Phylogenetic relationship of dengue virus type 3 isolated in Brazil and Paraguay and global evolutionary divergence dynamics

Background: Dengue is the most important mosquito-borne viral disease worldwide. Dengue virus comprises four antigenically related viruses named dengue virus type 1 to 4 (DENV1-4). DENV-3 was re-introduced into the Americas in 1994 causing outbreaks in Nicaragua and Panama. DENV-3 was introduced in Brazil in 2000 and then spread to most of the Brazilian States, reaching the neighboring country, Paraguay in 2002. In this study, we have analyzed the phylogenetic relationship of DENV-3 isolated in Brazil and Paraguay with viruses isolated worldwide. We have also analyzed the evolutionary divergence dynamics of DENV-3 viruses. Results: The entire open reading frame (ORF) of thirteen DENV-3 isolated in Brazil (n = 9) and Paraguay (n = 4) were sequenced for phylogenetic analysis. DENV-3 grouped into three main genotypes (I, II and III). Several internal clades were found within each genotype that we called lineage and sub-lineage. Viruses included in this study belong to genotype III and grouped together with viruses isolated in the Americas within the lineage III. The Brazilian viruses were further segregated into two different sub-lineage, A and B, and the Paraguayan into the sub-lineage B. All three genotypes showed internal grouping. The nucleotide divergence was in average 6.7% for genotypes, 2.7% for lineages and 1.5% for sub-lineages. Phylogenetic trees constructed with any of the protein gene sequences showed the same segregation of the DENV-3 in three genotypes. Conclusion: Our results showed that two groups of DENV-3 genotypes III circulated in Brazil during 2002-2009, suggesting different events of introduction of the virus through different regions of the country. In Paraguay, only one group DENV-3 genotype III is circulating that is very closely related to the Brazilian viruses of sub-lineage B. Different degree of grouping can be observed for DENV-3 and each group showed a characteristic evolutionary divergence. Finally, we have observed that any protein gene sequence can be used to identify the virus genotype.

Genotypes and antibiotic resistance of Campylobacter coli in fattening pigs

Campylobacter coli is a food-borne zoonotic pathogen causing human gastroenteritis worldwide. The organism is a commensal in the intestine of many food production animals including fattening pigs. The role of the pig as a potential reservoir for C. coli affecting human either directly or via poultry has hardly been investigated and genetic characterization of porcine strains is needed to address this question. For this aim multilocus sequence typing (MLST) and flaB typing was applied to 256 C. coli isolates from faeces of fattening pig collected during 2009 at different slaughterhouses in Switzerland. In addition genotypic resistances towards macrolides and quinolones based on point mutations in the 23S rRNA and gyrA genes, respectively, were determined. Of the 67 sequence types (STs) obtained by MLST, 37 were found for the first time. flaB typing revealed 46 different types with 14 of them being novel and was useful to further differentiate strains with an identical ST. Quinolone resistance was detected in 33.6% and macrolide resistance was found in 10.6% of isolates. Comparison with 99 C. coli pig isolates from 2001 revealed a significant decrease in antibiotic resistance towards both groups of antibiotics and there was high overlap between genotypes of 2001 and 2009. Little overlap of porcine genotypes was found with 97 C. coli isolates from poultry collected 2008, however, macrolide resistance was significantly higher in pig isolates. In conclusion, C. coli from Swiss pig are heterogeneous containing many novel STs, findings that could reflect the partitioned Swiss pig production with almost no international breed exchange. The antibiotic resistance echoes the use of corresponding drugs in the Swiss livestock production and indicates the efficacy of restrictive application of antibiotics in order to reduce resistances.

Phylogeny of Pasteurellaceae

This chapter provides an overview on the DNA based phylogeny of the family Pasteurellaceae and the genetic relatedness between taxa taking into account the various gene targets and approaches applied in the literature. The classical 16S rRNA gene based phylogeny as well as phylogenies based on house-keeping genes are described. Moreover, strength and weakness of the different trees and their topology are discussed based on the phylogenetic groups resolved. The data should help to get a clearer picture on the recent, current and future classification and also provide information to genetic characterization of members of the family. The history of phylogeny applied to the family as well as the phylogenetic history of the family is thereby presented. In this way it is the story of the search for the optimal phylogenetic marker without giving a final conclusive suggestion but it is also a resource for choosing the appropriate gene target(s) for people investigating the phylogeny of groups of Pasteurellaceae.

