Molecular phylogenetics of Lentibulariaceae inferred from plastid rps16 Intron and trnL-F DNA sequences: implications for character evolution and biogeography

Phylogenetic relationships among 75 species of Lentibulariaceae, representing the three recognized genera, were assessed by cladistic analysis of DNA sequences from the plastid rps16 intron and the trnL-F region. Sequence data from the two loci were analyzed both separately and in combination. Consensus trees from all analyses are congruent, and parsimony jackknife results demonstrate strong support for relationships both between and within each of the three demonstrably monophyletic genera. The genus Pinguicula is sister to a Genlisea-Utricularia clade, the phylogenetic structure within this clade closely follows Taylor's recent sectional delimitations based on morphology. Three principal clades are shown within Utricularia, with the basal sections Polypoinpholyx and Pleiochasia together forming the sister lineage of the remaining Utricularia species. Of the fundamental morphological specializations, the stoloniferous growth form apparently arose independently within Genlisea and Utricularia three times, and within Utricularia itself, perhaps more than once. The epiphytic habit has evolved independently at least three times, in Pinguicula, in Utricularia section Phyllaria, and within the two sections Orchidioides and Iperua (in the latter as bromeliad tank-epiphytes). The suspended aquatic habit may have evolved independently within sections Utricularia and Vesiculina. Biogeographic optimization on the phylogeny demonstrates patterns commonly associated with the boreotropics hypothesis and limits the spatial origin of Lentibulariaceae to temperate Eurasia or tropical America.

Inferring genome trees by using a filter to eliminate phylogenetically discordant sequences and a distance matrix based on mean normalized BLASTP scores

Darwin's paradigm holds that the diversity of present-day organisms has arisen via a process of genetic descent with modification, as on a bifurcating tree. Evidence is accumulating that genes are sometimes transferred not along lineages but rather across lineages. To the extent that this is so, Darwin's paradigm can apply only imperfectly to genomes, potentially complicating or perhaps undermining attempts to reconstruct historical relationships among genomes (i.e., a genome tree). Whether most genes in a genome have arisen via treelike (vertical) descent or by lateral transfer across lineages can be tested if enough complete genome sequences are used. We define a phylogenetically discordant sequence (PDS) as an open reading frame (ORF) that exhibits patterns of similarity relationships statistically distinguishable from those of most other ORFs in the same genome. PDSs represent between 6.0 and 16.8% (mean, 10.8%) of the analyzable ORFs in the genomes of 28 bacteria, eight archaea, and one eukaryote (Saccharomyces cerevisiae). In this study we developed and assessed a distance-based approach, based on mean pairwise sequence similarity, for generating genome trees. Exclusion of PDSs improved bootstrap support for basal nodes but altered few topological features, indicating that there is little systematic bias among PDSs. Many but not all features of the genome tree from which PDSs were excluded are consistent with the 16S rRNA tree.

C-terminal sequences in R-Ras are involved in integrin regulation and in plasma membrane microdomain distribution

The small GTPases R-Ras and H-Ras are highly homologous proteins with contrasting biological properties, for example, they differentially modulate integrin affinity: H-Ras suppresses integrin activation in fibroblasts whereas R-Ras can reverse this effect of H-Ras. To gain insight into the sequences directing this divergent phenotype, we investigated a panel of H-Ras/R-Ras chimeras and found that sequences in the R-Ras hypervariable C-terminal region including amino acids 175-203 are required for the R-Ras ability to increase integrin activation in CHO cells; however, the proline-rich site in this region, previously reported to bind the adaptor protein Nck, was not essential for this effect. In addition, we found that the GTPase TC21 behaved similarly to R-Ras. Because the C-termini of Ras proteins can control their subcellular localization, we compared the localization of H-Ras and R-Ras. In contrast to H-Ras, which migrates out of lipid rafts upon activation, we found that activated R-Ras remained localized to lipid rafts. However, functionally distinct H-Ras/R-Ras chimeras containing different C-terminal R-Ras segments localized to lipid rafts irrespective of their integrin phenotype. (C) 2003 Elsevier Inc. All rights reserved.

