek na strukturu a vlastnosti amfifilních nanovláken ze směsi želatiny a syntetického polykaprolaktonu. Přídavek HNT zlepšil tepelnou stabilitu, mechanické vlastnosti (jak tuhost, tak prodloužení) a snížil krystalinitu nanovláken. HNT z různých zdrojů neměl vliv na chování buněk, ale mírně ovlivnil proliferaci a životaschopnost buněk na povrchu nanovláken.

Cardiovascular disease is the leading cause of mortality in the western world. The pro-inflammatory and pro-proliferative etiology of vascular proliferative diseases is well characterized, while much less is known about the mechanisms of anti-inflammatory and anti-proliferative processes. Interleukin-19 (IL-19) is a newly described member of the IL-10 family of anti-inflammatory interleukins, and our group was the first to discover IL-19 expression in activated, synthetic, but not quiescent, contractile human vascular smooth muscle cells (hVSMC). We also found that IL-19 is anti-inflammatory and anti-proliferative for hVSMC. IL-19 is able to reduce the abundance of COX-2, IL-1β, IL-8, and Cyclin D1 transcripts which contain AU-rich elements (ARE) in their 3'-untranslated regions (3'-UTR). IL-19 is able to reduce the abundance of HuR, a stabilizing RNA-binding protein, which we feel provides a mechanism for these effects. The overall goal of this study is to elucidate IL-19's anti-inflammatory and anti-proliferative mechanism(s) in hVSMC in the context of vascular proliferative diseases. This goal has directed our overall hypothesis: IL-19's anti-proliferative and anti-inflammatory effects in hVSMC are mediated, at least in part, by modulation of HuR abundance and translocation, resulting in decreased stability of mRNA transcripts. HuR functions through a translocation mechanism, and IL-19 is able to reduce HuR cytoplasmic abundance. IL-19 also reduces HuR phosphorylation, which is a pre-requisite for HuR translocation, possibly through a PKCα-dependent mechanism. The stability of ARE-containing transcripts is reduced with IL-19 treatment, and reducing HuR expression by siRNA has the same inhibitory effect. VSMC are important mediators in the initiation of atherosclerosis. Oxidized low-density lipoprotein (ox-LDL) is able to induce IL-19 expression in these cells. VSMC are known to express scavenger receptors that take up ox-LDL. IL-19 is able to reduce the uptake of ox-LDL and the abundance of ox-LDL induced LOX-1 and CX-CL16 scavenger receptors. Interestingly, these scavenger receptors also have ARE in their 3'-UTR. IL-19 is able to reduce ox-LDL induced HuR cytoplasmic abundance. HuR knockdown by siRNA reduces the uptake of ox-LDL by hVSMC. These data suggest that IL-19 reduced scavenger receptor abundance may be due to decreased total and cytoplasmic HuR abundance. IL-19 reduces the abundance of ox-LDL induced COX-2 expression. Taken together, these results demonstrate that IL-19 down-regulates vital steps in vascular proliferative disease processes through an HuR-dependent mechanism. / Molecular and Cellular Physiology

The thesis entitled “New Supramolecular Ion Sensing Probes and their Application in the Detection of Environmentally Relevant Ions” deals with the design and synthesis of several small molecular probes which can specifically sense environmentally relevant ions of (anion or cation) particularly in aqueous or biological medium. The probes have been designed using four different molecular entities which include anthraquinone, oxidized bis-indolyl system, pyrene and rhodamine. The probes afford naked eye detection of a particular ion in the aqueous medium. This work has been divided into six chapters. Chapter 1. Introduction The first chapter gives a brief idea of ion sensor. It provides the description of various approaches used for designing molecular sensors. The chapter further presents an overview of the four different dyes (anthraquinone, oxidized-bis-indole, pyrene and rhodamine) used for designing probes in this work. The properties of these probes, their advantages and disadvantages to use as a signaling subunit have been discussed. This chapter also describes the use of micellar medium