Novel pseudo-staphylococcal cassette chromosome mec element (ψSCCmec57395) in methicillin-resistant Staphylococcus pseudintermedius CC45

Genetic characterization of methicillin-resistant Staphylococcus pseudintermedius (MRSP) from Thailand and Israel revealed the presence of a predominant atypical clonal lineage which was not typeable by SmaI-PFGE and SCCmec typing. All the atypical isolates (n = 34) belonged to CC45 (30 ST45 and 2 ST179 isolates, 1 ST57 isolate, and 1 ST85 isolate). The isolates originated from healthy and diseased dogs and cats, as well as from the environment of one clinic. Cfr9I-pulsed-field gel electrophoresis (Cfr9I-PFGE) and dru typing permitted the further distinction of CC45 isolates from the two different countries. Microarray analysis identified genes that confer resistance to β-lactams (mecA; blaZ), aminoglycosides [aac(6')-Ie-aph(2')-Ia; aph(3')-III; ant(6)-Ia], macrolides and lincosamides [erm(B)], tetracyclines [tet(M)], trimethoprim [dfr(G)], streptothricin (sat4), and chloramphenicol (catpC221). Fluoroquinolone resistance was attributed to specific amino acid substitutions, i.e., Ser84Leu in GyrA and Ser80Ile and Asp84Asn in GrlA. A novel pseudo-staphylococcal cassette chromosome (ΨSCCmec57395) element was identified in MRSP strain 57395 (sequence type ST45) by whole-genome sequencing. The 12,282-bp ΨSCCmec57395 element contained a class C1 mec gene complex but no ccr genes. In addition to the methicillin resistance gene mecA, ΨSCCmec57395 also carried determinants of resistance to heavy metals, such as arsenic, cadmium, and copper. Bsu36I restriction analysis of the ΨSCCmec57395 element amplified by long-range PCR revealed the presence of ΨSCCmec57395 in the 33 additional isolates of MRSP CC45. The ΨSCCmec57395 element represents a new class of SCCmec and has been identified in MRSP of CC45, which is a predominant clonal lineage in Israel and Thailand.

TECPR2 Associated Neuroaxonal Dystrophy in Spanish Water Dogs.

Clinical, pathological and genetic examination revealed an as yet uncharacterized juvenile-onset neuroaxonal dystrophy (NAD) in Spanish water dogs. Affected dogs presented with various neurological deficits including gait abnormalities and behavioral deficits. Histopathology demonstrated spheroid formation accentuated in the grey matter of the cerebral hemispheres, the cerebellum, the brain stem and in the sensory pathways of the spinal cord. Iron accumulation was absent. Ultrastructurally spheroids contained predominantly closely packed vesicles with a double-layered membrane, which were characterized as autophagosomes using immunohistochemistry. The family history of the four affected dogs suggested an autosomal recessive inheritance. SNP genotyping showed a single genomic region of extended homozygosity of 4.5 Mb in the four cases on CFA 8. Linkage analysis revealed a maximal parametric LOD score of 2.5 at this region. By whole genome re-sequencing of one affected dog, a perfectly associated, single, non-synonymous coding variant in the canine tectonin beta-propeller repeat-containing protein 2 (TECPR2) gene affecting a highly conserved region was detected (c.4009C>T or p.R1337W). This canine NAD form displays etiologic parallels to an inherited TECPR2 associated type of human hereditary spastic paraparesis (HSP). In contrast to the canine NAD, the spinal cord lesions in most types of human HSP involve the sensory and the motor pathways. Furthermore, the canine NAD form reveals similarities to cases of human NAD defined by widespread spheroid formation without iron accumulation in the basal ganglia. Thus TECPR2 should also be considered as candidate gene for human NAD. Immunohistochemistry and the ultrastructural findings further support the assumption, that TECPR2 regulates autophagosome accumulation in the autophagic pathways. Consequently, this report provides the first genetic characterization of juvenile canine NAD, describes the histopathological features associated with the TECPR2 mutation and provides evidence to emphasize the association between failure of autophagy and neurodegeneration.

