Development and validation of a method for the analysis of Ochratoxin A in roasted coffee by liquid chromatography/electrospray-mass spectrometry in Tandem (LC/ESI-MS/MS)

A method using LC/ESI-MS/MS for the quantitative analysis of Ochratoxin A in roasted coffee was described. Linearity was demonstrated (r = 0.9175). The limits of detection and quantification were 1.0 and 3.0 ng g-1, respectively. Trueness, repeatability and intermediate precision values were 89.0-108.8%; 2.4-13.7%; 12.5-17.8%, respectively. To the best of our knowledge, this is the first report in which Ochratoxin A in roasted coffee is analysed by LC/ESI-MS/MS, contributing to the field of mycotoxin analysis, and it will be used for future production of Certified Reference Material.

Parametric sensitivity analysis for the helium dimers on a model potential

Potential parameters sensitivity analysis for helium unlike molecules, HeNe, HeAr, HeKr and HeXe is the subject of this work. Number of bound states these rare gas dimers can support, for different angular momentum, will be presented and discussed. The variable phase method, together with the Levinson's theorem, is used to explore the quantum scattering process at very low collision energy using the Tang and Toennies potential. These diatomic dimers can support a bound state even for relative angular momentum equal to five, as in HeXe. Vibrational excited states, with zero angular momentum, are also possible for HeKr and HeXe. Results from sensitive analysis will give acceptable order of magnitude on potentials parameters.

Comparative analysis of different meat traceability systems using multiple criteria and a social network approach

The adoption of a proper traceability system is being incorporated into meat production practices as a method of gaining consumer confidence. The various partners operating in the chain of meat production can be considered a social network, and they have the common goal of generating a communication process that can ensure each characteristic of the product, including safety. This study aimed to select the most appropriate meat traceability system “from farm to fork” that could be applied to Brazilian beef and pork production for international trade. The research was done in three steps. The first used the analytical hierarchy process (AHP) for selecting the best on-farm livestock traceability. In the second step, the actors in the meat production chain were identified to build a framework and defined each role in the network. In the third step, the selection of the traceability system was done. Results indicated that with an electronic traceability system, it is possible to acquire better connections between the links in the chain and to provide the means for managing uncertainties by creating structures that facilitate information flow more efficiently.

Multiple scales analysis of nonlinear oscillations of a portal frame foundation for several machines

An analytical study of the nonlinear vibrations of a multiple machines portal frame foundation is presented. Two unbalanced rotating machines are considered, none of them resonant with the lower natural frequencies of the supporting structure. Their combined frequencies is set in such a way as to excite, due to nonlinear behavior of the frame, either the first anti-symmetrical mode (sway) or the first symmetrical mode. The physical and geometrical characteristics of the frame are chosen to tune the natural frequencies of these two modes into a 1:2 internal resonance. The problem is reduced to a two degrees of freedom model and its nonlinear equations of motions are derived via a Lagrangian approach. Asymptotic perturbation solutions of these equations are obtained via the Multiple Scales Method.

Structural analysis of the vegetation on a highland granitic rock outcrop in Southeast Brazil

Granitic rock outcrops of the Brazilian southeast are either coastal or inland. The latter can often have high altitudes, such as in the summits of "Serra do Mar" and "Serra da Mantiqueira", where they are known as "Campos de Altitude". The landscape on these high altitude plateaux is often a mosaic of shrubs and treelets within a bunchgrass matrix, with sparse pteridophytes and other herbs, interspersed with variable extensions of rock outcrops. Despite the pervasiveness of rock outcrops in the Brazilian landscape, studies on the structural analysis of the vegetation on such formations are scarce. This study aimed to analyze the structure of the vegetation on a highland granitic rock outcrop in "Parque Estadual da Serra do Brigadeiro", state of Minas Gerais, Southeast Brazil (42°20' - 42°40' S and 20°20' - 21°00' W, 1,722 m of elevation ). Quantitative parameters of absolute and relative frequency and dominance (cover) were estimated. The group analysis used the Jaccard similarity index. Trilepis lhotzkiana, Panicum sp. 1, and Vellozia variegata presented the highest relative frequencies, relative dominances and importance values. These three species, along with Dyckia bracteata, Rhynchospora emaciate, and Tibouchina cf. manicata, represented 98.3% of the relative dominance. The remaining 1.7% referred to 22 remaining species. The distinction among quadrats within formed groups by cluster analysis was due to the great number of low frequency species.

