Charakterystyka ekspresji receptorów scavenger w błonach komórkowych monocytów ludzkich u pacjentów z chorobą niedokrwienną serca

Wydział Biologii: Instytut Biologii Eksperymentalnej

Inibidores do co-transportador renal de sódio-glicose 2

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Inibidores PCSK9

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

A genome-wide association study for blood lipid phenotypes in the Framingham Heart Study

BACKGROUND:Blood lipid levels including low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglycerides (TG) are highly heritable. Genome-wide association is a promising approach to map genetic loci related to these heritable phenotypes.METHODS:In 1087 Framingham Heart Study Offspring cohort participants (mean age 47 years, 52% women), we conducted genome-wide analyses (Affymetrix 100K GeneChip) for fasting blood lipid traits. Total cholesterol, HDL-C, and TG were measured by standard enzymatic methods and LDL-C was calculated using the Friedewald formula. The long-term averages of up to seven measurements of LDL-C, HDL-C, and TG over a ~30 year span were the primary phenotypes. We used generalized estimating equations (GEE), family-based association tests (FBAT) and variance components linkage to investigate the relationships between SNPs (on autosomes, with minor allele frequency [greater than or equal to]10%, genotypic call rate [greater than or equal to]80%, and Hardy-Weinberg equilibrium p [greater than or equal to] 0.001) and multivariable-adjusted residuals. We pursued a three-stage replication strategy of the GEE association results with 287 SNPs (P < 0.001 in Stage I) tested in Stage II (n ~1450 individuals) and 40 SNPs (P < 0.001 in joint analysis of Stages I and II) tested in Stage III (n~6650 individuals).RESULTS:Long-term averages of LDL-C, HDL-C, and TG were highly heritable (h2 = 0.66, 0.69, 0.58, respectively; each P < 0.0001). Of 70,987 tests for each of the phenotypes, two SNPs had p < 10-5 in GEE results for LDL-C, four for HDL-C, and one for TG. For each multivariable-adjusted phenotype, the number of SNPs with association p < 10-4 ranged from 13 to 18 and with p < 10-3, from 94 to 149. Some results confirmed previously reported associations with candidate genes including variation in the lipoprotein lipase gene (LPL) and HDL-C and TG (rs7007797; P = 0.0005 for HDL-C and 0.002 for TG). The full set of GEE, FBAT and linkage results are posted at the database of Genotype and Phenotype (dbGaP). After three stages of replication, there was no convincing statistical evidence for association (i.e., combined P < 10-5 across all three stages) between any of the tested SNPs and lipid phenotypes.CONCLUSION:Using a 100K genome-wide scan, we have generated a set of putative associations for common sequence variants and lipid phenotypes. Validation of selected hypotheses in additional samples did not identify any new loci underlying variability in blood lipids. Lack of replication may be due to inadequate statistical power to detect modest quantitative trait locus effects (i.e., < 1% of trait variance explained) or reduced genomic coverage of the 100K array. GWAS in FHS using a denser genome-wide genotyping platform and a better-powered replication strategy may identify novel loci underlying blood lipids.

Capillary electrophoresis with boron doped diamond electrode for amperometric detection of low molecular weight biological substrate