for solubilizing different organic dyes in water. Chapter 2. Colorimetric Probes based on Anthraimidazolediones for Selective Sensing of Fluoride and Cyanide ion via Intramolecular Charge Transfer. The second chapter describes the design and synthesis of four different probes based on anthra [1, 2-d] imidazole-6, 11-dione. The anthraquinone part of each molecule has an acceptor moiety whereas substituted nitrogen linked aromatic unit forms the donor site. Each probe acted as strong colorimetric sensor for fluoride and cyanide ion detection and exhibited intramolecular charge transfer (ICT) band which showed significant red-shifts after addition of either the F¯ or CN¯ ion. One of the probes 2 showed selective colorimetric sensing for both cyanide and fluoride ions. In organic medium 2 showed selective color change with fluoride and cyanide, whereas in aqueous organic medium it showed a selective ratiometric response towards cyanide ion. The effect of anionic charge (on the donor moiety) on ICT has been discussed. Among the various donor moieties, the donor site having negative charges on them was found to disperse greater electron density on them. Figure 1. Molecular structures of the sensors Chapter 3 deals with chemodosimetric detection of cyanide ion in water using various oxidized bis-indole based compounds. Chapter 3A. A Chemodosimetric Probe based on a Conjugated and oxidized Bis¬ indolyl System for Selective Naked Eye Sensing of Cyanide ion in Water. The chapter 3A describes the design and synthesis of a new water-soluble bis-indolyl based probe, 5 which possesses two –COOH groups. This probe specifically reacted with the CN¯ ion in pure water at ambient temperature and produced a remarkable change in color from red to colorless. The mechanism of this process was investigated by NMR (1H, 13C and DEPT-135) spectroscopy, mass spectrometry and kinetic studies. The mechanism investigation showed that the cyanide ion reacts with the probe and removes the conjugation of the bis-indolyl moiety of the probe with that of the 4-substituted aromatic ring which renders the probe colorless. Taken together a plausible mechanism of the reaction was presented which showed to operate via a Michael type adduct formation under ambient conditions of pH and temperature in water. The probe gave a detection limit of 0.38 ppm for detection of cyanide ion in water. Figure 2. Molecular structure of the probe 5. Chapter 3B. Micelle Assisted ppb level Detection of Cyanide ion in Water by Chemodosimetry and Visual detection of the Endogenous Cyanide. The chapter 3B deals with the synthesis of a bis-indole based colorimetric probe 6. The probe showed selective detection of the cyanide ion in water at ppb level and a visible detection of endogenous cyanide from cassava (a major staple food in the developing world) by chemodosimetry. The cyanide ion binds with the probe 6 in a chemodosimetric fashion and follows pseudo first-order kinetics in water under appropriate conditions. It showed a highly sensitive detection of the cyanide ion in water with a detection limit of 0.33 ppm. The use of the micellar medium improved the detection limit drastically and a ppb level detection limit was achieved. The probe also showed the detection of the endogenously bound cyanide in cassava both visually and by spectrophotometer. Figure 3. Molecular structure of the probe 6. Chapter 3C. Ratiometric Cyanide ion probe in Water and for the detection of the Endogenously bound cyanide. Chapter 3C presents the synthesis of two new bis-indolyl (7 and 8) based probes for colorimetric detection of cyanide ion in pure water. Compound 8 showed a ratiometric response with cyanide in water and a visual detection of the endogenously bound cyanide ion in cassava. Using compound 8 the selective detection of the cyanide ion in water was achieved with a detection limit of ~ 17 ppb which is almost 13 times lower than the permitted limit as specified by EPA, United States. 