Utilization of microsatellites for the analysis of genomic alterations in colorectal cancers in Brazil

Two different pathogenetic mechanisms are proposed for colorectal cancers. One, the so-called "classic pathway", is the most common and depends on multiple additive mutational events (germline and/or somatic) in tumor suppressor genes and oncogenes, frequently involving chromosomal deletions in key genomic regions. Methodologically this pathway is recognizable by the phenomenon of loss of heterozygosity. On the other hand, the "mutator pathway" depends on early mutational loss of the mismatch repair system (germline and/or somatic) leading to accelerated accumulation of gene mutations in critical target genes and progression to malignancy. Methodologically this second pathway is recognizable by the phenomenon of microsatellite instability. The distinction between these pathways seems to be more than academic since there is evidence that the tumors emerging from the mutator pathway have a better prognosis. We report here a very simple methodology based on a set of tri-, tetra- and pentanucleotide repeat microsatellites allowing the simultaneous study of microsatellite instability and loss of heterozygosity which could allocate 70% of the colorectal tumors to the classic or the mutator pathway. The ease of execution of the methodology makes it suitable for routine clinical typing

Analysis of polymorphism at site -174 G/C of interleukin-6 promoter region in multiple myeloma

It is well established that interleukin-6 (IL-6) is an essential growth factor for multiple myeloma (MM) and patients with increased IL-6 levels have a poor prognosis. In healthy subjects, the presence of the C allele at a polymorphic site (-174 G/C) of the IL-6 gene is related to low IL-6 levels. In view of the potential association of this particular polymorphism with IL-6 concentration, and the relevance of IL-6 in MM pathogenesis, the objective of the present study was to investigate the prevalence of IL-6 (-174 G/C) promoter polymorphism and its association with development of MM in Brazilian individuals. We investigated the prevalence of these alleles in 52 patients and 60 healthy subjects (matched by age, sex, and race) of a Brazilian population. Thirty patients were male (42.4%), 24 (46.2%) were white and the median age at diagnosis was 58.5 years (range: 28 to 84 years). To determine the IL-6 (-174 G/C) polymorphism, molecular analysis was performed by polymerase chain reaction followed by endonuclease restriction digestion. The genotype distributions observed in the group of patients were 4% CC, 42% GC and 54% GG. The C allele frequency was 0.25. These results were similar to the control group, suggesting no impact of this polymorphism on the susceptibility to MM.

Serum levels of brain-derived neurotrophicfactor correlate with the number of T2 MRI lesions in multiple sclerosis

The objective of the present study was to determine if there is a relationship between serum levels of brain-derived neurotrophic factor (BDNF) and the number of T2/fluid-attenuated inversion recovery (T2/FLAIR) lesions in multiple sclerosis (MS). The use of magnetic resonance imaging (MRI) has revolutionized the study of MS. However, MRI has limitations and the use of other biomarkers such as BDNF may be useful for the clinical assessment and the study of the disease. Serum was obtained from 28 MS patients, 18-50 years old (median 38), 21 women, 0.5-10 years (median 5) of disease duration, EDSS 1-4 (median 1.5) and 28 healthy controls, 19-49 years old (median 33), 19 women. BDNF levels were measured by ELISA. T1, T2/FLAIR and gadolinium-enhanced lesions were measured by a trained radiologist. BDNF was reduced in MS patients (median [range] pg/mL; 1160 [352.6-2640]) compared to healthy controls (1640 [632.4-4268]; P = 0.03, Mann-Whitney test) and was negatively correlated (Spearman correlation test, r = -0.41; P = 0.02) with T2/FLAIR (11-81 lesions, median 42). We found that serum BDNF levels were inversely correlated with the number of T2/FLAIR lesions in patients with MS. BDNF may be a promising biomarker of MS.

