Diversidade genética do Anaplasma marginale em condições de transmissão natural

Pós-graduação em Medicina Veterinária - FCAV

Isolamento de sequências repetitivas do genoma de espécies do gênero Ancistrus (Siluriformes: Loricariidae)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

eNOS Tag SNP Haplotypes in Hypertensive Disorders of Pregnancy

Haplotypes formed by polymorphisms (T-786C, rs2070744; a variable number of tandem repeats in intron 4, and Glu298Asp, rs1799983) of the eNOS gene were associated previously with gestational hypertension (GH) and preeclampsia (PE). However, no study has explored the Tag SNPs rs743506 and rs7830 in these disorders. The aim of the current study was to compare the distribution of the genotypes and haplotypes formed by the five eNOS polymorphisms mentioned among healthy pregnant (HP, n = 122), GH (n = 138), and PE (n = 157). The haplotype formed by "C b G G C" was more frequent in HP compared to GH and PE (p = 0.0071), which is supported by previous findings that demonstrated the association of the combination "C b G" with a higher level of nitrite (NO marker). Our results suggest a protective effect of the haplotype "C b G G C" against the development of hypertensive disorders of pregnancy.

Large-Scale Population Analysis Challenges the Current Criteria for the Molecular Diagnosis of Fascioscapulohumeral Muscular Dystrophy

Facioscapulohumeral muscular dystrophy (FSHD) is a common hereditary myopathy causally linked to reduced numbers (<= 8) of 3.3 kilobase D4Z4 tandem repeats at 4q35. However, because individuals carrying D4Z4-reduced alleles and no FSHD and patients with FSHD and no short allele have been observed, additional markers have been proposed to support an FSHD molecular diagnosis. In particular a reduction in the number of D4Z4 elements combined with the 4A(159/161/168)PAS haplotype (which provides the possibility of expressing DUX4) is currently used as the genetic signature uniquely associated with FSHD. Here, we analyzed these DNA elements in more than 800 Italian and Brazilian samples of normal individuals unrelated to any FSHD patients. We find that 3% of healthy subjects carry alleles with a reduced number (4-8) of D4Z4 repeats on chromosome 4q and that one-third of these alleles, 1.3%, occur in combination with the 4A161PAS haplotype. We also systematically characterized the 4q35 haplotype in 253 unrelated FSHD patients. We find that only 127 of them (50.1%) carry alleles with 1-8 D4Z4 repeats associated with 4A161PAS, whereas the remaining FSHD probands carry different haplotypes or alleles with a greater number of D4Z4 repeats. The present study shows that the current genetic signature of FSHD is a common polymorphism and that only half of FSHD probands carry this molecular signature. Our results suggest that the genetic basis of FSHD, which is remarkably heterogeneous, should be revisited, because this has important implications for genetic counseling and prenatal diagnosis of at-risk families.

The contrasting role of heterochromatin in the differentiation of sex chromosomes: an overview from Neotropical fishes

During the evolutionary process of the sex chromosomes, a general principle that arises is that cessation or a partial restriction of recombination between the sex chromosome pair is necessary. Data from phylogenetically distinct organisms reveal that this phenomenon is frequently associated with the accumulation of heterochromatin in the sex chromosomes. Fish species emerge as excellent models to study this phenomenon because they have much younger sex chromosomes compared to higher vertebrates and many other organisms making it possible to follow their steps of differentiation. In several Neotropical fish species, the heterochromatinization, accompanied by amplification of tandem repeats, represents an important step in the morphological differentiation of simple sex chromosome systems, especially in the ZZ/ZW sex systems. In contrast, multiple sex chromosome systems have no additional increase of heterochromatin in the chromosomes. Thus, the initial stage of differentiation of the multiple sex chromosome systems seems to be associated with proper chromosomal rearrangements, whereas the simple sex chromosome systems have an accumulation of heterochromatin. In this review, attention has been drawn to this contrasting role of heterochromatin in the differentiation of simple and multiple sex chromosomes of Neotropical fishes, highlighting their surprising evolutionary dynamism.