7; R = H 8; R = -(OCH2CH2)3CH3 Figure 4. Molecular structures of the probes 1 and 2. Chapter 4 deals with the colorimetric and ratiometric detection of the Cu2+and Hg2+ions using different small synthetic molecular probes. Chapter 4A. Colorimetric Sensors for Ratiometric Detection of Copper and Mercury ions in Biological media and below ppm level in Water. The chapter 4A deals with the synthesis of two novel colorimetric probes (9, 10) using bispicolyl unit as the binding moiety and anthraimidazolediones and bis-indolyl system as a signaling sub-unit. Using the two sensors, Cu2+ion can be detected below the permitted limit (1.3 ppm) in both drinking water and at physiological pH 7.4. Sensor 9 can detect both Cu2+and Hg2+ in water with very low detection limit. It showed specific binding with Cu2+ at physiological pH 7.4 and in presence of serum albumins. Chemosensor 10 can be used for the specific detection of both Cu2+and Hg2in water as well as for the contamination in microorganisms. Figure 5. Molecular structure of the sensors 9 and 10. Chapter 4B. A New Molecular Probe for the Selective Sensing of Cu2+ and Hg2+ ions in Micellar Media and in Live ells.This chapter describes a synthesis of a novel bispicolyl based sensor 11 which can detect Cu2+ ion specifically in water medium and both Cu2+ and Hg2+ ions selectivelyin Brij-58 micellar medium. In micellar medium both the ions can be detected in the ppb level. Using fluorescence spectroscopy these two metal ions can be discriminated.The probe is also be useful for checking metal ion contamination in cellular samples. Figure 6. Molecular structure of the sensor 11. Chapter 4C. Rhodamine based Sensors for Cu2+ and Hg2+ ions in Water and in Biological media. The chapter 4C presents the synthesis and the sensing properties of the three positional isomers of the pyridine end of the rhodamine-pyridine compounds (12-14). The three isomers only differ in the position of nitrogen of the pyridine moiety. Sensor 12, which contains the pyridine nitrogen at the ortho-position showed selective sensing toward Cu2+ ion in both pure water and in buffered physiological media of pH 7.4. It gave a detection limit of ~13 ppb which is 100 times lesser than the EPA permitted limit. The other two sensors 13 and 14, which possessed the pyridine ends with the nitrogen atom at the meta- and the para- positions respectively showed the selective sensing of Hg2+ ion in water and did not show any interaction with the Cu2+ ion. Probes 2 and 3 showed ‘turn-on’ detection of Hg2+ ion both in the UV-vis and the fluorescence emission spectroscopy. Compound 2 and 3 showed a detection limit of ~ 9 and 4 ppb respectively. The NMR titration showed the change in color was due to the opening of the spirolactam ring of the rhodamine. The sensors can also be used for the detection of Cu2+ and Hg2+ ion in real life water samples and in the live cells. Figure 7. Molecular structure of the sensors 12, 13 and 14. Chapter 5. Ratiometric and ppb level Detection of Toxic Transition Metal ions using a Single Probe in Micellar media. This chapter describes the selective sensing of multiple ions using a single probe 15. The probe incorporates pyrene and pyridine as signaling and interacting moiety respectively. The sensor showed different responses towards different metal ions just by varying the medium of detection. In organic solvent (acetonitrile), the probe showed selective detection of Hg2+ ion. In water the fluorescence quenching was observed with three metal ions, Cu2+, Hg2+ and Ni2+. Further just by varying the surface charge of different micellar media, the probe showed selective interaction with Hg2+ ion in neutral micelles (Brij-58). However, in anionic micellar medium (SDS), the probe showed selective changes with both Cu2+ and Ni2+ in the UV-vis spectroscopy. The discrimination between these two ions was achieved by emission spectroscopy, where it showed selective quenching only with Cu2+. Thus using a single probe all the three metal ions Cu2+, Hg2+ and Ni2+ can be detected and discriminated just by varying the surface charge of the micellar medium. Figure 8. Molecular structure of the sensors 15. Chapter 6. Highly sensitive Rhodamine Based Dual Probes for the Visual detection of F¯ and Hg2+ ions in