The research work in this thesis included the sensitive and selective separation of biological substance by capillary electrophoresis with a boron doped diamond electrode for amperometric detection. Chapter 1 introduced the capillary electrophoresis and electrochemical detection. It included the different modes of capillary electrophoresis, polyelectrolyte multilayers coating for open tubular capillary electrochromatography, different modes of electrochemical detection and carbon based electrodes. Chapter 2 showed the synthesized and electropolymerized N-acetyltyramine with a negatively charged sulfobutylether-β-cyclodextrin on a boron doped diamond (BDD) electrode followed by the electropolymerzation of pyrrole to form a stable and permselective film for selective dopamine detection. For comparison, a glassy carbon (GC) electrode with a combined electropolymerized permselective film of polytyramine and polypyrrole-1-propionic acid was used for selective detection of dopamine. The detection limit of dopamine was improved from 100 nM at a GC electrode to 5 nM at a BDD electrode. Chapter 3 showed field-amplified sample stacking using a fused silica capillary coated with gold nanoparticles embedded in poly(diallyldimethylammonium) chloride, which has been investigated for the electrophoretic separation of indoxyl sulphate, homovanillic acid and vanillylmandelic acid. The detection limit of the three analytes obtained by using a boron doped diamond electrode was around 75 nM, which was significantly below their normal physiological levels in biological fluids. This combined separation and detection scheme was applied to the direct analysis of these analytes and other interfereing chemicals including uric and ascorbic acids in urine samples without off-line sample treatment or preconcentration. Chapter 4 showed the selective detection of Pseudomonas Quinolone Signal, PQS for quorum sensing from its precursor HHQ, using a simply boron doped diamond electrode. Furthermore, by combining poly(diallyldimethylammonium) chloride modified fused silica capillary with a BDD electrode for amperometric detection, PQS was separated from HHQ and other analogues. The detection limit of PQS was as low as 65 nM. Different P. aeruginosa mutant strains were studied. Chapter 5 showed the separation of aminothiols by layer-by-layer coating of silica capillary with a boron doped diamond electrode. The capillary was layer-by-layer coated with the polycation poly(diallyldimethylammonium) chloride and negatively charged silica nanoparticles. All the aminothiols was separated and detected using a BDD electrode in an acidic electrolyte. It was a novel scheme for the separation and detection of glutathione reduced and oxidized forms, which is important for estimated overstressed level in the human system.

Epidemiology and comparative analysis of Yersinia in Ireland

Yersiniosis is an acute or chronic enteric zoonosis caused by enteropathogenic Yersinia species. Although yersiniosis is predominantly associated with gastroenteric forms of infection, extraintestinal forms are often reported from the elderly or patients with predisposing factors. Yersiniosis is often reported in countries with cold and mild climates (Northern and Central Europe, New Zealand and North of Russian Federation). The Irish Health Protection Surveillance Centre (HPSC) currently records only 3-7 notified cases of yersiniosis per year. At the same time pathogenic Yersinia enterocolitica is recovered from pigs (main source of pathogenic Y. enterocolitica) at the levels similar to that observed in Yersinia endemic countries. Introduction of Yersinia selective culture procedures may increase Yersinia isolation rates. To establish whether the small number of notifications of human disease was an underestimate due to lack of specific selective culture for Yersinia we carried out a prospective culture study of faecal samples from outpatients with diarrhoea, with additional culture of appendix and throat swabs. Higher levels of anti-Yersinia seroprevalence than yersiniosis notification rates in endemic countries suggests that most yersiniosis cases are unrecognised by culture. Subsequently, in addition to a prospective culture study of clinical specimens, we carried out serological screening of Irish blood donors and environmental screening of human sewage. Pathogenic Yersinia strains were not isolated from 1,189 faeces samples, nor from 297 throat swabs, or 23 appendix swabs. This suggested that current low notification rates in Ireland are not due to the lack of specific Yersinia culture procedures. Molecular screening detected a wider variety of Y. enterocolitica-specific targets in pig slurry than in human sewage. A serological survey for antibodies against Yersinia YOP (Yersinia Outer Proteins) proteins in Irish blood donors found antibodies in 25%, with an age-related trend to increased seropositivity, compatible with the hypothesis that yersiniosis may have been more prevalent in Ireland in the recent past. Y. enterocolitica is a heterogeneous group of microorganisms that comprises strains with different degree of pathogenicity. Although non-pathogenic Y. enterocolitica lack conventional virulence factors, these strains can be isolated from patients with diarrhoea. Insecticidal Toxin Complex (ITC) and Cytolethal Distending Toxins can potentially contribute to the virulence of non-pathogenic Y. enterocolitica in the absence of other virulence factors. We compared distribution of ITC and CDT loci among pathogenic and non-pathogenic Y. enterocolitica. Additionally, to demonstrate potential pathogenicity of non-pathogenic Y. enterocolitica we compared their virulence towards Galleria mellonella larvae (a non-mammalian model of human bacterial infections) with the virulence of highly and mildly pathogenic Y. enterocolitica strains. Surprisingly, virulence of pathogenic and non-pathogenic Y. enterocolitica in Galleria mellonella larvae observed at 37°C did not correlate with their pathogenic potential towards humans. Comparative phylogenomic analysis detects predicted coding sequences (CDSs) that define host-pathogen interactions and hence providing insights into molecular evolution of bacterial virulence. Comparative phylogenomic analysis of microarray data generated in Y. enterocolitica strains isolated in the Great Britain from humans with diarrhoea and domestic animals revealed high genetic heterogeneity of these species. Because of the extensive human, animal and food exchanges between the UK and Ireland the objective of this study was to gain further insight into genetic heterogeneity and relationships among clinical and non-clinical Y. enterocolitica strains of various pathogenic potential isolated in Ireland and Great Britain. No evidence of direct transfer of strains between the two countries was found.