Analysis of acylcarnitine profiles in umbilical cord blood and during the early neonatal period by electrospray ionization tandem mass spectrometry

Acylcarnitine profiling by electrospray ionization tandem mass spectrometry (ESI-MS/MS) is a potent tool for the diagnosis and screening of fatty acid oxidation and organic acid disorders. Few studies have analyzed free carnitine and acylcarnitines in dried blood spots (DBS) of umbilical cord blood (CB) and the postnatal changes in the concentrations of these analytes. We have investigated these metabolites in healthy exclusively breastfed neonates and examined possible effects of birth weight and gestational age. DBS of CB were collected from 162 adequate for gestational age neonates. Paired DBS of heel-prick blood were collected 4-8 days after birth from 106 of these neonates, the majority exclusively breastfed. Methanol extracts of DBS with deuterium-labeled internal standards were derivatized before analysis by ESI-MS/MS. Most of the analytes were measured using a full-scan method. The levels of the major long-chain acylcarnitines, palmitoylcarnitine, stearoylcarnitine, and oleoylcarnitine, increased by 27, 12, and 109%, respectively, in the first week of life. Free carnitine and acetylcarnitine had a modest increase: 8 and 11%, respectively. Propionylcarnitine presented a different behavior, decreasing 9% during the period. The correlations between birth weight or gestational age and the concentrations of the analytes in DBS were weak (r £ 0.20) or nonsignificant. Adaptation to breast milk as the sole source of nutrients can explain the increase of these metabolites along the early neonatal period. Acylcarnitine profiling in CB should have a role in the early detection of metabolic disorders in high-risk neonates.

ChIP-seq analysis of histone H3K9 trimethylation in peripheral blood mononuclear cells of membranous nephropathy patients

Membranous nephropathy (MN), characterized by the presence of diffuse thickening of the glomerular basement membrane and subepithelial in situimmune complex disposition, is the most common cause of idiopathic nephrotic syndrome in adults, with an incidence of 5-10 per million per year. A number of studies have confirmed the relevance of several experimental insights to the pathogenesis of human MN, but the specific biomarkers of MN have not been fully elucidated. As a result, our knowledge of the alterations in histone methylation in MN is unclear. We used chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) to analyze the variations in a methylated histone (H3K9me3) in peripheral blood mononuclear cells from 10 MN patients and 10 healthy subjects. There were 108 genes with significantly different expression in the MN patients compared with the normal controls. In MN patients, significantly increased activity was seen in 75 H3K9me3 genes, and decreased activity was seen in 33, compared with healthy subjects. Five positive genes, DiGeorge syndrome critical region gene 6 (DGCR6), sorting nexin 16 (SNX16), contactin 4 (CNTN4), baculoviral IAP repeat containing 3 (BIRC3), and baculoviral IAP repeat containing 2 (BIRC2), were selected and quantified. There were alterations of H3K9me3 in MN patients. These may be candidates to help explain pathogenesis in MN patients. Such novel findings show that H3K9me3 may be a potential biomarker or promising target for epigenetic-based MN therapies.

Prevalence of overweight and obesity in children from urban and rural Niagara

Purpose: The influence of environment in the development of overweight and obesity is an ongoing concern. This investigation examined the influence of urbanization on the rates of childhood overweight and obesity. Method: 2167 (1090M, 1077F) grade four children from 75 schools in Ontario's Niagara Region were sampled. A sophisticated algorithm overlaying electoral boundaries, population densities, and the knowledge of community members was used to classify schools into one of three location categories: urban {N= 1588), urban fringe {N= 379), and rural (A^= 234). Each subject was measured for: height, weight, and aerobic performance (Leger). Physical activity was evaluated with the self-report Participation Questionnaire (free-time and organized sport activities), and teacher's evaluations of student activity. Overweight (overweight and obesity combined) was measured both as a continuous (BMI) and categorical variable (BMI category), to evaluate the prevalence by location. A multivariate analysis was used to test for a suppression effect. Results: BMI and BMI category did not differ significantly by location or gender, and no evidence of a gender interaction existed. According to both a linear and logistic regression, physical activity or fitness levels did not suppress the influence of location on BMI and BMI category. Age, gender, free-time activity, organized sports, fitness level, and number of siblings, were all found to significantly influence overweight. Conclusions: It is plausible that the prevalence of overweight does not differ in urban and rural children from the Niagara Region. Further investigation is recommended, examining subjects by individual location of residence, in multiple regions throughout Ontario.