Water. This chapter deals with the design and synthesis of two new rhodamine based probes (16-17) which act as dual probes for the ppb level selective detection of Hg2+ and F¯ ions in water and at physiological pH 7.4. The two probes were synthesized by coupling tert-butyldiphenylsilyl (TBDPS) protected forms of 4-hydroxybenzaldehyde and 2, 4- dihydroxy benzaldehyde with rhodamine hydrazone. The F¯ ion detection is based on the desilylation of the probe, whereas the spirolactam ring opening leads to the detection of Hg2+ ion. The two probes gave turn-on detection of both Hg2+ and F¯ ion selectively in aqueous medium with the detection limit well below the EPA permitted limits. The probes showed detection of both the ions by dual mode with visibly different color and fluorescence under UV-lamp. The F¯ ion interacts with the silyl bond of probe and the cleavage results into yellow color whereas; the addition of Hg2+ ion to the probe solution opened the spirolactam ring and resulted into appearance of pink color. Figure 9. Molecular structure of the probes 16 and 17. (For structural formula pl see the abstract file)

A aterosclerose é uma doença crônica onde duas características marcantes são observadas: retenção de lipídios e inflamação. Compreender as interações entre as células do sistema imunológico e as lipoproteínas envolvidas na aterogênese são desafios urgentes, uma vez que as doenças cardiovasculares são a principal causa de morte no mundo. Os macrófagos são cruciais para o desenvolvimento de placas ateroscleróticas e para a perpetuação da inflamação em tais lesões; estas células também estão diretamente envolvidas na ruptura de placa instável. Recentemente diferentes populações de macrófagos estão sendo identificadas nas lesões ateroscleróticas. Embora macrófagos M2 tenham sido identificados, a função destas células na aterosclerose ainda não está definida. Neste projeto, avaliamos se a adição de LDLox altera a função de macrófagos M2. Resultados: 1- Foi possível observar que os M2 se mantem viáveis após o estímulo com as lipoproteínas. 2- Quando avaliamos a expressão de moléculas co-estimulatórias, receptores Scavenger, lectinas e integrinas na superfície das células, observamos que a adição de LDLn ou LDLox em 2 concentrações diferentes (5 e 50ug/ml), por diferentes períodos de tempo não alterou a expressão de nenhum dos marcadores avaliados. A presença de LDL também não alterou outra função primordial dos M2, a capacidade de fagocitose. 3- Quando investigamos a presença de citocinas no sobrenadante das culturas estimuladas ou não com as lipoproteínas, identificamos um aumento na secreção de IL-8, uma citocina pró-inflamatória, na presença de LDLox, semelhante ao observado com a população de macrófagos M1. 4- Avaliamos se os macrófagos M2 estimulados ou não com LDL mantem sua capacidade de favorecer a angiogênese. Observamos que nas culturas estimuladas com o sobrenadante das culturas dos M2 mantidos na presença de LDLox houve uma inibição significativa da formação de túbulos pelas HUVECs. 5- Observamos que na presença do meio condicionado dos M2 estimulados com LDLox ocorreu uma intensa degradação dos filamentos de matriz extracelular produzida por MEFs. 6- Avaliamos a expressão gênica de componentes de matriz, membrana basal, moléculas de adesão, proteases e também inibidores de protease nestas células. Dos 96 genes avaliados, observamos que a adição de LDLox reduziu a expressão de 10 genes de maneira significativa, entre eles: beta-Actina (ACTB), Colágeno 6A2 (Col6A2), Integrina alfa 6 (ITGA6), Metaloproteinase 15 (MMP15), molécula de adesão celular endotelial plaquetária (PECAM) e Inibidor de metalopeptidase 2 (TIMP2). A adição de LDLox aumentou significativamente somente a expressão de trombospondina (TSP1). A adição de LDLn não alterou a expressão de nenhum gene de forma significativa. 