DNA-lipid systems: A physical chemistry study

It is well known that the interaction of polyelectrolytes with oppositely charged surfactants leads to an associative phase separation; however, the phase behavior of DNA and oppositely charged surfactants is more strongly associative than observed in other systems. A precipitate is formed with very low amounts of surfactant and DNA. DNA compaction is a general phenomenon in the presence of multivalent ions and positively charged surfaces; because of the high charge density there are strong attractive ion correlation effects. Techniques like phase diagram determinations, fluorescence microscopy, and ellipsometry were used to study these systems. The interaction between DNA and catanionic mixtures (i.e., mixtures of cationic and anionic surfactants) was also investigated. We observed that DNA compacts and adsorbs onto the surface of positively charged vesicles, and that the addition of an anionic surfactant can release DNA back into solution from a compact globular complex between DNA and the cationic surfactant. Finally, DNA interactions with polycations, chitosans with different chain lengths, were studied by fluorescence microscopy, in vivo transfection assays and cryogenic transmission electron microscopy. The general conclusion is that a chitosan effective in promoting compaction is also efficient in transfection.

Skin secretion of Siphonops paulensis (Gymnophiona, Amphibia) forms voltage-dependent ionic channels in lipid membranes

The effect of the skin secretion of the amphibian Siphonops paulensis was investigated by monitoring the changes in conductance of an artificial planar lipid bilayer. Skin secretion was obtained by exposure of the animals to ether-saturated air, and then rinsing the animals with distilled water. Artificial lipid bilayers were obtained by spreading a solution of azolectin over an aperture of a Delrin cup inserted into a cut-away polyvinyl chloride block. In 9 of 12 experiments, the addition of the skin secretion to lipid bilayers displayed voltage-dependent channels with average unitary conductance of 258 ± 41.67 pS, rather than nonspecific changes in bilayer conductance. These channels were not sensitive to 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid or tetraethylammonium ion, but the experimental protocol used does not permit us to specify their characteristics.

Lipid peroxidation and total radical-trapping potential of the lungs of rats submitted to chronic and sub-chronic stress

Exposure to stress induces a cluster of physiological and behavioral changes in an effort to maintain the homeostasis of the organism. Long-term exposure to stress, however, has detrimental effects on several cell functions such as the impairment of antioxidant defenses leading to oxidative damage. Oxidative stress is a central feature of many diseases. The lungs are particularly susceptible to lesions by free radicals and pulmonary antioxidant defenses are extensively distributed and include both enzymatic and non-enzymatic systems. The aim of the present study was to determine lipid peroxidation and total radical-trapping potential (TRAP) changes in lungs of rats submitted to different models of chronic stress. Adult male Wistar rats weighing 180-230 g were submitted to different stressors (variable stress, N = 7) or repeated restraint stress for 15 (N = 10) or 40 days (N = 6) and compared to control groups (N = 10 each). Lipid peroxidation levels were assessed by thiobarbituric acid reactive substances (TBARS), and TRAP was measured by the decrease in luminescence using the 2-2'-azo-bis(2-amidinopropane)-luminol system. Chronic variable stress induced a 51% increase in oxidative stress in lungs (control group: 0.037 ± 0.002; variable stress: 0.056 ± 0.007, P < 0.01). No difference in TBARS was observed after chronic restraint stress, but a significant 57% increase in TRAP was presented by the group repeatedly restrained for 15 days (control group: 2.48 ± 0.42; stressed: 3.65 ± 0.16, P < 0.05). We conclude that different stressors induce different effects on the oxidative status of the organism.