Development and characterization of concentric capillary nebulizer used in inductively coupled plasma analysis /

A simple, low-cost concentric capillary nebulizer (CCN) was developed and evaluated for ICP spectrometry. The CCN could be operated at sample uptake rates of 0.050-1.00 ml min'^ and under oscillating and non-oscillating conditions. Aerosol characteristics for the CCN were studied using a laser Fraunhofter diffraction analyzer. Solvent transport efficiencies and transport rates, detection limits, and short- and long-term stabilities were evaluated for the CCN with a modified cyclonic spray chamber at different sample uptake rates. The Mg II (280.2nm)/l\/lg 1(285.2nm) ratio was used for matrix effect studies. Results were compared to those with conventional nebulizers, a cross-flow nebulizer with a Scott-type spray chamber, a GemCone nebulizer with a cyclonic spray chamber, and a Meinhard TR-30-K3 concentric nebulizer with a cyclonic spray chamber. Transport efficiencies of up to 57% were obtained for the CCN. For the elements tested, short- and long-term precisions and detection limits obtained with the CCN at 0.050-0.500 ml min'^ are similar to, or better than, those obtained on the same instrument using the conventional nebulizers (at 1.0 ml min'^). The depressive and enhancement effects of easily ionizable element Na, sulfuric acid, and dodecylamine surfactant on analyte signals with the CCN are similar to, or better than, those obtained with the conventional nebulizers. However, capillary clog was observed when the sample solution with high dissolved solids was nebulized for more than 40 min. The effects of data acquisition and data processing on detection limits were studied using inductively coupled plasma-atomic emission spectrometry. The study examined the effects of different detection limit approaches, the effects of data integration modes, the effects of regression modes, the effects of the standard concentration range and the number of standards, the effects of sample uptake rate, and the effect of Integration time. All the experiments followed the same protocols. Three detection limit approaches were examined, lUPAC method, the residual standard deviation (RSD), and the signal-to-background ratio and relative standard deviation of the background (SBR-RSDB). The study demonstrated that the different approaches, the integration modes, the regression methods, and the sample uptake rates can have an effect on detection limits. The study also showed that the different approaches give different detection limits and some methods (for example, RSD) are susceptible to the quality of calibration curves. Multicomponents spectral fitting (MSF) gave the best results among these three integration modes, peak height, peak area, and MSF. Weighted least squares method showed the ability to obtain better quality calibration curves. Although an effect of the number of standards on detection limits was not observed, multiple standards are recommended because they provide more reliable calibration curves. An increase of sample uptake rate and integration time could improve detection limits. However, an improvement with increased integration time on detection limits was not observed because the auto integration mode was used.

Frequency-dependent selection at the amylase locus in Drosophila melanogaster

A. strain of Drosophila melanog-aster deficient in null amylase activity (Amylase ) was isolated from a wild null population of flies. The survivorship of Amylase homozygous flies is very low when the principal dietary carbohydrate source is starch. However, the survivorship of the null Amylase genotype is comparable to the wild type when the dietary starch is replaced by glucose. In addition, the null viability of the amylase-producing and Amylase strains is comparable v and very lm<] f on a medium with no carbohydrates . Furthermore, amylase-producing genotypes were shovm to excrete enzymatically active amylase protein into the food medium. The excreted amylase causes the external breakdown of dietary starch to sugar. These results led to the following null prediction: the viability of the A.mvlase genotype (fed on a starch rich diet) might increase in the presence of individuals which were amylase-producing. It was shown experimentally that such an increase in viability did in fact occur and that this increase v\Tas proportional to the number of mnylase..::producing fli.es present. These results provide a unique example of a non-"competi ti ve inter-genotype interaction, and one where the underlying physio~ logical and biochemical mechanism has been fully understood.