7- A adição de LDLox induziu aumento da expressão da TSP1 e redução da expressão de colágeno 6, quando comparadas aos macrófagos M2 sem estímulo. Nossos resultados indicam que a adição de LDLox altera diversas funções dos macrófagos M2 in vitro. Em especial detectamos uma inibição significativa na angiogênese e também a secreção de mediadores que induzem a degradação da matriz extracelular. A adição de LDLox também inibiu a expressão de genes envolvidos com a estabilização da matriz extracelular. Nossos resultados sugerem que esta população de células pode contribuir para a perpetuação do processo inflamatório e degradação tecidual observados na lesão dos pacientes. Assim, acreditamos que este projeto contribuiu para o esclarecimento da participação dos M2 na patologia da aterosclerose / Atherosclerosis is a chronic disease where two key characteristics are observed: lipid retention and inflammation. Understanding the interactions between the cells of the immune system and the lipoproteins involved in atherogenesis are urgent challenges, since cardiovascular diseases are the leading cause of death in the world. Macrophages are crucial for the development of atherosclerotic plaques and for the inflammation in such lesions; These cells are also directly involved in unstable plaque rupture. Recently different populations of macrophages are being identified in atherosclerotic lesions. Although M2 macrophages has been identified, the function of these cells in atherosclerosis has not yet been defined. This project, we evaluated whether the addition of OxLDL alters the function of M2 macrophages. Results: 1- M2 macrophages remain viable after stimulation with the lipoproteins. 2- When evaluated the expression of co-stimulatory molecules, Scavenger receptors, lectins and integrins on the surface of the cells. We observed that the addition of LDLn or OxLDL at 2 different concentrations (5 and 50 ?g / ml) for different time periods did not alter the expression of any of the evaluated markers. 3- The presence of LDL also did not alter other primordial function of M2 cells, phagocytosis. 4- Was observed that cultures stimulated with conditioned medium of OxLDL-stimulated M2 there was a significant inhibition of tubule formation by HUVECs. 5- We observed that in the presence of OxLDL-stimulated M2 cells conditioned médium an intense degradation of the matrix filaments occurred. 6- We evaluated the gene expression of matrix components, basement membrane, adhesion molecules, proteases and also protease inhibitors in these cells. Of the 96 evaluated genes, we observed that the addition of OxLDL significantly reduced the expression of 10 genes, among them: Actin-beta (ACTB), Collagen 6A2 (Col6A2), Integrin alfa 6 (ITGA6), Metaloproteinase 15 (MMP15), Platelet endothelial cell adhesion molecule (PECAM) and metallopeptidase 2 inhibitor (TIMP2). The addition of OxLDL significantly increased only the expression, thrombospondin-1 (TSP1). Addition of LDLn did not significantly alter the expression of any gene. 7- That OxLDL addition induced increased TSP1 expression and reduced collagen 6 expression, when compared to M2 macrophages without stimulation. Our results indicate that the addition of OxLDL alters several M2 macrophages functions in vitro. In particular we detected a significant inhibition in angiogenesis and also the secretion of mediators that induce the degradation of the extracellular matrix. The addition of OxLDL also inhibited the expression of genes involved in extracellular matrix stabilization. Our results suggest that this cell population may contribute to the perpetuation of the inflammatory process and tissue degradation observed in the lesion of the patients. Thus, we believe that this project contributed to better understand the participation of M2 in the pathology of atherosclerosis