Early biomarkers to predict grade of encephalopathy following hypoxic ischaemic injury

The standard early markers for identifying and grading HIE severity, are not sufficient to ensure all children who would benefit from treatment are identified in a timely fashion. The aim of this thesis was to explore potential early biomarkers of HIE. Methods: To achieve this a cohort of infants with perinatal depression was prospectively recruited. All infants had cord blood samples drawn and biobanked, and were assessed with standardised neurological examination, and early continuous multi-channel EEG. Cord samples from a control cohort of healthy infants were used for comparison. Biomarkers studied included; multiple inflammatory proteins using multiplex assay; the metabolomics profile using LC/MS; and the miRNA profile using microarray. Results: Eighty five infants with perinatal depression were recruited. Analysis of inflammatory proteins consisted of exploratory analysis of 37 analytes conducted in a sub-population, followed by validation of all significantly altered analytes in the remaining population. IL-6 and IL-6 differed significantly in infants with a moderate/severely abnormal vs. a normal-mildly abnormal EEG in both cohorts (Exploratory: p=0.016, p=0.005: Validation: p=0.024, p=0.039; respectively). Metabolomic analysis demonstrated a perturbation in 29 metabolites. A Cross- validated Partial Least Square Discriminant Analysis model was developed, which accurately predicted HIE with an AUC of 0.92 (95% CI: 0.84-0.97). Analysis of the miRNA profile found 70 miRNA significantly altered between moderate/severely encephalopathic infants and controls. miRNA target prediction databases identified potential targets for the altered miRNA in pathways involved in cellular metabolism, cell cycle and apoptosis, cell signaling, and the inflammatory cascade. Conclusion: This thesis has demonstrated that the recruitment of a large cohortof asphyxiated infants, with cord blood carefully biobanked, and detailed early neurophysiological and clinical assessment recorded, is feasible. Additionally the results described, provide potential alternate and novel blood based biomarkers for the identification and assessment of HIE.

Geochemistry, mineralogy, and chemical modeling of the acid crater lake of Kawah Ijen volcano, Indonesia