Ex vivo analysis of T-cell responses to Epstein-Barr virus-encoded oncogene latent membrane protein 1 reveals highly conserved epitope sequences in virus isolates from diverse geographic regions

Epstein-Barr virus (EBV)-encoded oncogene latent membrane protein (LMP) 1, which is consistently expressed in multiple EBV-associated malignancies, has been proposed as a potential target antigen for any future vaccine designed to control these malignancies. However, the high degree of genetic variation in the LMP1 sequence has been considered a major impediment for its use as a potential immunotherapeutic target for the treatment of EBV-associated malignancies. In the present study, we have employed a highly efficient strategy, based on ex vivo functional assays, to conduct an extensive sequence-wide analysis of LMP1-specific T-cell responses in a large panel of healthy virus carriers of diverse ethnic origin and nasopharyngeal carcinoma patients. By comparing the frequencies of T cells specific for overlapping peptides spanning LMP1, we mapped a number of novel HLA class I- and class II-restricted LMP1 T-cell epitopes, including an epitope with dual HLA class I restriction. More importantly, extensive sequence analysis of LMP1 revealed that the majority of the T-cell epitopes were highly conserved in EBV isolates from Caucasian, Papua New Guinean, African, and Southeast Asian populations, while unique geographically constrained genetic variation was observed within one HLA A2 supertype-restricted epitope. These findings indicate that conserved LMP1 epitopes should be considered in designing epitope-based immunotherapeutic strategies against EBV-associated malignancies in different ethnic populations.

The value of idiosyncratic markers and changes to conserved tRNA sequences from the mitochondrial genome of hard ticks (Acari: Ixodida: Ixodidae) for phylogenetic inference

Idiosyncratic markers are features of genes and genomes that are so unusual that it is unlikely that they evolved more than once in a lineage of organisms. Here we explore further the potential of idiosyncratic markers and changes to typically conserved tRNA sequences for phylogenetic inference. Hard ticks were chosen as the model group because their phylogeny has been studied extensively. Fifty-eight candidate markers from hard ticks ( family Ixodidae) and 22 markers from the subfamily Rhipicephalinae sensu lato were mapped onto phylogenies of these groups. Two of the most interesting markers, features of the secondary structure of two different tRNAs, gave strong support to the hypothesis that species of the Prostriata ( Ixodes spp.) are monophyletic. Previous analyses of genes and morphology did not strongly support this relationship, instead suggesting that the Prostriata is paraphyletic with respect to the Metastriata ( the rest of the hard ticks). Parallel or convergent evolution was not found in the arrangements of mitochondrial genes in ticks nor were there any reversals to the ancestral arthropod character state. Many of the markers identified were phylogenetically informative, whereas others should be informative with study of additional taxa. Idiosyncratic markers and changes to typically conserved nucleotides in tRNAs that are phylogenetically informative were common in this data set, and thus these types of markers might be found in other organisms.

Effects of additional sequences directly downstream from the AUG on the expression of GFP gene

We have studied the expression of the green fluorescent protein (GFP) gene to gain more understanding of the effects of additional nucleotide triplets (codons) downstream from the initiation codon on the translation of the GFP mRNA in CHO and Cos1 cells. A leader sequence of six consecutive identical codons (GUG, CUC, AGU or UCA) was introduced into a humanized GFP (hm gfp) gene downstream from the AUG to produce four GFP gene variants. Northern blot and RT-PCR analysis indicated that mRNA transcription from the GFP gene was not significantly affected by any of the additional sequences. However, immunoblotting and FACS analysis revealed that AGU and UCA GFP variants produced GFP at a mean level per cell 3.5-fold higher than the other two GFP variants and the hm gfp gene. [35S]-Methionine labeling and immunoprecipitation demonstrate that GFP synthesis was very active in UCA variant transfected-cells, but not in GUG variant and hm gfp transfected-cells. Moreover, proteasome inhibitor MG-132 treatment indicated that the GFPs encoded by each of the GFP variants and the hm gfp were equally stable, and this together with the comparable mRNA levels observed for each construct suggested that the different steady-state GFP concentrations observed reflected different translation efficiencies of the various GFP genes. In addition, the CUC GFP variant, when transiently transfected into CHO or COS-1 cells, did not produce any GFP expressing cells (fully green cells), and the GUG variant produced GFP expressing cells less than 10%, while AGU and UCA GFP variants up to 30–35% in a time course study from 8 to 36 h posttransfection. Analysis of the potential secondary structure of the GFP variant mRNAs especially in the translation initiation region suggested that the secondary structure of the GFP mRNAs was unlikely to explain the different translation efficiencies of the GFP variants. The present findings indicate that a change of the initiation context of the GFP gene by addition of extra coding sequence can alter the translation efficiency of GFP mRNA, providing a means of more efficient expression of GFP in eukaryotic cells.