A enzima Paraoxonase-1 (PON1) possui atividades paraoxonase, arilestearase e lactonase, entre outras. É a mais estuda da família das PONs que é composta pela PON1, PON2 e PON3. Sugere-se, que todas atuam inibindo o processo de peroxidação lipídica de moléculas como a lipoproteína de baixa densidade (LDL) e alta densidade (HDL), caracterizando assim um possível papel anti-aterogênico. O gene da PON1 apresenta dois sítios polimórficos, com a troca de uma Gln192Arg (Q/R) e Met55Leu, que estão associados com diferenças na atividade e concentrações séricas da enzima. Por sua vez, indivíduos soropositivos para o HIV-1 apresentam alterações do metabolismo lipídico, que poderiam estar associados a alterações na atividade da PON1 e a terapia antirretroviral (TARV) com inibidores de protease (IP). O objetivo do estudo foi determinar as atividades séricas da PON1 e da arilestearase (ARE), e as freqüências alélicas dos polimorfismos genéticos da PON1 192QR e 55LM, e ainda, avaliar a correlação destes parâmetros com as alterações lipídicas em indivíduos soropositivos para o HIV-1 tratados com IP. No período de Setembro de 2009 até Junho de 2012, 174 indivíduos soropositivos e 46 soronegativos para o HIV-1 foram estudados. Foi realizada a genotipagem dos polimorfismos da PON1 192QR e 55LM através de PCR-RFLP. A atividade sérica da PON1/ARE foi avaliada por espectrofotometria empregando-se como substratos o paraoxon e o fenilacetato, respectivamente. O RNA-HIV-1 foi quantificado pelo método NASBA, e os linfócitos T-CD4+ e T-CD8+ por citometria de fluxo. Os níveis séricos de colesterol total, HDL, LDL, triglicérides (TG), ApoA1 e ApoB100 foram determinados e os anticorpos IgG anti-oxLDL por ELISA. A atividade sérica da PON1 foi inferior nos grupos de soropositivos, p<0,05, porém, a atividade ARE não apresentou diferenças entre os grupos estudados, p>0,05. Ambas as atividades não apresentaram relação com os genótipos PON1 192QR e 55LM, e estes genótipos apresentaram uma freqüência alélica semelhante ao grupo de soronegativos. Os níveis séricos de TG foram superiores nos grupos de soropositivos com TARV, p<0,05, enquanto o grupo tratado com IP apresentou níveis séricos de HDL e Apo-A1 inferiores aos demais grupos, p<0,05. Níveis séricos de Apo-B100, IgG anti-oxLDL, e o índice de risco aterogênico foram superiores no grupo tratado com IP, p<0,05. Concluí-se, que indivíduos soropositivos para o HIV-1 apresentaram alterações no metabolismo lipídico, principalmente nos tratados com IP, que adicionalmente apresentaram um maior índice de risco aterogênico e maiores níveis de anticorpos IgG anti-oxLDL. Estas alterações não apresentaram relação com os polimorfismos PON1 192QR e 55LM da PON1, e demonstraram que a atividade da enzima PON-1 esta diminuída em indivíduos soropositivos para o HIV-1 / The enzyme Paraoxonase-1 (PON1) has paraoxonase (PON), arylesterase (ARE) and lactonase activities, among others. It is the most studied member of PON family which is composed of PON1, PON2 and PON3. It is suggested that all members acts by inhibiting the peroxidation of lipid molecules as the low-density lipoprotein (LDL) and high-density lipoprotein (HDL), characterizing a potential anti-atherogenic effect. The PON1 gene has two mainly polymorphic sites, with an exchange of Gln192Arg (Q/R) and Met55Leu (L/M), which are associated with differences in activity and serum concentrations of the enzyme. In turn, seropositive individuals for HIV-1 show changes in lipid metabolism, which could be associated with changes in the activity of PON1 and highly active antiretroviral therapy (HAART) with protease inhibitors (PI). The aim of this study was to determinate the serum PON and ARE activities of PON1, the allele frequencies of PON1 192QR and PON1 55LM genetic polymorphisms and evaluate the correlation between these parameters and lipid abnormalities in seropositive patients for HIV-1 treated with IP. In the period from September 2009 until June 2012, 174 seropositive individuals and 46 soronegative individuals for HIV-1 were studied. We performed PON1 192QR and 55LM genotyping by PCR-RFLP. Serum activities PON and ARE of PON1 were evaluated by spectrophotometry using paraoxon and phenylacetate, respectively, as substrates. The HIV-1-RNA was quantified by the NASBA method, and lymphocytes T-CD4+ and T-CD8+, by flow cytometry. Serum levels of total cholesterol, HDL, LDL, triglycerides (TG), apoA1 and ApoB100 were determined. IgG anti-oxLDL antibodies were quantified by ELISA. The serum PON1 activity was lower in the seropositive group, p<0.05, however, ARE activity did not differ between groups, p>0.05. Both activities had no relation with the