Cloning and characterisation of a novel chromosome end repeat enriched with homopolymeric (dA)/(dT) DNA in Rhynchosciara americana (Diptera: Sciaridae)

Short tandem DNA repeats and telomerase compose the telomere structure in the vast majority of eukaryotic organisms. However, such a conserved organisation has not been found in dipterans. While telomeric DNA in Drosophila is composed of specific retrotransposons, complex terminal tandem repeats are present in chromosomes of Anopheles and chironomid species. In the sciarid Rhynchosciara americana, short repeats (16 and 22 bp long) tandemly arrayed seem to reach chromosome ends. Moreover, in situ hybridisation data using homopolymeric RNA probes suggested in this species the existence of a third putative chromosome end repeat enriched with (dA).(dT) homopolymers. In this work, chromosome micro-dissection and PCR primed by homopolymeric primers were employed to clone these repeats. Named T-14 and 93 % AT-rich, the repetitive unit is 14 bp long and appears organised in tandem arrays. It is localised in five non-centromeric ends and in four interstitial bands of R. americana chromosomes. To date, T-14 is the shortest repeat that has been characterised in chromosome ends of dipterans. As observed for short tandem repeats identified previously in chromosome ends of R. americana, the T-14 probe hybridised to bridges connecting non-homologous polytene chromosome ends, indicative of close association of T-14 repeats with the very end of the chromosomes. The results of this work suggest that R. americana represents an additional example of organism provided with more than one DNA sequence that is able to reach chromosome termini.

Epistasis among eNOS, MMP-9 and VEGF maternal genotypes in hypertensive disorders of pregnancy

Polymorphisms of the endothelial nitric oxide synthase (eNOS), matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF) genes were shown to be associated with hypertensive disorders of pregnancy. However, epistasis is suggested to be an important component of the genetic susceptibility to preeclampsia (PE). The aim of this study was to characterize the interactions among these genes in PE and gestational hypertension (GH). Seven clinically relevant polymorphisms of eNOS (T-786C, rs2070744, a variable number of tandem repeats in intron 4 and Glu298Asp, rs1799983), MMP-9 (C-1562T, rs3918242 and -90(CA)(13-25), rs2234681) and VEGF (C-2578A, rs699947 and G-634C, rs2010963) were genotyped by TaqMan allelic discrimination assays or PCR and fragment separation by electrophoresis in 122 patients with PE, 107 patients with GH and a control group of 102 normotensive pregnant (NP) women. A robust multifactor dimensionality reduction analysis was used to characterize gene-gene interactions. Although no significant genotype combinations were observed for the comparison between the GH and NP groups (P>0.05), the combination of MMP-9-1562CC with VEGF-634GG was more frequent in NP women than in women with PE (P<0.05). Moreover, the combination of MMP-9-1562CC with VEGF-634CC or MMP-9-1562CT with VEGF-634CC or-634GG was more frequent in women with PE than in NP women (P<0.05). These results are obscured when single polymorphisms in these genes are considered and suggest that specific genotype combinations of MMP-9 and VEGF contribute to PE susceptibility. Hypertension Research (2012) 35, 917-921; doi:10.1038/hr.2012.60; published online 10 May 2012

Structural and functional analysis of centromeric chromatin

Animal neocentromeres are defined as ectopic centromeres that have formed in non-centromeric locations and avoid some of the features, like the DNA satellite sequence, that normally characterize canonical centromeres. Despite this, they are stable functional centromeres inherited through generations. The only existence of neocentromeres provide convincing evidence that centromere specification is determined by epigenetic rather than sequence-specific mechanisms. For all this reasons, we used them as simplified models to investigate the molecular mechanisms that underlay the formation and the maintenance of functional centromeres. We collected human cell lines carrying neocentromeres in different positions. To investigate the region involved in the process at the DNA sequence level we applied a recent technology that integrates Chromatin Immuno-Precipitation and DNA microarrays (ChIP-on-chip) using rabbit polyclonal antibodies directed against CENP-A or CENP-C human centromeric proteins. These DNA binding-proteins are required for kinetochore function and are exclusively targeted to functional centromeres. Thus, the immunoprecipitation of DNA bound by these proteins allows the isolation of centromeric sequences, including those of the neocentromeres. Neocentromeres arise even in protein-coding genes region. We further analyzed if the increased scaffold attachment sites and the corresponding tighter chromatin of the region involved in the neocentromerization process still were permissive or not to transcription of within encoded genes. Centromere repositioning is a phenomenon in which a neocentromere arisen without altering the gene order, followed by the inactivation of the canonical centromere, becomes fixed in population. It is a process of chromosome rearrangement fundamental in evolution, at the bases of speciation. The repeat-free region where the neocentromere initially forms, progressively acquires extended arrays of satellite tandem repeats that may contribute to its functional stability. In this view our attention focalized to the repositioned horse ECA11 centromere. ChIP-on-chip analysis was used to define the region involved and SNPs studies, mapping within the region involved into neocentromerization, were carried on. We have been able to describe the structural polymorphism of the chromosome 11 centromeric domain of Caballus population. That polymorphism was seen even between homologues chromosome of the same cells. That discovery was the first described ever. Genomic plasticity had a fundamental role in evolution. Centromeres are not static packaged region of genomes. The key question that fascinates biologists is to understand how that centromere plasticity could be combined to the stability and maintenance of centromeric function. Starting from the epigenetic point of view that underlies centromere formation, we decided to analyze the RNA content of centromeric chromatin. RNA, as well as secondary chemically modifications that involve both histones and DNA, represents a good candidate to guide somehow the centromere formation and maintenance. Many observations suggest that transcription of centromeric DNA or of other non-coding RNAs could affect centromere formation. To date has been no thorough investigation addressing the identity of the chromatin-associated RNAs (CARs) on a global scale. This prompted us to develop techniques to identify CARs in a genome-wide approach using high-throughput genomic platforms. The future goal of this study will be to focalize the attention on what strictly happens specifically inside centromere chromatin.