Phylogeny of the Sporobolus indicus complex, based on internal transcribed spacer (ITS) sequences

The entire internal transcribed spacer ( ITS) region, including the 5.8S subunit of the nuclear ribosomal DNA ( rDNA), was sequenced by direct double-stranded sequencing of polymerase chain reaction (PCR) amplified fragments. The study included 40 Sporobolus ( Family Poaceae, subfamily Chloridoideae) seed collections from 14 putative species ( all 11 species from the S. indicus complex and three Australian native species). These sequences, along with those from two out-group species [ Pennisetum alopecuroides ( L.) Spreng. and Heteropogon contortus ( L.) P. Beauv. ex Roemer & Schultes, Poaceae, subfamily Panicoideae], were analysed by the parsimony method (PAUP; version 4.0b4a) to infer phylogenetic relationships among these species. The length of the ITS1, 5.8S subunit and ITS2 region were 222, 164 and 218 base pairs ( bp), respectively, in all species of the S. indicus complex, except for the ITS2 region of S. diandrus P. Beauv. individuals, which was 217 bp long. Of the 624 characters included in the analysis, 245 ( 39.3%) of the 330 variable sites contained potential phylogenetic information. Differences in sequences among the members of the S. pyramidalis P. Beauv., S. natalensis (Steud.) Dur & Schinz and S. jacquemontii Kunth. collections were 0%, while differences ranged from 0 to 2% between these and other species of the complex. Similarly, differences in sequences among collections of S. laxus B. K. Simon, S. sessilis B. K. Simon, S. elongatus R. Br. and S. creber De Nardi were 0%, compared with differences of 1-2% between these four species and the rest of the complex. When comparing S. fertilis ( Steud.) Clayton and S. africanus (Poir.) Robyns & Tourney, differences between collections ranged from 0 to 1%. Parsimony analysis grouped all 11 species of the S. indicus complex together, indicating a monophyletic origin. For the entire data set, pair-wise distances among members of the S. indicus complex varied from 0.00 to 1.58%, compared with a range of 20.08-21.44% among species in the complex and the Australian native species studied. A strict consensus phylogenetic tree separated 11 species of the S. indicus complex into five major clades. The phylogeny, based on ITS sequences, was found to be congruent with an earlier study on the taxonomic relationship of the weedy Sporobolus grasses revealed from random amplified polymorphic DNA ( RAPD). However, this cladistic analysis of the complex was not in agreement with that created on past morphological analyses and therefore gives a new insight into the phylogeny of the S. indicus complex.

Modelo agrometeorológico-espectral de estimativa de rendimento de grãos de trigo no Rio Grande do Sul