The Kawah Ijen volcano-with a record of phreatic eruptions-has its 1000 m wide crater filled with a lake that has existed for at least one century. At present, the lake waters are hot (T ≈ 37°C), strongly mineralized (TDS = 105 g/L) and extremely acidic (pH ≈ 0.4). By its volume, the Javanese lake is probably the largest accumulation in the world of such acidic waters. Mineralogy of the suspended solids within the lake waters suggests that concentrations of Si, Ca, Ti, and Ba are controlled by precipitation of silica, gypsum, anatase, and barite. Lake sediment is composed of chemical precipitates with composition similar to the suspended solids. Thermodynamic calculations predict that the lake waters have reached equilibrium with respect to α-cristobalite, barite, gypsum, anglesite, celestite, and amorphous silica, in agreement with the analytical observations. Significant concentrations of ferric iron suggest that the current lake waters are fairly oxidized. Sulfides are absent in the water column but are always present in the native S spherules that form porous aggregates which float on the lake. The presence of native S provides direct evidence of more reduced conditions at the lake floor where H2S is probably being injected into the lake. With progressive addition of H2S to the acid waters, native S, pyrite, and enargite are theoretically predicted to be saturated. Reactions between upward streaming H2S-bearing gases discharged by subaqueous fumaroles, and metals dissolved in the acidic waters could initiate precipitation of these sulfides. A model of direct absorption of hot magmatic gases into cool water accounts for the extreme acidity of the crater lake. Results show that strongly acidic, sulfate-rich solutions are formed under oxidizing conditions at high gas/water ratios. Reactions between the acidic fluids and the Ijen andesite were modeled to account for elevated cation concentrations in lake water. Current concentrations of conservative rockforming elements are produced by dissolution of approximately 60 g of andesite per kg of acid solution. Complete neutralization of the acid lake waters by reaction with the wallrock produces a theoretical alteration assemblage equivalent to that observed in volcano-hosted, acid-sulfate epithermal ore deposits. © 1994.

Mass spectrometry-based thermal shift assay for protein-ligand binding analysis.

Described here is a mass spectrometry-based screening assay for the detection of protein-ligand binding interactions in multicomponent protein mixtures. The assay utilizes an oxidation labeling protocol that involves using hydrogen peroxide to selectively oxidize methionine residues in proteins in order to probe the solvent accessibility of these residues as a function of temperature. The extent to which methionine residues in a protein are oxidized after specified reaction times at a range of temperatures is determined in a MALDI analysis of the intact proteins and/or an LC-MS analysis of tryptic peptide fragments generated after the oxidation reaction is quenched. Ultimately, the mass spectral data is used to construct thermal denaturation curves for the detected proteins. In this proof-of-principle work, the protocol is applied to a four-protein model mixture comprised of ubiquitin, ribonuclease A (RNaseA), cyclophilin A (CypA), and bovine carbonic anhydrase II (BCAII). The new protocol's ability to detect protein-ligand binding interactions by comparing thermal denaturation data obtained in the absence and in the presence of ligand is demonstrated using cyclosporin A (CsA) as a test ligand. The known binding interaction between CsA and CypA was detected using both the MALDI- and LC-MS-based readouts described here.

Study protocol: the Adherence and Intensification of Medications (AIM) study--a cluster randomized controlled effectiveness study.

BACKGROUND: Many patients with diabetes have poor blood pressure (BP) control. Pharmacological therapy is the cornerstone of effective BP treatment, yet there are high rates both of poor medication adherence and failure to intensify medications. Successful medication management requires an effective partnership between providers who initiate and increase doses of effective medications and patients who adhere to the regimen. METHODS: In this cluster-randomized controlled effectiveness study, primary care teams within sites were randomized to a program led by a clinical pharmacist trained in motivational interviewing-based behavioral counseling approaches and authorized to make BP medication changes or to usual care. This study involved the collection of data during a 14-month intervention period in three Department of Veterans Affairs facilities and two Kaiser Permanente Northern California facilities. The clinical pharmacist was supported by clinical information systems that enabled proactive identification of, and outreach to, eligible patients identified on the basis of poor BP control and either medication refill gaps or lack of recent medication intensification. The primary outcome is the relative change in systolic blood pressure (SBP) measurements over time. Secondary outcomes are changes in Hemoglobin A1c, low-density lipoprotein cholesterol (LDL), medication adherence determined from pharmacy refill data, and medication intensification rates. DISCUSSION: Integration of the three intervention elements--proactive identification, adherence counseling and medication intensification--is essential to achieve optimal levels of control for high-risk patients. Testing the effectiveness of this intervention at the team level allows us to study the program as it would typically be implemented within a clinic setting, including how it integrates with other elements of care. TRIAL REGISTRATION: The ClinicalTrials.gov registration number is NCT00495794.