Synthese von tumor-assoziierten MUC1-Mucin-Glycopeptid-Vakzinen und deren immunologische Evaluierung

Eine alternative Methode zur Therapie von Tumorerkrankungen bestünde in einer Immuntherapie ausgelöst durch synthetische Antitumor-Vakzine. Ein vielversprechendes Zielmolekül für eine solche Aktivimmunisierung ist das Glycoprotein MUC1, das auf nahezu allen Epithelgeweben exprimiert und auf Tumorgeweben stark überexprimiert wird. Seine extrazelluläre Domäne enthält eine Vielzahl von Tandem-Repeat-Sequenzen der Art: HGVTSAPDTRPAPGSTAPPA mit fünf potentiellen O-Glycosylierungs-Positionen. Da die Form der Glycosylierung des MUC1 in Tumorzellen stark von der auf normalen Zellen abweicht, liegen auf Tumorzellen eine Reihe tumor-assoziierter Saccharidantigene und Peptidepitope vor.rnIn dieser Arbeit wurden tumor-assoziierte Glycopeptidantigene aus der MUC1-Tandem-Repeat-Region hergestellt. Die synthetisierten MUC1-Glycopeptide tragen in verschiedenen Positionen eine Glycosylierung mit den tumor-assoziierten Tn- und STn-Saccharid-Antigenen. Zur Gewinnung von Vakzinen wurden diese Glycopeptid-Antigene über einen Spacer mit immunstimulierenden Komponenten verknüpft. Als Immunstimulanzien wurden ein T-Zell-Epitop aus dem Ovalbumin (OVA323-339) sowie die Carrier-Proteine Rinderserumalbumin (BSA) und Tetanus-Toxoid (TTox) verwendet. rnDie synthetischen MUC1-Glycopeptide wurden durch Immunisierung von Mäusen einer immunologischen Evaluierung unterzogen. Insbesondere die synthetischen MUC1-Glycopeptid-TTox-Vakzine lösen sehr starke Immunantworten aus. Es konnte gezeigt werden, dass die induzierten Antikörper stark an Tumorzellen und auch an Mammakarzinom-Gewebe binden, was für die Entwicklung von Antitumor-Vakzinen als vielversprechend einzustufen ist.

Synthese von tumorassoziierten Glycopeptiden basierend auf der epithelialen Struktur des MUC1

