Species-specific kinetics and zonation of hepatic DNA synthesis induced by ligands of PPARα

PPARα ligands evoke a profound mitogenic response in rodent liver, and the aim of this study was to characterise the kinetics of induction of DNA synthesis. The CAR ligand, 1,4-bis[2-(3,5- dichoropyridyloxy)]benzene, caused induction of hepatocyte DNA synthesis within 48 hours in 129S4/SvJae mice, but the potent PPARα ligand, ciprofibrate, induced hepatocyte DNA synthesis only after 3 or 4 days dosing; higher or lower doses did not hasten the DNA synthesis response. This contrasted with the rapid induction (24 hours) reported by Styles et al. (Carcinogenesis 9:1647-1655). C57BL/6 and DBA/2J mice showed significant induction of DNA synthesis after 4, but not 2, days ciprofibrate treatment. Alderley Park and 129S4/SvJae mice dosed with methylclofenapate induced hepatocyte DNA synthesis at 4, but not 2, days after dosing, and proved that inconsistency with prior work was not due to a difference in mouse strain or PPARα ligand. Ciprofibrate-induced liver DNA synthesis and growth was absent in PPARα- null mice, and are PPARα-dependent. In the Fisher344 rat, hepatocyte DNA synthesis was induced at 24 hours after dosing, with a second peak at 48 hours. Lobular localisation of hepatocyte DNA synthesis showed preferential periportal induction of DNA synthesis in rat, but panlobular zonation of hepatocyte DNA synthesis in mouse. These results characterise a markedly later hepatic induction of panlobular DNA synthesis by PPARα ligands in mouse, compared to rapid induction of periportal DNA synthesis in rat.

Computing the structural and vibrational properties of polymorphic organic molecular crystals through van der Waals corrected density functional theory and the electronic properties of organic thin films through microelectrostatic calculations.

The present Thesis reports on the various research projects to which I have contributed during my PhD period, working with several research groups, and whose results have been communicated in a number of scientific publications. The main focus of my research activity was to learn, test, exploit and extend the recently developed vdW-DFT (van der Waals corrected Density Functional Theory) methods for computing the structural, vibrational and electronic properties of ordered molecular crystals from first principles. A secondary, and more recent, research activity has been the analysis with microelectrostatic methods of Molecular Dynamics (MD) simulations of disordered molecular systems. While only very unreliable methods based on empirical models were practically usable until a few years ago, accurate calculations of the crystal energy are now possible, thanks to very fast modern computers and to the excellent performance of the best vdW-DFT methods. Accurate energies are particularly important for describing organic molecular solids, since they often exhibit several alternative crystal structures (polymorphs), with very different packing arrangements but very small energy differences. Standard DFT methods do not describe the long-range electron correlations which give rise to the vdW interactions. Although weak, these interactions are extremely sensitive to the packing arrangement, and neglecting them used to be a problem. The calculations of reliable crystal structures and vibrational frequencies has been made possible only recently, thanks to development of some good representations of the vdW contribution to the energy (known as “vdW corrections”).

Implicações imunobiológicas da infecção a herpesvirus em tumores malignos : pesquisa e análise quantitativa de herpesvirus humanos no astrocitoma pilocítico