Effect of dietary fat saturation on lipid metabolism, arachidonic acid turnover and peritoneal macrophage oxidative stress in mice

We investigated the effects of a saturated fat diet on lipid metabolism and arachidonic acid (AA) turnover in mouse resident peritoneal macrophages. The pro-oxidative effect of this diet was also studied. Female C57BL/6 mice were weaned at 21 days of age and assigned to either the experimental diet containing coconut oil (COCO diet), or the control diet containing soybean oil as fat source (10 mice per group). The fat content of each diet was 15% (w/w). Mice were fed for 6 weeks and then sacrificed. The concentration of total lipids, triglycerides, (LDL + VLDL)-cholesterol, thiobarbituric acid-reactive substances (TBARS) and reduced glutathione were increased in the plasma of mice fed the COCO diet, without changes in phospholipid or total cholesterol concentrations compared to control. The concentrations of total cholesterol, free and esterified cholesterol, triglycerides, and TBARS were increased in the macrophages of COCO-fed mice, while the content of total phospholipids did not change. The phospholipid composition showed an increase of phosphatidylcholine and a decrease of phosphatidylethanolamine. The [³H]-AA distribution in the phospholipid classes showed an increase in phosphatidylcholine and phosphatidylethanolamine. Incorporation of [³H]-cholesterol into the macrophages of COCO-fed mice and into the cholesterol ester fraction was increased. The COCO diet did not affect [³H]-AA uptake but induced an increase in [³H]-AA release. The COCO diet also enhanced AA mobilization induced by lipopolysaccharide. These results indicate that the COCO diet, high in saturated fatty acids, alters the lipid metabolism and AA turnover of peritoneal macrophages in female mice and also produces a significant degree of oxidative stress.

Nanoparticle-Assisted Immunoassays for Point-of-Care Testing - With Specific Interest in Minimally Interference-Prone Assays for Cardiac Troponin I

Cardiac troponins (cTn) I and T are the current golden standard biochemical markers in the diagnosis and risk stratification of patients with suspected acute coronary syndrome. During the past few years, novel assays capable of detecting cTn‐concentrations in >50% of apparently healthy individuals have become readily available. With the emerging of these high sensitivity cTn assays, reductions in the assay specificity have caused elevations in the measured cTn levels that do not correlate with the clinical picture of the patient. The increased assay sensitivity may reveal that various analytical interference mechanisms exist. This doctoral thesis focused on developing nanoparticle‐assisted immunometric assays that could possibly be applied to an automated point‐of‐care system. The main objective was to develop minimally interference‐prone assays for cTnI by employing recombinant antibody fragments. Fast 5‐ and 15‐minute assays for cTnI and D‐dimer, a degradation product of fibrin, based on intrinsically fluorescent nanoparticles were introduced, thus highlighting the versatility of nanoparticles as universally applicable labels. The utilization of antibody fragments in different versions of the developed cTnI‐assay enabled decreases in the used antibody amounts without sacrificing assay sensitivity. In addition, the utilization of recombinant antibody fragments was shown to significantly decrease the measured cTnI concentrations in an apparently healthy population, as well as in samples containing known amounts of potentially interfering factors: triglycerides, bilirubin, rheumatoid factors, or human anti‐mouse antibodies. When determining the specificity of four commercially available antibodies for cTnI, two out of the four cross‐reacted with skeletal troponin I, but caused crossreactivity issues in patient samples only when paired together. In conclusion, the results of this thesis emphasize the importance of careful antibody selection when developing cTnI assays. The results with different recombinant antibody fragments suggest that the utilization of antibody fragments should strongly be encouraged in the immunoassay field, especially with analytes such as cTnI that require highly sensitive assay approaches.

Elevated levels of erythrocyte-conjugated dienes indicate increased lipid peroxidation in schistosomiasis mansoni patients

Schistosoma mansoni causes liver disease by inducing granulomatous inflammation. This favors formation of reactive oxygen species, including superoxide ions, hydrogen peroxide and hydroxyl radicals all of which may induce lipid peroxidation. We have evaluated lipid peroxidation in 18 patients with hepatosplenic schistosomiasis mansoni previously treated with oxamniquine followed by splenectomy, ligature of the left gastric vein and auto-implantation of spleen tissue, by measuring levels of erythrocyte-conjugated dienes and plasma malondialdehyde (MDA). Age-matched, healthy individuals (N = 18) formed the control group. Erythrocyte-conjugated dienes were extracted with dichloromethane/methanol and quantified by UV spectrophotometry, while plasma MDA was measured by reaction with thiobarbituric acid. Patient erythrocytes contained two times more conjugated dienes than control cells (584.5 ± 67.8 vs 271.7 ± 20.1 µmol/l, P < 0.001), whereas the increase in plasma MDA concentration (about 10%) was not statistically significant. These elevated conjugated dienes in patients infected by S. mansoni suggest increased lipid peroxidation in cell membranes, although this was not evident when a common marker of oxidative stress, plasma MDA, was measured. Nevertheless, these two markers of lipid peroxidation, circulating MDA and erythrocyte-conjugated dienes, correlated significantly in both patient (r = 0.62; P < 0.01) and control (r = 0.57; P < 0.05) groups. Our data show that patients with schistosomiasis have abnormal lipid peroxidation, with elevated erythrocyte-conjugated dienes implying dysfunctional cell membranes, and also imply that this may be attenuated by the redox capacity of antioxidant agents, which prevent accumulation of plasma MDA.

