Synthesis and structural analysis of the N-terminal domain of the thyroid hormone-binding protein transthyretin

Transthyretin (TTR) is a 55 kDa protein responsible for the transport of thyroid hormones and retinol in human serum. Misfolded forms of the protein are implicated in the amyloid diseases familial amyloidotic polyneuropathy and senile systemic amyloidosis. Its folding properties and stabilization by ligands are of current interest due to their importance in understanding and combating these diseases. To assist in such studies we developed a method for the solid phase synthesis of the monomeric unit of a TTR analogue and its folding to form a functional 55 kDa tetramer. The monomeric unit of the protein was chemically synthesized in three parts, comprising amino acid residues 151, 5499 and 102127, and ligated using chemoselective thioether ligation chemistry. The synthetic protein was folded and assembled to a tetrameric structure in the presence of the TTRs native ligand, thyroxine, as shown by gel filtration chromatography, native gel electrophoresis, TTR antibody recognition and thyroid hormone binding. In the current study the solution structure of the first of these fragment peptides, TTR(151) is examined to determine its intrinsic propensity to form beta-sheet structure, potentially involved in amyloid fibril formation by TTR. Despite the presence of extensive beta-structure in the native form of the protein, the Nterminal fragment adopts an essentially random coil conformation in solution.

Isolation and characterization of nine microsatellite loci from the Hawaiian grouper Epinephelus quernus (Serranidae) for population genetic analyses

The availability of variable genetic markers for groupers (Serranidae) has generally been limited to mitochondrial DNA. For studies of population genetic structure, more loci are usually required; particularly useful are those that are nuclear in origin such as microsatellites. Here, we isolated and characterized 9 microsatellite loci from the endemic Hawaiian grouper Epinephelus quernus using a biotin-labeled oligonucleotide-streptavidin-coated magnetic bead approach. Of the 20 repeat-containing fragments isolated, 15 had sufficient flanking region in which to design primers. Among these, 9 produced consistent polymerase chain reaction product, and 6 were highly variable. These 6 loci were all composed of dinucleotide repeats, with the number of alleles ranging from 6 to 18, and heterozygosities from 33.3% to 91.7%. The high levels of variability observed should make these markers useful for population genetic studies of E. quernus, and potentially other epinephelines.

Molecular and functional analyses of Kunjin virus infectious cDNA clones demonstrate the essential roles for NS2A in virus assembly and for a nonconservative residue in NS3 in RNA replication

A number of full-length cDNA clones of Kunjin virus (KUN) were previously prepared; it was shown that two of them, pAKUN and FLSDX, differed in specific infectivities of corresponding in vitro transcribed RNAs by similar to100,000-fold (A. A. Khromykh et al., J. Virol. 72:7270-7279, 1998). In this study, we analyzed a possible genetic determinant(s) of the observed differences in infectivity initially by sequencing the entire cDNAs of both clones and comparing them with the published sequence of the parental KUN strain MRM61C. We found six common amino acid residues in both cDNA clones that were different from those in the published MRM61C sequence but were similar to those in the published sequences of other flaviviruses from the same subgroup. pAKUN clone had four additional codon changes, i.e., Ile59 to Asn and Arg175 to Lys in NS2A and Tyr518 to His and Ser557 to Pro in NS3. Three of these substitutions except the previously shown marker mutation, Arg175 to Lys in NS2A, reverted to the wild-type sequence in the virus eventually recovered from pAKUN RNA-transfected BHK cells, demonstrating the functional importance of these residues in viral replication and/or viral assembly. Exchange of corresponding DNA fragments between pAKUN and FLSDX clones and site-directed mutagenesis revealed that the Tyr518-to-His mutation in NS3 was responsible for an similar to5-fold decrease in specific infectivity of transcribed RNA, while the Ile59-to-Asn mutation in NS2A completely blocked virus production. Correction of the Asn59 in pAKUN NS2A to the wild-type lie residue resulted in complete restoration of RNA infectivity. Replication of KUN replicon RNA with an Ile59-to-Asn substitution in NS2A and with a Ser557-to-Pro substitution in NS3 was not affected, while the Tyr518-to-His substitution in NS3 led to severe inhibition of RNA replication. The impaired function of the mutated NS2A in production of infectious virus was complemented in trans by the helper wild-type NS2A produced from the KUN replicon RNA. However, replicon RNA with mutated NS2A could not be packaged in trans by the KUN structural proteins. The data demonstrated essential roles for the KUN nonstructural protein NS2A in virus assembly and for NS3 in RNA replication and identified specific single-amino-acid residues involved in these functions.