RESUMO: As infecções virais podem contribuir para o desenvolvimento do cancro, estando vários tumores malignos associados aos Herpesvirus (HHV). O vírus de Epstein-Barr (EBV) e o Herpesvirus 8, dois Herpesvirus, foram reconhecidos como agentes etiológicos de várias neoplasias. O astrocitoma pilocítico do cerebelo é um dos tumores cerebrais mais frequentes na criança, adolescentes e jovens adultos e a proliferação astrocitária ocorre geralmente após vários tipos de agressão, nomeadamente a infecção viral. Para investigar esta eventual interligação, estudámos 35 astrocitomas pilocíticos, pesquisando a presença dos 8 Herpesvirus. Neste estudo, foram utilizadas 10 amostras de biópsias do cerebelo de doentes que faleceram por doenças não relacionadas com infecção ou patologia tumoral. A maioria dos astrocitomas (33) eram tumores de baixa malignidade. As amostras foram analisadas por PCR (Polymerase Chain Reaction) quantitativa em tempo real (qPCR), com amplificação do gene da DNA polimerase viral. Treze astrocitomas e 7 controles revelaram pequenas quantidades de DNA viral (1-100 cópias/100ng DNA) de todos os Herpesvirus, com excepção do HHV6 A e B que estava ausente nas amostras. O EBV foi identificado em 9 dos 35 astrocitomas (26%) e em 7 dos 10 controles (70%) estando muito mais presente nos controles. As amostras positivas para o EBV foram também analisadas por imunohistoquímica, não tendo sido imunoreactivas para os anticorpos utilizados. A PCR com CODEHOP (consensus-degenerated hybrid oligonucleotide primers) foi utilizada para investigar a presença de um eventual Herpesvirus novo nestas amostras. Não foi identificada nenhuma sequência indicativa de um novo HHV por este método. 24. Em conclusão, os dados apontam para a presença de Herpesvirus, com particular relevância para o EBV, em tecido de cerebelo normal e em tumores cerebrais, embora em níveis demasiado baixos para poderem ser responsabilizados pela indução tumoral. A presença de sequências de DNA de Herpesvirus, nomeadamente do EBV, no Sistema Nervoso Central vem enriquecer a discussão sobre o significado da infecção viral na oncogénese humana, particularmente na neuro-oncogénese. ABSTRACT: Viral infections can contribute to the development of human cancer. Several human malignancies are linked with Human Herpesviruses (HHVs). Epstein-Barr virus and HHV8, two hHerpesvirus, have been recognized as etiologic agents of several neoplasms. Pilocytic astrocytoma of the cerebellum is one of the most common brain tumour in children, adolescents and young adults and astrocytary proliferation generally occurs after several types of injury, namely viral infection. To further explore this association, we have searched the tissue from 35 pilocytic astrocytoma, for all the 8 HHV. In this study, ten brain biopsies (cerebellum) from patients who died of unrelated diseases were used as controls. Most of the astrocytomas (33) were of low grade malignity. Samples were assessed by Real-time quantitative Polymerase Chain Reaction (q PCR) amplification of viral DNA polymerase gene. Thirteen astrocytoma and 7 controls showed low viral DNA levels (1-100 copies/100ng DNA) for all HHVs, with the exception of HHV6 that was absent. EBV was identified in 9 of the 35 astrocytoma (26 %) and in 7 of the 10 controls (70%) being more present in controls. EBV positive samples were also assessed by Immunohistochemistry (IHC) but none showed immunoreactivity for the antibodies used. PCR with consensus-degenerated hybrid oligonucleotide primers (CODEHOP) were also used to look for novel HHVs in these samples and no sequence indicative of a new HHV was detected. 26 Altogether the data indicate the presence of HHVs, with relevance for EBV in normal cerebellum tissue and also in brain tumours but at too low levels to be considered responsible for tumour induction. The presence of HHV DNA sequences, particularly EBV, in the studied brain tumours and control samples, further enriches the discussion about the relevance of viral infection in human oncogenesis, particularly neuro-oncogenesis.RÉSUMÉ: Les infections virales peuvent contribuer au développement du cancer. Les vírus de type Herpès sont associés à plusieurs néoplasies. Il est par exemple établi que les vírus Epstein-Barr et « human Herpesvirus 8 » (HHV-8) sont responsables de plusieurs tumeurs malignes. L´astrocytome pilocitique du cervelet est l’une des tumeurs les plus fréquentes chez les enfants, adolescents et adultes jeunes. En général la prolifération des astrocytes se produit en réponse à une agression. Posant l’hypothèse d’une agression d’origine virale, nous avons recherché la présence des 8 vírus Herpès dans les tissus de 35 astrocytomes. Dans cette étude, 10 échantillons de biopsie de cervelet de patients décédés suite à d’autres pathologies, ont été utilisés comme contrôles. La majorité des astrocytomes étaient de très basse malignité. Les échantillons ont été étudiés par PCR quantitative en temps réel, en amplifiant le gène de l’ADN-polymérase virale. Treize astrocytomes sur 35 (37%) et 7 contrôles sur 10 (70%) ont été trouvés positifs pour tous les HHV sauf l´HHV6, toujours avec un nombre de copies de polymérase virale bas (< 100 copies/100 ng d’ADN). Notamment l’EBV a été identifié 7 fois dans les contrôles (70%) et 9 fois dans les astrocytomes (26%). Les échantillons positifs pour l`EBV ont aussi été étudiés par immuno-histochimie. Aucun signal n’a été observé avec les anticorps utilisés. Enfin, une technique de PCR avec oligonucléotides dégénérés (CODEHOP ou consensus degenerated hybrid oligonucleotide primers) a été utilisée pour rechercher la présence d´un éventuel nouveau vírus Herpès dans les échantillons d’astrocytome. Aucun nouveau vírus n’a été identifié. 28 En résumé, nous avons établi la présence de vírus Herpès, en particulier l´EBV, dans le cervelet normal et dans les tumeurs du cerveau. Les quantités d’ADN viral retrouvées sont faibles et ne permettent pas d’attribuer à ces vírus la responsabilité de l’induction des tumeurs. Cependant, la présence d’ADN de vírus Herpès dans le cerveau sain ou pathologique vient enrichir la discussion sur le signification de l´infection virale dans les processus d´oncogenèse en général, et dans la neuroonco-genèse en particulier.