Ordenaciones de material genético a partir de información multidimensional

Nuevas biotecnologías permiten obtener información para caracterizar materiales genéticos a partir de múltiples marcadores, ya sean éstos moleculares y/o morfológicos. La ordenación del material genético a través de la exploración de patrones de variabilidad multidimensionales se aborda mediante diversas técnicas de análisis multivariado. Las técnicas multivariadas de reducción de dimensión (TRD) y la representación gráfica de las mismas cobran sustancial importancia en la visualización de datos multivariados en espacios de baja dimensión ya que facilitan la interpretación de interrelaciones entre las variables (marcadores) y entre los casos u observaciones bajo análisis. Tanto el Análisis de Componentes Principales, como el Análisis de Coordenadas Principales y el Análisis de Procrustes Generalizado son TRD aplicables a datos provenientes de marcadores moleculares y/o morfológicos. Los Árboles de Mínimo Recorrido y los biplots constituyen técnicas para lograr representaciones geométricas de resultados provenientes de TRD. En este trabajo se describen estas técnicas multivariadas y se ilustran sus aplicaciones sobre dos conjuntos de datos, moleculares y morfológicos, usados para caracterizar material genético fúngico.

Caracterización genética de germoplasma de maní cultivado (Arachis hypogaea L.) mediante el empleo de marcadores microsatélites

El maní cultivado (Arachis hypogaea L.), es una especie de gran importancia económica, nativo de América del Sur. Se divide en dos subespecies y seis variedades botánicas. Genéticamente es alotetraploide, constituido por dos juegos genómicos duplicados. El empleo de marcadores microsatélites resulta más apropiado para realizar la caracterización genética de esta especie, puesto que permiten detectar un elevado nivel de polimorfismo. El objetivo del presente trabajo fue caracterizar la diversidad genética existente en las entradas de germoplasma de maní cultivado pertenecientes al Banco Activo de Germoplasma de Maní del Instituto Nacional de Tecnología Agropecuaria (INTA). Veinticinco entradas fueron genotipificadas con 23 marcadores microsatélites, de los cuales, 17 resultaron polimórficos. Se observaron 75 fragmentos polimórficos amplificados, con un promedio de 4,41 alelos por locus y un rango de 1 a 9 alelos. El contenido de información polimórfica osciló entre 0,15 y 0,58. El valor de la diversidad genética promedio fue de 0,165. Tanto el análisis de conglomerados como el de coordenadas principales evidenciaron dos grupos, uno formado por los materiales representantes de la subespecie fastigiata y otro por los de la subespecie hypogaea. Los resultados del análisis molecular de la varianza mostraron varianza tanto dentro como entre, las subespecies analizadas.

Metabolic rates and tissue composition of the coral Pocillopora verrucosa over 12 latitudes in the Red Sea characterized by strong temperature and nutrient gradient

Global warming was reported to cause growth reductions in tropical shallow water corals in both, cooler and warmer, regions of the coral species range. This suggests regional adaptation with less heat-tolerant populations in cooler and more thermo-tolerant populations in warmer regions. Here, we investigated seasonal changes in the in situ metabolic performance of the widely distributed hermatypic coral Pocillopora verrucosa along 12 degrees latitudes featuring a steep temperature gradient between the northern (28.5 degrees N, 21-27 degrees C) and southern (16.5 degrees N, 28-33 degrees C) reaches of the Red Sea. Surprisingly, we found little indication for regional adaptation, but strong indications for high phenotypic plasticity: Calcification rates in two seasons (winter, summer) were found to be highest at 28-29 degrees C throughout all populations independent of their geographic location. Mucus release increased with temperature and nutrient supply, both being highest in the south. Genetic characterization of the coral host revealed low inter-regional variation and differences in the Symbiodinium clade composition only at the most northern and most southern region. This suggests variable acclimatization potential to ocean warming of coral populations across the Red Sea: high acclimatization potential in northern populations, but limited ability to cope with ocean warming in southern populations already existing at the upper thermal margin for corals