Mean anisotropy of homogeneous Gaussian random fields and anisotropic norms of linear translation-invariant operators on multidimensional integer lattices

Sensitivity of output of a linear operator to its input can be quantified in various ways. In Control Theory, the input is usually interpreted as disturbance and the output is to be minimized in some sense. In stochastic worst-case design settings, the disturbance is considered random with imprecisely known probability distribution. The prior set of probability measures can be chosen so as to quantify how far the disturbance deviates from the white-noise hypothesis of Linear Quadratic Gaussian control. Such deviation can be measured by the minimal Kullback-Leibler informational divergence from the Gaussian distributions with zero mean and scalar covariance matrices. The resulting anisotropy functional is defined for finite power random vectors. Originally, anisotropy was introduced for directionally generic random vectors as the relative entropy of the normalized vector with respect to the uniform distribution on the unit sphere. The associated a-anisotropic norm of a matrix is then its maximum root mean square or average energy gain with respect to finite power or directionally generic inputs whose anisotropy is bounded above by a���0. We give a systematic comparison of the anisotropy functionals and the associated norms. These are considered for unboundedly growing fragments of homogeneous Gaussian random fields on multidimensional integer lattice to yield mean anisotropy. Correspondingly, the anisotropic norms of finite matrices are extended to bounded linear translation invariant operators over such fields.

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.

Gene flow in great bustard populations across the Strait of Gibraltar as elucidated from excremental PCR and mtDNA sequencing

Recent advances in molecular biology have made it possible to use the trace amounts of DNA in faeces to non-invasively sample endangered species for genetic studies. Here we use faeces as a source of DNA and mtDNA sequence data to elucidate the relationship among Spanish and Moroccan populations of great bustards. 834 bp of combined control region and cytochrome-b mtDNA fragments revealed four variable sites that defined seven closely related haplotypes in 54 individuals. Morocco was fixed for a single mtDNA haplotype that occurs at moderate frequency (28%) in Spain. We could not differentiate among the sampled Spanish populations of Caceres and Andalucia but these combined populations were differentiated from the Moroccan population. Estimates of gene flow (Nm = 0.82) are consistent with extensive observations on the southern Iberian peninsular indicating that few individuals fly across the Strait of Gibraltar. We demonstrate that both this sea barrier and mountain barriers in Spain limit dispersal among adjacent great bustard populations to a similar extent. The Moroccan population is of high ornithological significance as it holds the only population of great bustards in Africa. This population is critically small and genetic and observational data indicate that it is unlikely to be recolonised via immigration from Spain should it be extirpated. In light of the evidence presented here it deserves the maximum level of protection.

Survey of the Hymenoptera parasitoids in Eucalyptus grandis and in a native vegetation area in Ipaba, State of Minas Gerais, Brazil

Foi feito um levantamento da fauna de Hymenoptera parasit��ides em um transecto eucalipto/vegeta����o nativa/eucalipto, em Ipaba, Minas Gerais, no per��odo de mar��o de 1997 a mar��o de 1998, com armadilhas Malaise. Foram coletados indiv��duos de nove superfam��lias (Ceraphronoidea, Chalcidoidea, Chrysidoidea, Cynipoidea, Evanioidea, Ichneumonoidea, Proctotrupoidea, Platygastroidea e Vespoidea), distribu��dos em 26 fam��lias.