HFE gene mutations in coronary atherothrombotic disease

Although iron can catalyze the production of free radicals involved in LDL lipid peroxidation, the contribution of iron overload to atherosclerosis remains controversial. The description of two mutations in the HFE gene (Cys282Tyr and His63Asp) related to hereditary hemochromatosis provides an opportunity to address the question of the association between iron overload and atherosclerosis. We investigated the prevalence of HFE mutations in 160 survivors of myocardial infarction with angiographically demonstrated severe coronary atherosclerotic disease, and in 160 age-, gender- and race-matched healthy control subjects. PCR amplification of genomic DNA followed by RsaI and BclI restriction enzyme digestion was used to determine the genotypes. The frequency of the mutant Cys282Tyr allele was identical among patients and controls (0.022; carrier frequency, 4.4%), whereas the mutant His63Asp allele had a frequency of 0.143 (carrier frequency, 27.5%) in controls and of 0.134 (carrier frequency, 24.5%) in patients. Compound heterozygotes were found in 2 of 160 (1.2%) controls and in 1 of 160 (0.6%) patients. The finding of a similar prevalence of Cys282Tyr and His63Asp mutations in the HFE gene among controls and patients with coronary atherothrombotic disease, indirectly questions the possibility of an association between hereditary hemochromatosis and atherosclerosis.

HLA-DQA1 allele typing by nonisotopic PCR-LIS-SSCP

In the present study we used a simple and reliable method for HLA-DQA1 allele typing based on the single-stranded conformation polymorphism (SSCP) properties of DNA molecules obtained by PCR. The technique consists of PCR amplification of a DNA fragment comprising the second exon of the HLA-DQA1 gene, amplicon denaturation using a low ionic strength solution (LIS), and electrophoresis on a small native polyacrylamide gel, followed by a rapid silver staining procedure. In order to validate the technique and to obtain the allele patterns for the DQA1 gene, 50 cervical samples were typed using this methodology and the commercial Amplitype® HLA DQA1 Amplification and Typing kit. All the alleles detected with the kit were characterized by the LIS-SSCP approach. This procedure proved to be useful for population screening and typing of the DQA1 gene as well as for detecting new alleles or mutations in the donor-recipient molecular matching of HLA class II genes.

Surfactant protein B gene polymorphism in preterm babies with respiratory distress syndrome

The etiology of respiratory distress syndrome (RDS) is multifactorial and multigenic. Studies have suggested that polymorphisms and mutations in the surfactant protein B (SP-B) gene are associated with the pathogenesis of RDS. The objectives of this study were to determine and compare the frequencies of SP-B gene polymorphisms in preterm babies with and without RDS. We studied 151 neonates: 79 preterm babies without RDS and 72 preterm newborns with RDS. The following four SP-B gene polymorphisms were analyzed: A/C at -18, C/T at 1580, A/G at 9306, and G/C at nucleotide 8714. The polymorphisms were detected by PCR amplification of genomic DNA and genotyping. The genotypes were determined using PCR-based converted restriction fragment length polymorphisms. The control group consisted of 42 (53%) girls and 37 (47%) boys. Weight ranged from 1170 to 3260 g and mean gestational age (GA) was 33.9 weeks (range: 29 to 35 weeks and 6 days). The RDS group consisted of 31 (43%) girls and 41 (57%) boys. Weight ranged from 614 to 2410 g and mean GA was 32 weeks (range: 26 to 35 weeks). The logistic regression model showed that GA was the variable that most contributed to the occurrence of RDS. The AG genotype of the A/G polymorphism at position 9306 of the SP-B gene was a protective factor in this population (OR = 0.1681; 95%CI = 0.0426-0.6629). We did not detect differences in the frequencies of the other polymorphisms between the two groups of newborns.