Este trabalho teve como objetivo construir uma regressão linear múltipla, empregando variáveis agrometeorológicas e espectrais, para estimativa de rendimento de grãos de trigo, em municípios pertencentes à região de atuação da Cooperativa Cotrijal (norte do Rio Grande do Sul). Para isso, foram empregados dados de rendimento (1991 a 2006), dados agrometeorológicos mensais (1991 a 2006) e dados espectrais (imagens NDVI/MODIS, 2000 a 2006). Foi analisada existência de aumento significativo no rendimento de grãos, decorrente da incorporação de novas tecnologias (tendência tecnológica). Para escolha das variáveis independentes da regressão linear, foi analisada a correlação dos dados agrometeorológicos e espectrais com os dados de rendimento. Definidas as variáveis, foi construída uma regressão linear múltipla de estimativa de rendimento de grãos de trigo. Os resultados mostraram que não houve aumento significativo no rendimento de grãos de trigo da Cotrijal, no período analisado. Foram escolhidas as seguintes variáveis independentes para construção da regressão linear múltipla: precipitação pluvial (outubro), índice de dano por geadas (setembro), graus-dia (acumulados de maio a outubro) e índice de vegetação por diferença normalizada (integrado de junho a outubro). As regressões lineares múltiplas apresentaram resultados satisfatórios, com erros de estimativa inferiores a 10%, na maior parte dos anos analisados. As características de precisão, fácil execução e baixo custo das regressões apontaram para possibilidade de uso conjunto de dados agrometeorológicos e espectrais, na estimativa de rendimento de grãos de trigo. Mais estudos são necessários para verificação dos resultados dos modelos, quando da incorporação de uma série mais longa de dados espectrais.

Desenvolvimento de modelo agrometeorológico espectral para estimativa de rendimento do milho na Província de Manica-Moçambique

Moçambique é um país localizado ao longo da costa Leste da África Austral, com a economia baseada essencialmente na agricultura. A cultura do milho (Zea mays L.) é a mais importante, cultivada em regime de sequeiro, com rendimentos dependentes das condições meteorológicas. Este trabalho teve como objetivo o ajuste de um modelo agrometeorológico espectral, para estimativa de rendimentos do milho, na província de Manica. A área de estudo envolveu os distritos de Gondola, Manica, Mossurize e Sussundenga, responsáveis por mais de 80% da produção de milho na província de Manica, nos anos de 2000 a 2009. ETr e ETm e ISNA foram as variáveis agrometeorológicas testadas no ajuste, obtidas a partir de estimativas de elementos meteorológicos do modelo do ECMWF. As variáveis espectrais foram os índices EVI e NDVI, provenientes do produto MOD13Q1, e o índice LSWI, calculado utilizando-se as bandas de refletância contidas neste produto. O modelo agrometeorológico espectral, ajustado por meio de regressão linear múltipla, teve como variáveis independentes os índices meteorológicos e espectrais e, como variável dependente, o rendimento médio ou o rendimento relativo. O modelo regional, que incluiu os distritos de Gondola, Manica e Sussundenga, e considerou o rendimento relativo, foi o mais recomendado para estimativa de rendimentos do milho, na região, com r² = 0,762 e RMSE de 9,46%.

Robust temporal alignment of multimodal cardiac sequences

Given the dynamic nature of cardiac function, correct temporal alignment of pre-operative models and intraoperative images is crucial for augmented reality in cardiac image-guided interventions. As such, the current study focuses on the development of an image-based strategy for temporal alignment of multimodal cardiac imaging sequences, such as cine Magnetic Resonance Imaging (MRI) or 3D Ultrasound (US). First, we derive a robust, modality-independent signal from the image sequences, estimated by computing the normalized crosscorrelation between each frame in the temporal sequence and the end-diastolic frame. This signal is a resembler for the left-ventricle (LV) volume curve over time, whose variation indicates di erent temporal landmarks of the cardiac cycle. We then perform the temporal alignment of these surrogate signals derived from MRI and US sequences of the same patient through Dynamic Time Warping (DTW), allowing to synchronize both sequences. The proposed framework was evaluated in 98 patients, which have undergone both 3D+t MRI and US scans. The end-systolic frame could be accurately estimated as the minimum of the image-derived surrogate signal, presenting a relative error of 1:6 1:9% and 4:0 4:2% for the MRI and US sequences, respectively, thus supporting its association with key temporal instants of the cardiac cycle. The use of DTW reduces the desynchronization of the cardiac events in MRI and US sequences, allowing to temporally align multimodal cardiac imaging sequences. Overall, a generic, fast and accurate method for temporal synchronization of MRI and US sequences of the same patient was introduced. This approach could be straightforwardly used for the correct temporal alignment of pre-operative MRI information and intra-operative US images.