Light hydrocarbons in sediments of ODP Site 124-768

Geochemical investigations on gases and interstitial waters from ODP Site 768 (Sulu Trench/Philippines) demonstrate the application of molecular gas composition in combination with stable isotope analyses to the genetic classification of light hydrocarbons. 13C/12C and D/H ratios of methane from gas pockets in cores and gases desorbed from frozen sediments by a vacuum/acid treatment suggest a microbial generation of methane by a CO2 reducing process in sediments with low sulfate concentrations. Isotope data and molecular composition of sediment gases liberated by the vacuum/acid treatment seem to be affected by a secondary desorption process during sampling. A comparison between the D/H ratios of methane from gas pockets and interstitial H2O points to an in-situ generation of methane down to a sub-bottom depth of approx. 720 m. Below this depth hydrogen isotope data indicate a migration of light hydrocarbons into pyroclastic sediments at this site. The occurrence of higher hydrocarbons (propane to pentane) in gases from gas pockets coincides with the vertical distribution of mature organic matter. Gases within the zone of mature organic matter are gases of a mixed microbial and thermal origin.

Molecular tools to improve chestnut management: El Bierzo as a case study

The European chestnut (Castanea sativa Mill.) is a multipurpose species that has been widely cultivated around the Mediterranean basin since ancient times. New varieties were brought to the Iberian Peninsula during the Roman Empire, which coexist since then with native populations that survived the last glaciation. The relevance of chestnut cultivation has being steadily growing since the Middle Ages, until the rural decline of the past century put a stop to this trend. Forest fires and diseases were also major factors. Chestnut cultivation is gaining momentum again due to its economic (wood, fruits) and ecologic relevance, and represents currently an important asset in many rural areas of Europe. In this Thesis we apply different molecular tools to help improve current management strategies. For this study we have chosen El Bierzo (Castile and Leon, NW Spain), which has a centenary tradition of chestnut cultivation and management, and also presents several unique features from a genetic perspective (next paragraph). Moreover, its nuts are widely appreciated in Spain and abroad for their organoleptic properties. We have focused our experimental work on two major problems faced by breeders and the industry: the lack of a fine-grained genetic characterization and the need for new strategies to control blight disease. To characterize with sufficient detail the genetic diversity and structure of El Bierzo orchards, we analyzed DNA from 169 trees grafted for nut production covering the entire region. We also analyzed 62 nuts from all traditional varieties. El Bierzo constitutes an outstanding scenario to study chestnut genetics and the influence of human management because: (i) it is located at one extreme of the distribution area; (ii) it is a major glacial refuge for the native species; (iii) it has a long tradition of human management (since Roman times, at least); and (iv) its geographical setting ensures an unusual degree of genetic isolation. Thirteen microsatellite markers provided enough informativeness and discrimination power to genotype at the individual level. Together with an unexpected level of genetic variability, we found evidence of genetic structure, with three major gene pools giving rise to the current population. High levels of genetic differentiation between groups supported this organization. Interestingly, genetic structure does not match with spatial boundaries, suggesting that the exchange of material and cultivation practices have strongly influenced natural gene flow. The microsatellite markers selected for this study were also used to classify a set of 62 samples belonging to all traditional varieties. We identified several cases of synonymies and homonymies, evidencing the need to substitute traditional classification systems with new tools for genetic profiling. Management and conservation strategies should also benefit from these tools. The avenue of high-throughput sequencing technologies, combined with the development of bioinformatics tools, have paved the way to study transcriptomes without the need for a reference genome. We took advantage of RNA sequencing and de novo assembly tools to determine the transcriptional landscape of chestnut in response to blight disease. In addition, we have selected a set of candidate genes with high potential for developing resistant varieties via genetic engineering. Our results evidenced a deep transcriptional reprogramming upon fungal infection. The plant hormones ET and JA appear to orchestrate the defensive response. Interestingly, our results also suggest a role for auxins in modulating such response. Many transcription factors were identified in this work that interact with promoters of genes involved in disease resistance. Among these genes, we have conducted a functional characterization of a two major thaumatin-like proteins (TLP) that belongs to the PR5 family. Two genes encoding chestnut cotyledon TLPs have been previously characterized, termed CsTL1 and CsTL2. We substantiate here their protective role against blight disease for the first time, including in silico, in vitro and in vivo evidence. The synergy between TLPs and other antifungal proteins, particularly endo-p-1,3-glucanases, bolsters their interest for future control strategies based on biotechnological approaches.