Ehrlichiosis in anemic, thrombocytopenic, or tick-infested dogs from a hospital population in South Brazil

Ehrlichia canis has a worldwide distribution, but clinical manifestations may vary geographically. We selected 129 dogs to determine prevalence of ehrlichiosis in dogs with anemia, thrombocytopenia, or ticks presented to a Veterinary Teaching Hospital in South Brazil. Of the 129 dogs, 68 carried the brown dog tick (Rhipicephalus sanguineus), 61 had thrombocytopenia (platelet count <150,000/μl), and 19 had anemia (PCV < 22%). Twenty dogs fulfilled more than one inclusion criteria. Ehrlichiosis was diagnosed by positive amplification of ehrlichial DNA by PCR using primers ECC and ECB that amplify a sequence of the 16S rRNA gene. Presence of E. canis was confirmed by cleavage of the amplified DNA using endonucleases HaeIII and AvaI. Fourteen of 68 (21%) dogs with ticks had ehrlichiosis, whereas 12 of 61 (20%) dogs presented with thrombocytopenia and 4 of 19 (21%) anemic dogs had ehrlichiosis. Similar results were obtained in dogs with thrombocytopenia and anemia (one of eight positive) and in dogs with thrombocytopenia and ticks (two of seven positive). All four dogs with anemia and ticks, and the dog that fulfilled all inclusion criteria yield no amplification of ehrlichial DNA by PCR. Based on our results, one in each five dogs infested by the brown dog tick, with anemia or thrombocytopenia had ehrlichosis. Contrary to widespread believe, ehrlichiosis was not the main cause for thrombocytopenia in our region. © 2003 Elsevier B.V. All rights reserved.

Detecção de Mycobacterium lepra por PCR em "SWAB" nasal e "SWAB" da linfa do lóbulo da orelha de pacientes hansenianos

Recentemente vários estudos têm usado a técnica Reação em Cadeia de Polimerase (PCR) para detecção do DNA do Mycobacterium leprae, em diversas amostras biológicas, demonstrando alta sensibilidade. O objetivo deste trabalho foi avaliar a sensibilidade da PCR na detecção de M. leprae em “swab” nasal e “swab” da linfa do lóbulo da orelha de pacientes hansenianos e comparar os resultados da PCR com a baciloscopia e histopatologia e formas multibacilares (MBs) e paucibacilares (PBs) da hanseníase. Foram coletadas amostras de secreção nasal e linfa do lóbulo da orelha de 24 pacientes hansenianos. Para amplificação do DNA foram testados três pares de primers: S13 e S62, R1 e R2, LP1 e LP2 que amplificam fragmentos de DNA de 531 pb, 372pb e 129pb, respectivamente. Os iniciadores LP1 e LP2 expressaram maior sensibilidade, independente das amostras clínicas. Os resultados da PCR foram altamente significativos para as amostras de secreção nasal (p<0.0000) e significativos para os espécimes de linfa do lóbulo da orelha (p=0.0000). Comparando os resultados da PCR, usando os primers LP1 e LP2 e conservante lise 1, com a baciloscopia e histopatologia, os estudos apontaram que a PCR, em amostras de secreção nasal, obteve maior sensibilidade para as formas MBs (41,67%), seguida da baciloscopia (25%) e histopatologia (8,33%). Nas formas PBs, a sensibilidade foi considerada a mesma entre a PCR e Histopatologia (8,33%). A baciloscopia não apresentou sensibilidade (0%). Nas amostras da linfa do lóbulo da orelha, a baciloscopia demonstrou maior sensibilidade para as formas MBs (25%), seguido da PCR (20,83%) e histopatologia (16,7%). Nas formas PBs, a PCR e Histopatologia apresentaram a mesma sensibilidade (4,17%). Não houve sensibilidade na baciloscopia (0%). A PCR, apesar de não demonstrar uma sensibilidade de 100% é uma ferramenta com perspectivas futuras para auxiliar no monitoramento do tratamento e cura dos pacientes hansenianos.

Ocorrência de microrganismos oportunistas e superinfectantes em crianças com 6, 12 e 18 meses de idade e suas mães

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Avaliação de protozoários em tilápias (Oreochromis niloticus) e possíveis riscos em saúde pública

Pós-graduação em Medicina Veterinária - FMVZ

Patógenos periodontais: comparação entre cultura bacteriana e PCR em tempo real para teste diagnóstico

The correct distinguishment of microorganisms involved in the periodontal disease pathogen, it is important in the understanding of its progression and adequate treatment planning. Considering this fact, some molecular methods of identification and quantification were developed and are extremely sensitive and precise in the characterization of different bacteria species. The present study aimed to realize a literature review, including studies that realized a comparative analysis between bacterial culture and real time PCR methods in the identification of pathogens. The bacterial culture method can possibly identify new microorganisms and realize antibiotics sensitivity tests. The real time PCR is a microbiologic test that identifies and quantifies bacterial species, through gene amplification of predetermined DNA fragments, with high sensitivity and specificity, and need a shorter operation time of the operator when compared to the bacterial culture method. In this way, to determine a specific diagnostic test, should be considered not only its precision in the identification of microorganisms, but the cost-benefit relationship as well.