Effectiveness of Self-Instructional Mediator Training in Applied Behaviour Analysis

Interventions using applied behaviour analysis (ABA) are widely used with children with autism spectrum disorders (ASD). Individuals who work with this population are important to target for ABA education. This study evaluated the efficacy of a self-directed program in increasing parent and student ABA knowledge and skills, self-efficacy, and new skill development in children with ASD. Study 1 was a pilot study of the newly developed evaluation materials. Study 2 tested the self-instructional package with three parents of children with ASD, three university students, and eight children diagnosed with ASD. Parents and students were given the Simple Steps ABA training package to use independently and were measured using a multiple baseline across participants and/or skills design. After training, ABA knowledge scores and self-efficacy showed variable improvement as did children’s appropriate behaviours. These results suggest that more research is needed to determine the efficacy of a self-instructional ABA package.

HUMAN GENOME VARIATIONS AND EVOLUTION WITH A FOCUS ON THE ANALYSIS OF TRANSPOSABLE ELEMENTS

Genome sequence varies in numerous ways among individuals although the gross architecture is fixed for all humans. Retrotransposons create one of the most abundant structural variants in the human genome and are divided in many families, with certain members in some families, e.g., L1, Alu, SVA, and HERV-K, remaining active for transposition. Along with other types of genomic variants, retrotransponson-derived variants contribute to the whole spectrum of genome variants in humans. With the advancement of sequencing techniques, many human genomes are being sequenced at the individual level, fueling the comparative research on these variants among individuals. In this thesis, the evolution and functional impact of structural variations is examined primarily focusing on retrotransposons in the context of human evolution. The thesis comprises of three different studies on the topics that are presented in three data chapters. First, the recent evolution of all human specific AluYb members, representing the second most active subfamily of Alus, was tracked to identify their source/master copy using a novel approach. All human-specific AluYb elements from the reference genome were extracted, aligned with one another to construct clusters of similar copies and each cluster was analyzed to generate the evolutionary relationship between the members of the cluster. The approach resulted in identification of one major driver copy of all human specific Yb8 and the source copy of the Yb9 lineage. Three new subfamilies within the AluYb family – Yb8a1, Yb10 and Yb11 were also identified, with Yb11 being the youngest and most polymorphic. Second, an attempt to construct a relation between transposable elements (TEs) and tandem repeats (TRs) was made at a genome-wide scale for the first time. Upon sequence comparison, positional cross-checking and other relevant analyses, it was observed that over 20% of all TRs are derived from TEs. This result established the first connection between these two types of repetitive elements, and extends our appreciation for the impact of TEs on genomes. Furthermore, only 6% of these TE-derived TRs follow the already postulated initiation and expansion mechanisms, suggesting that the others are likely to follow a yet-unidentified mechanism. Third, by taking a combination of multiple computational approaches involving all types of genetic variations published so far including transposable elements, the first whole genome sequence of the most recent common ancestor of all modern human populations that diverged into different populations around 125,000-100,000 years ago was constructed. The study shows that the current reference genome sequence is 8.89 million base pairs larger than our common ancestor’s genome, contributed by a whole spectrum of genetic mechanisms. The use of this ancestral reference genome to facilitate the analysis of personal genomes was demonstrated using an example genome and more insightful recent evolutionary analyses involving the Neanderthal genome. The three data chapters presented in this thesis conclude that the tandem repeats and transposable elements are not two entirely distinctly isolated elements as over 20% TRs are actually derived from TEs. Certain subfamilies of TEs themselves are still evolving with the generation of newer subfamilies. The evolutionary analyses of all TEs along with other genomic variants helped to construct the genome sequence of the most recent common ancestor to all modern human populations which provides a better alternative to human reference genome and can be a useful resource for the study of personal genomics, population genetics, human and primate evolution.