APOA1/C3/A4 gene cluster variability and lipid levels in Brazilian children

Genetic studies have suggested that polymorphisms of genes coding for apolipoproteins are significant determinants of serum lipoprotein and lipid levels in adults. However, only a few studies have investigated the association of these polymorphisms in children. Therefore, in the present investigation we studied the distribution of APOA1 -75 G>A, +83 C>T, APOC3 -482 C>T, -455 T>C and 3238 C>G, and APOA4 Q360H and T347S polymorphisms and their influence on plasma lipoprotein levels in children from a Brazilian northeastern admixed population. The seven polymorphic sites were genotyped in 414 children aged 5 to 15 years (mean 8.9 ± 2.9). The genotypes of the seven polymorphic sites were assessed by PCR-RFLP methods. The frequencies of the less common alleles were, in general, intermediate among parental populations, as expected. Strong linkage disequilibrium was detected between polymorphisms at the APOA1, APOC3 and APOA4 loci in this admixed population sample. Overall the genotype effects seen in adults were weaker or absent in children. The APOC3/-455 and APOA4 T347S variants showed significant effects on HDL cholesterol in girls (P = 0.033 and P = 0.016, respectively). Significantly higher plasma total (P = 0.003) and LDL cholesterol (P = 0.004) levels were observed in boys who were carriers of the 3238G allele at the APOC3/3238 C>G site. These results disclosed an overall absence of associations between these polymorphisms and lipids in children. This finding is not unexpected because expression of the effect of these polymorphisms might depend on the interaction with environmental variables both internal and external to the individual.

Atherogenic lipid profile of Brazilian near-term newborns

Cardiovascular disease is the primary cause of death in Brazil. Recent studies have shown that low birth weight and preterm birth are linked to a higher prevalence of cardiovascular disease. The aim of the present study was to compare the levels of lipids and apolipoproteins and atherogenic indexes between term and near-term newborn infants. A sample of umbilical cord blood was obtained from 135 newborns (66 males) divided into two groups: 25 near-term neonates (35-36.6 weeks of gestational age) and 110 term neonates (37-42 weeks of gestational age). The total cholesterol concentrations were higher in the near-term neonates than in the term group (94.04 ± 8.02 vs 70.42 ± 1.63 mg/dl, P < 0.01), due to an increase in the LDL-cholesterol fraction in the near-term group (57.76 ± 6.39 vs 34.38 ± 1.29 mg/dl, P < 0.001). The atherogenic indexes (total cholesterol/HDL-cholesterol, LDL-cholesterol/HDL-cholesterol and apolipoprotein B/apolipoprotein A-I) were higher in the near-term group (P < 0.001, P < 0.001, and P < 0.05, respectively). The gestational age of the newborns was inversely correlated with total cholesterol and LDL-cholesterol, and also with the total cholesterol/HDL-cholesterol and LDL-cholesterol/HDL-cholesterol indexes. These findings demonstrate that the lipid profile is worse in the group of near-term neonates compared with the term group. Future studies are needed to determine if this atherogenic profile in near-term neonates can affect body metabolism, increasing the risk for cardiovascular diseases in adult life.