Polimosfismo genético da glicose-6-fosfato desidrogenase na população da região de Araraquara-SP

The most important role played by the enzyme Glucose- 6-Phosphate Dehydrogenase (G6PD) in erythrocyte metabolism is in generating energy and reducing power used to protect the cell against oxidative attack. G6PD deficiency is the erythroenzymopathy that most frequently causes hemolytic anemia, and more than 130 molecular variants have already been identified. The aim of this study was to analyze the genetic mutations in the G6PD-deficient adult males in the population of the region of Araraquara, São Paulo State. Out of 5087 male blood donors, 89 were deficient for G6PD, as confirmed by assaying the enzyme activity and electrophoresis on cellulose acetate. Thus, a frequency of 1.75% of G6PD-deficient patients was found, this value being similar to other investigations in São Paulo state. Molecular analysis was performed by amplification of genomic DNA with specific primers and digestion with restriction enzymes. In 96.6% of the patients, the G6PD A¯ variant was observed, with mutations at residues 376(A→G) and 202(G→A). Mean G6PD specific activity among the patients was 1.31 IU.g Hb-1.min-1 at 37ºC, that is 10.8% of the normal activity of the G6PD B enzyme. The variant forms G6PD A¯ 680(G→T) and 968(T→C) were not found. In 3.4% of the deficient individuals, the G6PD Mediterranean variant was found, with a mutation at 563(C→T). In these cases, mean enzymatic activity was 0.25 IU.g Hb-1.min-1 at 37ºC, or 2.1% of the enzymatic activity of G6PD B. The use of traditional techniques, allied to the identification of the different molecular variants, is important for the understanding of the structural and functional properties and hemolytic behavior of the red blood cells of the patient.

Bacillus cereus infection outbreak in captive psittacines

This study reports an uncommon epizootic outbreak of Bacillus cereus that caused the sudden death of 12 psittacines belonging to the species Anodorhynchus hyacinthinus (1 individual), Diopsittaca nobilis (1 individual), Ara severe (1 individual) and Ara ararauna (9 individuals) in a Brazilian zoo. Post-mortem examination of the animals reveled extensive areas of lung hemorrhage, hepatic congestion, hemorrhagic enteritis and cardiac congestion. Histopathological examination of the organs showed the presence of multiple foci of vegetative cells of Gram-positive bacilli associated with discrete and moderate mononuclear inflammatory cell infiltrate. Seventeen B. cereus strains isolated from blood and sterile organs of nine A. ararauna were analyzed in order to investigate the genetic diversity (assessed by Rep-PCR) and toxigenic profiles (presence of hblA, hblC and hblD; nheA, nheB and nheC as well as cytK, ces and entFM genes) of such strains. Amplification of genomic DNA by Rep-PCR of B. cereus strains generated two closely related profiles (Rep-PCR types A and B) with three bands of difference. All strains were classified as belonging to the toxigenic profile I which contained HBL and NHE gene complexes, entFM and cytK genes. Altogether, microbiological and histopathological findings and the evidence provided by the success of the antibiotic prophylaxis, corroborate that B. cereus was the causative agent of the infection that killed the birds. (C) 2012 Elsevier B.V. All rights reserved.

Differentiation and genetic analysis of Rhodnius prolixus and Rhodnius colombiensis by rDNA and RAPD amplification

Domiciliated Rhodnius prolixus and sylvatic R. colombiensis were analyzed in order to confirm their genetic divergence and verify the risk that the latter represents in the domiciliation process, and to provide tools for identifying the sources of possible reinfestation by triatomines in human dwellings allowing control programs to be undertaken. Comparison of random amplified polymorphic DNA amplification patterns and cluster analysis suggests reproductive discontinuity between the two species. The calculated statistical F value of 0.24 and effective migration rate of 0.6 individuals per generation are insufficient to maintain genetic homogeneity between them and confirm the absence of present genetic flow. R. colombiensis presents higher intrapopulation variability. Polymerase chain reaction of ribosomal DNA supports these findings. The low genetic flow between the two species implies that R. colombiensis do not represent an epidemiological risk for the domiciliary transmission of Trypanosoma cruzi in the Tolima Department. The lower variability of the domiciliated R. prolixus could result in greater susceptibility to the use of pesticides in control programs.