Genome-wide association study of Lp-PLA(2) activity and mass in the Framingham Heart Study.

Lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) is an emerging risk factor and therapeutic target for cardiovascular disease. The activity and mass of this enzyme are heritable traits, but major genetic determinants have not been explored in a systematic, genome-wide fashion. We carried out a genome-wide association study of Lp-PLA(2) activity and mass in 6,668 Caucasian subjects from the population-based Framingham Heart Study. Clinical data and genotypes from the Affymetrix 550K SNP array were obtained from the open-access Framingham SHARe project. Each polymorphism that passed quality control was tested for associations with Lp-PLA(2) activity and mass using linear mixed models implemented in the R statistical package, accounting for familial correlations, and controlling for age, sex, smoking, lipid-lowering-medication use, and cohort. For Lp-PLA(2) activity, polymorphisms at four independent loci reached genome-wide significance, including the APOE/APOC1 region on chromosome 19 (p = 6 x 10(-24)); CELSR2/PSRC1 on chromosome 1 (p = 3 x 10(-15)); SCARB1 on chromosome 12 (p = 1x10(-8)) and ZNF259/BUD13 in the APOA5/APOA1 gene region on chromosome 11 (p = 4 x 10(-8)). All of these remained significant after accounting for associations with LDL cholesterol, HDL cholesterol, or triglycerides. For Lp-PLA(2) mass, 12 SNPs achieved genome-wide significance, all clustering in a region on chromosome 6p12.3 near the PLA2G7 gene. Our analyses demonstrate that genetic polymorphisms may contribute to inter-individual variation in Lp-PLA(2) activity and mass.

Beta-arrestins regulate atherosclerosis and neointimal hyperplasia by controlling smooth muscle cell proliferation and migration.

Atherosclerosis and arterial injury-induced neointimal hyperplasia involve medial smooth muscle cell (SMC) proliferation and migration into the arterial intima. Because many 7-transmembrane and growth factor receptors promote atherosclerosis, we hypothesized that the multifunctional adaptor proteins beta-arrestin1 and -2 might regulate this pathological process. Deficiency of beta-arrestin2 in ldlr(-/-) mice reduced aortic atherosclerosis by 40% and decreased the prevalence of atheroma SMCs by 35%, suggesting that beta-arrestin2 promotes atherosclerosis through effects on SMCs. To test this potential atherogenic mechanism more specifically, we performed carotid endothelial denudation in congenic wild-type, beta-arrestin1(-/-), and beta-arrestin2(-/-) mice. Neointimal hyperplasia was enhanced in beta-arrestin1(-/-) mice, and diminished in beta-arrestin2(-/-) mice. Neointimal cells expressed SMC markers and did not derive from bone marrow progenitors, as demonstrated by bone marrow transplantation with green fluorescent protein-transgenic cells. Moreover, the reduction in neointimal hyperplasia seen in beta-arrestin2(-/-) mice was not altered by transplantation with either wild-type or beta-arrestin2(-/-) bone marrow cells. After carotid injury, medial SMC extracellular signal-regulated kinase activation and proliferation were increased in beta-arrestin1(-/-) and decreased in beta-arrestin2(-/-) mice. Concordantly, thymidine incorporation and extracellular signal-regulated kinase activation and migration evoked by 7-transmembrane receptors were greater than wild type in beta-arrestin1(-/-) SMCs and less in beta-arrestin2(-/-) SMCs. Proliferation was less than wild type in beta-arrestin2(-/-) SMCs but not in beta-arrestin2(-/-) endothelial cells. We conclude that beta-arrestin2 aggravates atherosclerosis through mechanisms involving SMC proliferation and migration and that these SMC activities are regulated reciprocally by beta-arrestin2 and beta-arrestin1. These findings identify inhibition of beta-arrestin2 as a novel therapeutic strategy for combating atherosclerosis and arterial restenosis after angioplasty.