PON1 192QR and PON1 55LM genotype, and these individuals showed an allele frequency similar to the seronegative group. Serum levels of TG were higher in groups of HIV-positive with HAART, p<0.05, while the IP-treated group showed serum levels of HDL and ApoA1 lower than other groups, p <0.05. Serum levels of ApoB100, IgG anti-oxLDL antibodies, and atherogenic risk indices were higher in the group treated with PI, p<0.05. It was concluded that individuals HIV-1-infected showed changes in lipid metabolism, especially in those treated with IP, which additionally showed a higher rate of atherogenic risk and higher levels of IgG anti-oxLDL antibodies. These changes did not correlated with PON1 192QR and 55LM polymorphisms and demonstrated that the activity of PON1enzyme is decreased in individuals seropositive for HIV-1

Le diabète de type 2 (DT2) est associé à un risque athéro-thrombotique élevé, en partie dû à l'hyperactivation plaquettaire et aux dyslipoprotéinémies. Les lipoprotéines de haute densité (HDL) possèdent des propriétés anti-athérogènes et subissent des modifications glycoxydatives lors du DT2. Notre objectif a été de déterminer les effets d'HDL glycoxydées in vitro ou de DT2 sur les plaquettes sanguines humaines et de déterminer leur contenu en lipides oxydés. Les HDL glycoxydées possèdent des proportions moindres d'acides linoléique et arachidonique dans les phospholipides (PL) et esters de cholestérol, des concentrations plus élevées de dialdéhyde malonique et des principaux acides gras hydroxylés (AGOH) dont les 9-HODE, 13- HODE et 15-HETE dans toutes les classes lipidiques, en particulier dans les PL ainsi que des concentrations très faibles de vitamine E comparativement aux HDL contrôles. Les HDL glycoxydées in vitro et de patients DT2 inhibent de façon dose-dépendante l'agrégation plaquettaire induite par le collagène via le récepteur SR-BI. Ces HDL glycoxydées diminuent la phosphorylation des p38 MAPK et cPLA2 plaquettaires. D'autre part, des HDL contrôles enrichies avec le PC(16:0/13-HODE) inhibent fortement l'agrégation comparativement aux HDL contrôles. De plus, les effets des sous-classes d'HDL, HDL 2 & 3, de DT2 et de témoins ont été testés sur l'agrégation plaquettaire. Les HDL2 de DT2 possèdent des concentrations d'AGOH plus élevées que les HDL3 de DT2 et tendent à inhiber plus l'agrégation plaquettaire. En conclusion, nos résultats montrent que les HDL glycoxydées de patients diabétiques ne perdent pas leurs propriétés anti-agrégantes, qui pourraient être médiées par certaines PL oxydés / Type 2 diabetes (T2D) is associated with a high athero-thrombotic risk, partly due to platelet hyperactivation and dyslipoproteinemia. High-density lipoproteins (HDL) possess antiatherogenic properties and undergo glycoxidation changes in T2D. Our objective was to determine the effects of glycoxidized HDL in vitro or from T2D patients on human blood platelets and to identify their oxidized lipid species. Compared to control HDL, glycoxidized HDL have lower proportions of linoleic and arachidonic acids in phospholipids (PL) and cholesteryl esters, higher concentrations of malondialdehyde and main hydroxylated fatty acid (HOFA) including 9-HODE, 13-HODE and 15-HETE in all lipid classes, especially in PL, and very low concentrations of vitamin E. In vitro glycoxidized and T2D HDL dose-dependently inhibit platelet aggregation induced by collagen via the SR-BI receptor. Glycoxidized HDL decrease the phosphorylation of platelet p38 MAPK and cPLA2. On the other hand, control HDL enriched with oxidized phospholipids i.e. PC(16:0/13-HODE) strongly inhibit platelet aggregation compared to controls. Moreover, the effects of HDL subclasses, HDL 2 & 3, from T2D patients and healthy controls were tested on platelet aggregation. T2D HDL2 have higher concentrations of HOFA than T2D HDL3 and tend to inhibit platelet aggregation to a greater extent. In conclusion, our results show that T2D glycoxidized HDL do not lose their anti-aggregatingproperties and are even more effective than control HDL. These anti-aggregatory effects could be partly due to some oxidized PL species