Efficient allelic exchange and transposon mutagenesis in Mycobacterium tuberculosis

A better understanding of Mycobacterium tuberculosis virulence mechanisms is highly dependent on the design of efficient mutagenesis systems. A system enabling the positive selection of insertional mutants having lost the delivery vector was developed. It uses ts-sacB vectors, which combine the counterselective properties of the sacB gene and a mycobacterial thermosensitive origin of replication and can therefore be efficiently counterselected on sucrose at 39°C. This methodology allowed the construction of M. tuberculosis transposition mutant libraries. Greater than 106 mutants were obtained, far exceeding the number theoretically required to obtain at least one insertion in every nonessential gene. This system is also efficient for gene exchange mutagenesis as demonstrated with the purC gene: 100% of the selected clones were allelic exchange mutants. Therefore, a single, simple methodology has enabled us to develop powerful mutagenesis systems, the lack of which was a major obstacle to the genetic characterization of M. tuberculosis.

Adaptor Complex-independent Clathrin Function in Yeast

Clathrin-associated adaptor protein (AP) complexes are major structural components of clathrin-coated vesicles, functioning in clathrin coat assembly and cargo selection. We have carried out a systematic biochemical and genetic characterization of AP complexes in Saccharomyces cerevisiae. Using coimmunoprecipitation, the subunit composition of two complexes, AP-1 and AP-2R, has been defined. These results allow assignment of the 13 potential AP subunits encoded in the yeast genome to three AP complexes. As assessed by in vitro binding assays and coimmunoprecipitation, only AP-1 interacts with clathrin. Individual or combined disruption of AP-1 subunit genes in cells expressing a temperature-sensitive clathrin heavy chain results in accentuated growth and α-factor pheromone maturation defects, providing further evidence that AP-1 is a clathrin adaptor complex. However, in cells expressing wild-type clathrin, the same AP subunit deletions have no effect on growth or α-factor maturation. Furthermore, gel filtration chromatography revealed normal elution patterns of clathrin-coated vesicles in cells lacking AP-1. Similarly, combined deletion of genes encoding the β subunits of the three AP complexes did not produce defects in clathrin-dependent sorting in the endocytic and vacuolar pathways or alterations in gel filtration profiles of clathrin-coated vesicles. We conclude that AP complexes are dispensable for clathrin function in S. cerevisiae under normal conditions. Our results suggest that alternative factors assume key roles in stimulating clathrin coat assembly and cargo selection during clathrin-mediated vesicle formation in yeast.

A essência do Arbutus unedo: caraterização morfológica e genética do medronheiro de Castelo de Paiva

O medronheiro é um arbusto da região mediterrânica que pode ser encontrada por todo o país. Ao contrário do que verifica na região sul do país, no concelho de Castelo de Paiva é atribuída uma reduzida importância económica a esta espécie. Com o intuito de preservar e potenciar a produção desta espécie e contribuir para a dinamização da economia do concelho, procedeu-se à caracterização morfológica e genética de uma amostra da população de medronheiros de Castelo de Paiva. A caracterização morfológica e genética foi realizada para um total de 10 genótipos. Para tal recolheram-se 70 folhas aleatoriamente em cada árvore. Em 40 folhas mediu-se o comprimento, largura, comprimento do pedúnculo, peso fresco, peso seco e determinou-se a área foliar. Dos caracteres morfológicos analisados, aqueles que se revelaram mais úteis na distinção dos vários genótipos foram: comprimento do pedúnculo, peso fresco e peso seco. As restantes 30 folhas foram utilizadas para a caracterização genética. Esta caracterização foi realizada recorrendo a um marcador de DNA, ISSR. Os 5 primeiros exemplaresutilizados na técnica de ISSR demonstraram-se polimórficos. Os resultados da caracterização genética sugerem que a variabilidade genética na população é média a alta.

Farmacogenética em toxicologia forense : estudo do gene CYP2D6 em amostras post mortem com tramadol

Tese de mestrado, Medicina Legal e Ciências Forenses, Universidade de Lisboa, Faculdade de Medicina, 2016

Estudo dos marcadores genéticos dos sistemas CODIS e ESS na população imigrante de Cabo Verde residente em Lisboa

Tese de mestrado, Medicina Legal e Ciências Forenses, Universidade de Lisboa, Faculdade de Medicina, 2016