Plasma lipoproteins of free-ranging howling monkeys (Alouatta palliata).

1. Plasma lipids and lipoproteins of free-ranging howling monkeys from Costa Rica (Alouatta palliata), aged 5 months to 23 years, were characterized. 2. High density lipoproteins were lipid-rich, similar to HDL2 of human plasma. 3. Fatty acid compositions of major lipid classes of very low, low and high density lipoproteins differed among social groups, possibly due to both dietary and genetic factors. 4. Low and high density lipoprotein phospholipids were enriched in phosphatidylethanolamine. 5. Howler plasma cross reacted with antihuman apoA-I antibodies but not with antihuman LDL antibodies. 6. No dimeric form of apoA-II was present, unlike human apoA-II.

Atherosclerosis: Viewing the problem from a different perspective including possible treatment options

This paper proposes that atherosclerosis is initiated by a signaling event that deposits calcium hydroxyapatite (Ca-HAP). This event is preceded by a loss of mechanical structure in the arterial wall. After Ca-HAP has been deposited, it is unlikely that it will be reabsorbed because the solubility product constant (K sp) is very small, and the large stores of Ca +2 and PO 4-3 in the bones oppose any attempts to dissolve Ca-HAP by decreasing the common ions. The hydroxide ion (OH -) of Ca-HAP can be displaced in nature by fluoride (F -) and carbonate (CO 3-2) ions, and it is proposed that anions associated with cholesterol ester hydrolysis and, in very small quantities, the enolate of 7-ketocholesterol could also displace the OH -of Ca-HAP, forming an ionic bond. The free energy of hydration of Ca-HAP at 310 K is most likely negative, and the ionic radii of the anions associated with the hydrolysis of cholesterol ester are compatible with the substitution. Furthermore, examination of the pathology of atherosclerotic lesions by Raman and NMR spectroscopy and confocal microscopy supports deposition of Ca-HAP associated with cholesterol. Investigating the affinity of intermediates of cholesterol hydrolysis for Ca-HAP compared to lipoproteins such as HDL, LDL, and VLDL using isothermic titration calorimetry could add proof of this concept and may lead to the development of a new class of medications targeted at the deposition of cholesterol within Ca-HAP. Treatment of acute ischemic events as a consequence of atherosclerosis with denitrogenation and oxygenation is discussed. © the author(s), publisher and licensee Libertas Academica Ltd.

Recombinant Adeno-Associated Virus-Mediated Gene Therapy for Treatment of Familial Hypercholesterolemia in Rabbits

Familial hypercholesterolemia (FH) is a genetic disorder characterized by abnormally high concentrations of low-density lipoprotein-cholesterol (LDLcholesterol) in the blood that can contribute to heart disease. FH can result from a defect in the gene for the LDL receptor (LDL-R). FH patients lacking functional LDL-R may benefit from viral-mediated transfer of a functional copy of the open reading frame (ORF) of the LDL-R. Since a recombinant adeno-associated virus (rAAV) is not immunogenic and can be mass-produced, it shows promise for gene therapy applications. AAV6 and AAV8 have been shown to specifically transduce hepatocytes in several species, which normally remove the majority of LDL-cholesterol from the blood via LDL-R-mediated endocytosis. Because of the potential of rAAV to treat FH by delivery of a correct LDL-R ORF to hepatocytes, the liver specificity of these two AAV serotypes was evaluated. Additionally, rabbits were chosen as the animal model for this study because a specific strain of rabbits, Watanabe heritable hyperlipidemic (WHHL), adequately mimics the pathology of FH in humans. Exposure of rabbit liver to rAAV with the marker LacZ and subsequent inspection of liver tissue showed that AAV8 transduced rabbit liver more efficiently than AAV6. To assess the feasibility of producing a rAAV capable of transferring the LDL-R ORF to rabbit hepatocytes in vivo, rAAV8-LDL-R was mass-produced by a baculovirus system in suspension grown insect cells.