In epithelialen Tumorzellen zeigen Membranglycoproteine ein charakteristisch verändertes Glycosylierungsmuster, das durch eine veränderte Aktivität von Glycosyltransferasen hervorgerufen wird. Diese veränderte Aktivität führt zur Expression von stark verkürzten und frühzeitig sialylierten Kohlenhydratseitenketten der mucinartigen Glycoproteine auf Tumorzellen, welche als tumorassoziierte Antigene bezeichnet werden. Die tumorassoziierten Peptidepitope stellen eine wichtige Zielstruktur für eine potentielle selektive Immuntherapie dar. Das Ziel ist es, dass das Immunsystem zwischen den tumorassoziierten und den normal exprimierten Glycoproteinen unterscheiden kann, und damit in der Lage ist, eine selektiv gegen Tumorzellen gerichtete Immunantwort auszulösen. Daher ist eine Synthese von strukturell exakt definierten synthetischen Glycopeptiden und deren Einbau in Vakzine ein entscheidender Schritt für eine angestrebte Immuntherapie gegen Krebs. Ein Ziel dieser Arbeit war in diesem Zusammenhang die Synthese exakt definierter tumorassoziierter Glycopeptide, wobei Peptide aus der Tandem Repeat-Domäne des MUC1 synthetisiert wurden. Die verschiedenen Saccharidantigene wurden als glycosylierte Aminosäurebausteine in die immundominante Domäne der MUC1-Peptidsequenz eingebaut (TN-Antigen sowie das Sialyl-TN-Antigen, das (2,6)-Sialyl-T- und das (2,3)-Sialyl-T-Antigen). Zum einen wurden die MUC1-Glycopeptide anschließend über einen nicht immunogenen Triethylenglycol-Spacer an Carrier-Proteine wie Ovalbumin-OVA323-339-Sequenz und Rinderserumalbumin (BSA) konjugiert, so dass potenzielle Tumorvakzine erhalten wurden. Diese wurden in ersten ELISA-Experimenten untersucht und haben gezeigt, dass mit den synthetischen Glycopeptidantigenen feine Strukturunterschiede immunologisch differenziert werden können, was für die Unterscheidung zwischen Tumorzellen und gesunden Zellen bedeutsam ist. rnFür Immunisierungen ohne Freunds Adjuvanz wurde der TLR2-Agonist Pam3CSKKKK synthetisiert, der in einer Fragmentkondensation mit einem OVA-Spacer-MUC1-Glycopeptid konjugiert wurde. Die immunologischen Evaluierungen stehen noch aus.rnrn

Interaction between serotonin 5-HT2A receptor gene and dopamine transporter (DAT1) gene polymorphisms influences personality trait of persistence in Austrian Caucasians

We examined 89 normal volunteers using Cloninger's Temperament and Character Inventory (TCI). Genotyping the 102T/C polymorphism of the serotonin 5HT2A receptor gene and the ser9gly polymorphism in exon 1 of the dopamine D3 receptor (DRD3) gene was performed using PCR-RFLP, whereas the dopamine transporter (DAT1) gene variable number of tandem repeats (VNTR) polymorphism was investigated using PCR amplification followed by electrophoresis in an 8% acrylamide gel with a set of size markers. We found a nominally significant association between gender and harm avoidance (P=0.017; women showing higher scores). There was no association of either DAT1, DRD3 or 5HT2A alleles or genotypes with any dimension of the TCI applying Kruskal-Wallis rank-sum tests. Comparing homozygote and heterozygote DAT1 genotypes, we found higher novelty seeking scores in homozygotes (P=0.054). We further found a nominally significant interaction between DAT1 and 5HT2A homo-/heterozygous gene variants (P=0.0071; DAT1 and 5HT2A genotypes P value of 0.05), performing multivariate analysis of variance (MANOVA). Examining the temperamental TCI subscales, this interaction was associated with persistence (genotypes: P=0.004; homo-/heterozygous gene variants: P=0.0004). We conclude that an interaction between DAT1 and 5HT2A genes might influence the temperamental personality trait persistence.

Effect of repetitiveness on the immunogenicity and antigenicity of Trypanosoma cruzi FRA protein

Repetitive proteins (RP) of Trypanosoma cruzi are highly present in the parasite and are strongly recognized by sera from Chagas' disease patients. Flagelar Repetitive Antigen (FRA), which is expressed in all steps of the parasite life cycle, is the RP that displays the greatest number of aminoacids per repeat and has been indicated as one of the most suitable candidate for diagnostic test because of its high performance in immunoassays. Here we analyzed the influence of the number of repeats on the immunogenic and antigenic properties of the antigen. Recombinant proteins containing one, two, and four tandem repeats of FRA (FRA1, FRA2, and FRA4, respectively) were obtained and the immune response induced by an equal amount of repeats was evaluated in a mouse model. The reactivity of specific antibodies present in sera from patients naturally infected with T. cruzi was also assessed against FRA1, FRA2, and FRA4 proteins, and the relative avidity was analyzed. We determined that the number of repeats did not increase the humoral response against the antigen and this result was reproduced when the repeated motifs were alone or fused to a non-repetitive protein. By contrast, the binding affinity of specific human antibodies increases with the number of repeated motifs in FRA antigen. We then concluded that the high ability of FRA to be recognized by specific antibodies from infected individuals is mainly due to a favorable polyvalent interaction between the antigen and the antibodies. In accordance with experimental results, a 3D model was proposed and B epitope in FRA1, FRA2, and FRA4 were predicted.