Solid-state reference and ion-selective electrodes : towards portable potentiometric sensing

Potentiometric sensors are very attractive tools for chemical analysis because of their simplicity, low power consumption and low cost. They are extensively used in clinical diagnostics and in environmental monitoring. Modern applications of both fields require improvements in the conventional construction and in the performance of the potentiometric sensors, as the trends are towards portable, on-site diagnostics and autonomous sensing in remote locations. The aim of this PhD work was to improve some of the sensor properties that currently hamper the implementation of the potentiometric sensors in modern applications. The first part of the work was concentrated on the development of a solid-state reference electrode (RE) compatible with already existing solid-contact ion-selective electrodes (ISE), both of which are needed for all-solid-state potentiometric sensing systems. A poly(vinyl chloride) membrane doped with a moderately lipophilic salt, tetrabutylammonium-tetrabutylborate (TBA-TBB), was found to show a satisfactory stability of potential in sample solutions with different concentrations. Its response time was nevertheless slow, as it required several minutes to reach the equilibrium. The TBA-TBB membrane RE worked well together with solid-state ISEs in several different situations and on different substrates enabling a miniature design. Solid contacts (SC) that mediate the ion-to-electron transduction are crucial components of well-functioning potentiometric sensors. This transduction process converting the ionic conduction of an ion-selective membrane to the electronic conduction in the circuit was studied with the help of electrochemical impedance spectroscopy (EIS). The solid contacts studied were (i) the conducting polymer (CP) poly(3,4-ethylienedioxythiophene) (PEDOT) and (ii) a carbon cloth having a high surface area. The PEDOT films were doped with a large immobile anion poly(styrene sulfonate) (PSS-) or with a small mobile anion Cl-. As could be expected, the studied PEDOT solid-contact mediated the ion-toelectron transduction more efficiently than the bare glassy carbon substrate, onto which they were electropolymerized, while the impedance of the PEDOT films depended on the mobility of the doping ion and on the ions in the electrolyte. The carbon cloth was found to be an even more effective ion-to-electron transducer than the PEDOT films and it also proved to work as a combined electrical conductor and solid contact when covered with an ion-selective membrane or with a TBA-TBB-based reference membrane. The last part of the work was focused on improving the reproducibility and the potential stability of the SC-ISEs, a problem that culminates to the stability of the standard potential E°. It was proven that the E° of a SC-ISE with a conducting polymer as a solid contact could be adjusted by reducing or oxidizing the CP solid contact by applying current pulses or a potential to it, as the redox state of the CP solid-contact influences the overall potential of the ISE. The slope and thus the analytical performance of the SC-ISEs were retained despite the adjustment of the E°. The shortcircuiting of the SC-ISE with a conventional large-capacitance RE was found to be a feasible instrument-free method to control the E°. With this method, the driving force for the oxidation/reduction of the CP was the potential difference between the RE and the SC-ISE, and the position of the adjusted potential could be controlled by choosing a suitable concentration for the short-circuiting electrolyte. The piece-to-piece reproducibility of the adjusted potential was promising, and the day-today reproducibility for a specific sensor was excellent. The instrumentfree approach to control the E° is very attractive considering practical applications.

Effects of metformin on the glycemic control, lipid profile, and arterial blood pressure of type 2 diabetic patients with metabolic syndrome already on insulin

Fifty-seven type 2 diabetic patients with metabolic syndrome and on insulin were assessed by a paired analysis before and 6 months after addition of metformin as combination therapy to evaluate the impact of the association on glycemic control, blood pressure, and lipid profile. This was a historical cohort study in which the files of type 2 diabetic patients with metabolic syndrome on insulin were reviewed. The body mass index (BMI), waist circumference, lipid profile, A1C level, fasting blood glucose level, daily dose of NPH insulin, systolic blood pressure, and diastolic blood pressure were assessed in each patient before the start of metformin and 6 months after the initiation of combination therapy. Glycemic control significantly improved (P < 0.001) after the addition of metformin (1404.4 ± 565.5 mg/day), with 14% of the 57 patients reaching A1C levels up to 7%, and 53% reaching values up to 8%. There was a statistically significant reduction (P < 0.05) of total cholesterol (229.0 ± 29.5 to 214.2 ± 25.0 mg/dL), BMI (30.7 ± 5.4 to 29.0 ± 4.0 kg/m²), waist circumference (124.6 ± 11.7 to 117.3 ± 9.3 cm), and daily necessity of insulin. The reduction of total cholesterol occurred independently of the reductions of A1C (9.65 ± 1.03 to 8.18 ± 1.01%) and BMI and the reduction of BMI and WC did not interfere with the improvement of A1C. In conclusion, our study showed the efficacy of the administration of metformin and insulin simultaneously without negative effects. No changes were detected in HDL-cholesterol or blood pressure.