Construção de uma base de dados espectral (IV) de toners de impressoras laser para identificação forense de documentos

Esta dissertação surgiu com o objectivo de se criar uma base de dados de Toners para impressoras e fotocopiadoras e um método fiável de comparação de espectros para estudos forenses e de investigação criminal. Para viabilizar o estudo, efectuou-se à priori a recolha de várias amostras de Toners, de diferentes marcas, de cartuchos originais e remanufacturados aplicando de seguida dois métodos de análise distintos: a Espectroscopia de infravermelho com transformada de Fourier (FT-IR) e a Espectroscopia de Absorção-Reflexão (ATR - Reflectância total atenuada). A espectroscopia FT-IR permitiu obter a base de dados de espectros em suporte KBr e usando o software OPUS, das várias amostras de Toners dos cartuchos originais e remanufacturados recolhidas. Para se criar o método de comparação entre espectros, realizou-se o mesmo procedimento mas agora para amostras obtidas por raspagem de um papel impresso /quadrados 2x2), bem como para o papel de impressão para se poder descontar este da amostra. Dado que o interesse desta análise se remetia ao estudo de textos escritos, efectuou-se a análise de um texto padrão por Espectroscopia FT-IR. O método foi utilizado à posteriori como método de comparação e uma alternativa (não destrutiva) ao método de FT-IR para se aplicar a casos reais. Os espectros foram obtidos num microscópio usando o silício como acessório ATR e o software OMNIC. Finalizado o estudo pode-se concluir, quanto à espectroscopia FT-IR, que as amostras da marca HP e Minolta apresentaram espectros muito semelhantes, quando se faz uma análise global dos mesmos. No entanto, as restantes marcas são também muito parecidas entre si, mas ainda é possível encontrar algumas distinções quando se efectua a comparação visual principalmente abaixo da zona de impressão digital (<6000 cm-1). Relativamente aos Toners remanufacturados, constata-se que são muito semelhantes e que diferem da marca original apenas pelo elevado grau de humidade que possuem. No que diz respeito ao método ATR, verificou-se que apesar de ser um método não destrutivo da amostra revela-se bastante limitado, devido ao uso do cristal de Silício como acessório que só atinge comprimentos de onda entre 4000-1500cm-1.

Genus and Braid Index Associated to Sequences of Renormalizable Lorenz Maps

We describe the Lorenz links generated by renormalizable Lorenz maps with reducible kneading invariant (K(f)(-), = K(f)(+)) = (X, Y) * (S, W) in terms of the links corresponding to each factor. This gives one new kind of operation that permits us to generate new knots and links from the ones corresponding to the factors of the *-product. Using this result we obtain explicit formulas for the genus and the braid index of this renormalizable Lorenz knots and links. Then we obtain explicit formulas for sequences of these invariants, associated to sequences of renormalizable Lorenz maps with kneading invariant (X, Y) * (S,W)*(n), concluding that both grow exponentially. This is specially relevant, since it is known that topological entropy is constant on the archipelagoes of renormalization.

Spatio-temporal alignment of pedobarographic image sequences

O documento em anexo encontra-se na versão post-print (versão corrigida pelo editor).

Gold electrode modified by self-assembled monolayers of thiols to determine DNA sequences hybridization

The process of immobilization of biological molecules is one of the most important steps in the construction of a biosensor. In the case of DNA, the way it exposes its bases can result in electrochemical signals to acceptable levels. The use of self-assembled monolayer that allows a connection to the gold thiol group and DNA binding to an aldehydic ligand resulted in the possibility of determining DNA hybridization. Immobilized single strand of DNA (ssDNA) from calf thymus pre-formed from alkanethiol film was formed by incubating a solution of 2-aminoethanothiol (Cys) followed by glutaraldehyde (Glu). Cyclic voltammetry (CV) was used to characterize the self-assembled monolayer on the gold electrode and, also, to study the immobilization of ssDNA probe and hybridization with the complementary sequence (target ssDNA). The ssDNA probe presents a well-defined oxidation peak at +0.158 V. When the hybridization occurs, this peak disappears which confirms the efficacy of the annealing and the DNA double helix performing without the presence of electroactive indicators. The use of SAM resulted in a stable immobilization of the ssDNA probe, enabling the hybridization detection without labels. This study represents a promising approach for molecular biosensor with sensible and reproducible results.