A novel isolation method of Brucella species and molecular tracking of Brucella suis biovar 2 in domestic and wild animals

Brucella suis biovar 2 is the most common aetiological agent of porcine brucellosis in Europe. B. suis biovar 2 is considered to have low zoonotic potential, but is a causative agent of reproductive losses in pigs, and it is thus economically important. The multilocus variable-number of tandem repeats genotyping analysis of 16 loci (MLVA-16) has proven to be highly discriminatory and is the most suitable assay for simultaneously identifying B. suis and tracking infections. The aim of this study was to investigate the relatedness between isolates of B. suis biovar 2 obtained during a brucellosis outbreak in domestic pigs and isolates from wild boars and hares collected from proximal or remote geographical areas by MLVA-16. A cluster analysis of the MLVA-16 data revealed that most of the isolates obtained from Switzerland clustered together, with the exception of one isolate. The outbreak isolates constituted a unique subcluster (with a genetic similarity >93.8%) distinct from that of the isolates obtained from wild animals, suggesting that direct transmission of the bacterium from wild boars to domestic pigs did not occur in this outbreak. To obtain a representative number of isolates for MLVA-16, alternative methods of Brucella spp. isolation from tissue samples were compared with conventional direct cultivation on a Brucella-selective agar. We observed an enhanced sensitivity when mechanical homogenisation was followed by host cell lysis prior to cultivation on the Brucella-selective agar. This work demonstrates that MLVA-16 is an excellent tool for both monitoring brucellosis and investigating outbreaks. Additionally, we present efficient alternatives for the isolation of Brucella spp.

Bacillus anthracis: Molecular taxonomy, population genetics, phylogeny and patho-evolution

Bacillus anthracis, the etiological agent of anthrax, manifests a particular bimodal lifestyle. This bacterial species alternates between short replication phases of 20-40 generations that strictly require infection of the host, normally causing death, interrupted by relatively long, mostly dormant phases as spores in the environment. Hence, the B. anthracis genome is highly homogeneous. This feature and the fact that strains from nearly all parts of the world have been analysed for canonical single nucleotide polymorphisms (canSNPs) and variable number tandem repeats (VNTRs) has allowed the development of molecular epidemiological and molecular clock models to estimate the age of major diversifications in the evolution of B. anthracis and to trace the global spread of this pathogen, which was mostly promoted by movement of domestic cattle with settlers and by international trade of contaminated animal products. From a taxonomic and phylogenetic point of view, B. anthracis is a member of the Bacillus cereus group. The differentiation of B. anthracis from B. cereus sensu strict, solely based on chromosomal markers, is difficult. However, differences in pathogenicity clearly differentiate B. anthracis from B. cereus and are marked by the strict presence of virulence genes located on the two virulence plasmids pXO1 and pXO2, which both are required by the bacterium to cause anthrax. Conversely, anthrax-like symptoms can also be caused by organisms with chromosomal features that are more closely related to B. cereus, but which carry these virulence genes on two plasmids that largely resemble the B. anthracis virulence plasmids. (C) 2011 Elsevier B.V. All rights reserved.

Evolution of the leukotoxin promoter in genus Mannheimia

BACKGROUND: The Mannheimia species encompass a wide variety of bacterial lifestyles, including opportunistic pathogens and commensals of the ruminant respiratory tract, commensals of the ovine rumen, and pathogens of the ruminant integument. Here we present a scenario for the evolution of the leukotoxin promoter among representatives of the five species within genus Mannheimia. We also consider how the evolution of the leukotoxin operon fits with the evolution and maintenance of virulence. RESULTS: The alignment of the intergenic regions upstream of the leukotoxin genes showed significant sequence and positional conservation over a 225-bp stretch immediately proximal to the transcriptional start site of the lktC gene among all Mannheimia strains. However, in the course of the Mannheimia genome evolution, the acquisition of individual noncoding regions upstream of the conserved promoter region has occurred. The rate of evolution estimated branch by branch suggests that the conserved promoter may be affected to different extents by the types of natural selection that potentially operate in regulatory regions. Tandem repeats upstream of the core promoter were confined to M. haemolytica with a strong association between the sequence of the repeat units, the number of repeat units per promoter, and the phylogenetic history of this species. CONCLUSION: The mode of evolution of the intergenic regions upstream of the leukotoxin genes appears to be highly dependent on the lifestyle of the bacterium. Transition from avirulence to virulence has occurred at least once in M. haemolytica with some evolutionary success of bovine serotype A1/A6 strains. Our analysis suggests that changes in cis-regulatory systems have contributed to the derived virulence phenotype by allowing phase-variable expression of the leukotoxin protein. We propose models for how phase shifting and the associated virulence could facilitate transmission to the nasopharynx of new hosts.