Weld joint characteristics by nano-materials addition

The need for industries to remain competitive in the welding business, has created necessity to develop innovative processes that can exceed customer’s demand. Significant development in improving weld efficiency, during the past decades, still have their drawbacks, specifically in the weld strength properties. The recent innovative technologies have created smallest possible solid material known as nanomaterial and their introduction in welding production has improved the weld strength properties and to overcome unstable microstructures in the weld. This study utilizes a qualitative research method, to elaborate the methods of introducing nanomaterial to the weldments and the characteristic of the welds produced by different welding processes. The study mainly focuses on changes in the microstructural formation and strength properties on the welded joint and also discusses those factors influencing such improvements, due to the addition of nanomaterials. The effect of nanomaterial addition in welding process modifies the physics of joining region, thereby, resulting in significant improvement in the strength properties, with stable microstructure in the weld. The addition of nanomaterials in the welding processes are, through coating on base metal, addition in filler metal and utilizing nanostructured base metal. However, due to its insignificant size, the addition of nanomaterials directly to the weld, would poses complications. The factors having major influence on the joint integrity are dispersion of nanomaterials, characteristics of the nanomaterials, quantity of nanomaterials and selection of nanomaterials. The addition of nanomaterials does not affect the fundamental properties and characteristics of base metals and the filler metal. However, in some cases, the addition of nanomaterials lead to the deterioration of the joint properties by unstable microstructural formations. Still research are ongoing to achieve high joint integrity, in various materials through different welding processes and also on other factors that influence the joint strength.

Relevance of apolipoprotein E4 for the lipid profile of Brazilian patients with coronary artery disease

Apolipoprotein E (apoE - e2, e3, e4 alleles) plays a role in the regulation of lipid metabolism, with the e4 considered to be a risk factor for coronary artery disease (CAD). We aimed to evaluate the apoE polymorphisms in Brazilians with CAD and their influence on the lipid profile and other risk factors (hypertension, diabetes mellitus, smoking). Two hundred individuals were examined: 100 patients with atherosclerosis confirmed by coronary angiography and 100 controls. Blood samples were drawn to determine apoE polymorphisms and lipid profile. As expected, the e3 allele was prevalent in the CAD (0.87) and non-CAD groups (0.81; P = 0.099), followed by the e4 allele (0.09 and 0.14, respectively; P = 0.158). The e3/3 (76 and 78%) and e3/4 (16 and 23%) were the most common genotypes for patients and controls, respectively. The lipid profile was altered in patients compared to controls (P < 0.05), independently of the e4 allele. However, in the controls this allele was prevalent in individuals with elevated LDL-cholesterol levels only (odds ratio = 2.531; 95% CI = 1.028-6.232). The frequency of risk factors was higher in the CAD group (P < 0.05), but their association with the lipid profile was not demonstrable in e4 carriers. In conclusion, the e4 allele is not associated with CAD or lipid profile in patients with atherosclerosis. However, its frequency in the non-CAD group is associated with increased levels of LDL-cholesterol, suggesting an independent effect of the e4 allele on lipid profile when the low frequency of other risk factors in this group is taken into account.

Lipoprotein lipase PvuII polymorphism is associated with variations in serum lipid levels in non-diabetic pregnant women

The aim of the present study was to determine if there is an association between the single nucleotide polymorphisms (SNPs) of the lipoprotein lipase (LPL) and apolipoprotein E (apo E) genes and the serum lipid profile in pregnancy and puerperium. Non-diabetic women of European descent in the third semester of pregnancy (N = 120) were selected. Those with diseases or other condition that could modify their lipid profile were excluded from the study (N = 32). Serum lipids were measured by routine laboratory procedures and genomic DNA was extracted by a salting out method. LPL (PvuII and HindIII) and apo E (HhaI) SNPs were detected by the polymerase chain reaction and restriction fragment length polymorphism. Categorical and continuous variables were compared by the chi-square test and Student t-test or ANOVA, respectively. Women carrying the LPL P1P1 genotype had higher serum LDL cholesterol (N = 21; 155 ± 45 mg/dL) than women carrying the P1P2/P2P2 genotypes (N = 67; 133 ± 45 mg/dL; P = 0.032). During the puerperium period, serum levels of triglycerides and VLDL cholesterol were significantly reduced in women carrying the P1P1 (73%, P = 0.006) and P1P2 (51%, P = 0.002) genotypes but not in women carrying the P2P2 genotype (23%, P > 0.05). On the other hand, serum concentrations of lipids did not differ between the LPL HindIII and apo E genotypes during pregnancy and after delivery. We conclude that LPL PvuII SNP is associated with variations in serum lipids during pregnancy and the puerperal period in non-diabetic women.

Ambient particulate air pollution from vehicles promotes lipid peroxidation and inflammatory responses in rat lung

Oxidative stress plays a major role in the pathogenesis of particle-dependent lung injury. Ambient particle levels from vehicles have not been previously shown to cause oxidative stress to the lungs. The present study was conducted to a) determine whether short-term exposure to ambient levels of particulate air pollution from vehicles elicits inflammatory responses and lipid peroxidation in rat lungs, and b) determine if intermittent short-term exposures (every 4 days) induce some degree of tolerance. Three-month-old male Wistar rats were exposed to ambient particulate matter (PM) from vehicles (N = 30) for 6 or 20 continuous hours, or for intermittent (5 h) periods during 20 h for 4 consecutive days or to filtered air (PM <10 µm; N = 30). Rats continuously breathing polluted air for 20 h (P-20) showed a significant increase in the total number of leukocytes in bronchoalveolar lavage compared to control (C-20: 2.61 x 105 ± 0.51;P-20: 5.01 x 105 ± 0.81; P < 0.05) and in lipid peroxidation ([MDA] nmol/mg protein: C-20: 0.148 ± 0.01; P-20: 0.226 ± 0.02; P < 0.05). Shorter exposure (6 h) and intermittent 5-h exposures over a period of 4 days did not cause significant changes in leukocytes. Lipid damage resulting from 20-h exposure to particulate air pollution did not cause a significant increase in lung water content. These data suggest oxidative stress as one of the mechanisms responsible for the acute adverse respiratory effects of particles, and suggest that short-term inhalation of ambient particulate air pollution from street with high automobile traffic represents a biological hazard.

Purification, partial characterization and role in lipid transport to developing oocytes of a novel lipophorin from the cowpea weevil, Callosobruchus maculatus

Lipid transport in arthropods is achieved by highly specialized lipoproteins, which resemble those described in vertebrate blood. Here we describe purification and characterization of the lipid-apolipoprotein complex, lipophorin (Lp), from adults and larvae of the cowpea weevil Callosobruchus maculatus. We also describe the Lp-mediated lipid transfer to developing oocytes. Lps were isolated from homogenates of C. maculatus larvae and adults by potassio bromide gradient and characterized with respect to physicochemical properties and lipid content. The weevil Lp (465 kDa) and larval Lp (585 kDa), with hydrated densities of 1.22 and 1.14 g/mL, contained 34 and 56% lipids and 9 and 7% carbohydrates, respectively. In both Lps, mannose was the predominant monosaccharide detected by paper chromatography. SDS-PAGE revealed two apolipoproteins in each Lp with molecular masses of 225 kDa (apolipoprotein-I) and 79 kDa (apolipoprotein-II). The lipids were extracted and analyzed by thin-layer chromatography. The major phospholipids found were phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine in adult Lp, and phosphatidylcholine, phosphatidylethanolamine and sphingomyelin in larval Lp. Hydrocarbons, fatty acids and triacylglycerol were the major neutral lipids found in both Lps. Lps labeled in the protein moiety with radioactive iodine (125I-iodine) or in the lipid moiety with fluorescent lipids revealed direct evidence of endocytic uptake of Lps in live oocytes of C. maculatus.