Pequenos mam��feros n��o voadores em fragmentos de Mata Atl��ntica e ��reas agr��colas em Viana, Esp��rito Santo, Brasil

N��s inventariamos os pequenos mam��feros n��o-voadores em Viana, Esp��rito Santo, sudeste do Brasil, em 1981-1982 e 2006-2007. Foram capturados 439 pequenos mam��feros n��o-voadores pertencentes a tr��s ordens (Didelphimorphia, Rodentia e Lagomorpha) e seis fam��lias (Didelphidae, Sciuridade, Cricetidae, Muridae, Echimyidae e Leporidae). As esp��cies mais abundantes foram os roedores Akodon cursor e Nectomys squamipes e o marsupial Metachirus nudicaudatus. Registramos pela primeira vez a ocorr��ncia do roedor equim��deo Euryzygomatomys spinosus no estado do Esp��rito Santo. A riqueza espec��fica (S = 21) e o ��ndice de diversidade de Shannon (H = 2,23) est��o entre os maiores registrados para pequenos mam��feros da Mata Atl��ntica no estado, mesmo quando comparados aos valores obtidos para unidades de conserva����o. Esses maiores valores de riqueza e diversidade encontrados est��o provavelmente associados �� heterogeneidade de h��bitat e evidenciam a necessidade de conserva����o dos fragmentos florestais de Viana, os quais s��o importantes deposit��rios da biodiversidade da Mata Atl��ntica.

Fun����es gerenciais na gest��o educacional : caracteriza����o do trabalho gerencial dos gestores da Secretaria Municipal de Educa����o de Santa Maria de Jetib��-ES

Esta pesquisa tem a inten����o de descrever as caracter��sticas do trabalho gerencial de gestores educacionais, iniciando-se de estudo realizado com gestores escolares, coordenadores pedag��gicos que atendem ��s escolas multisseriadas da secretaria de educa����o do munic��pio de Santa Maria de Jetib�� (ES). Buscou-se caracterizar a a����o gerencial desses gestores partindo das percep����es deles sobre os trabalhos realizados junto aos demais membros da comunidade escolar, equipes de gest��o e gest��o da secretaria municipal, n��o deixando de lado as particularidades da ��rea educacional, principalmente sobre gest��o democr��tica e autonomia. Para se entender a diversidade, a complexidade e as din��micas rela����es dos gestores, foram realizadas entrevistas semiestruturadas que contribu��ram para aprofundamento nessas din��micas. Documentos foram analisados para conhecimento e caracteriza����o da formata����o da estrutura organizacional da secretaria de educa����o. Os relatos das entrevistas apresentaram fragmentos que puderam nortear ideias para um melhor tratamento dos dados, que foram realizados analisando os conte��dos apresentados pelos gestores entrevistados. As conversas realizadas com esses entrevistados mostraram ambiguidade, dinamismo e pluralidade no desenvolvimento das ���fun����es gerenciais��� dos gestores educacionais, que s��o l��deres, companheiros e executores de trabalhos junto aos demais componentes das rela����es do ambiente educacional. Assim, as expectativas s��o de que esta pesquisa possa fazer com que novos di��logos surjam para discutir as rela����es dos gestores educacionais, principalmente sobre as a����es dos coordenadores que atuam junto ��s escolas multisseriadas.

Ecofisiologia, fenologia e adapta����o de esp��cies florestais arb��reas nas condi����es edafoclim��ticas de Viana-ES

A intensa atividade humana devasta grandes extens��es de florestas nativas, seja para expans��o da agricultura seja para suprir a crescente demanda do mercado por madeira, de uma forma ou de outra os pequenos fragmentos florestais remanescentes sofrem constantes press��es antr��picas, contudo, o uso de esp��cies arb��reas adaptadas pode contribuir para a prote����o desses recursos naturais. Buscou-se neste estudo avaliar os padr��es fenol��gicos de 109 esp��cies florestais arb��reas pertencentes a 37 fam��lias e 82 g��neros, dentre nativas e ex��ticas, com nove anos de idade, para diferenciar as mais adaptadas e com potencial de serem utilizadas em programas de reflorestamento. O estudo foi realizado na Fazenda Experimental do Incaper, em Jucuruaba, munic��pio de Viana-ES, (UTM E-345524, N- 7741039). Foram realizadas an��lises qu��micas do solo na ��rea plantada e os dados climatol��gicos obtidos na esta����o meteorol��gica de Viana. O estudo baseou-se na observa����o do n��mero de plantas sobreviventes de cada esp��cie e da avalia����o do seu crescimento. Foram realizadas observa����es das fenofases de brota����o, senesc��ncia de folhas, flora����o e frutifica����o. As avalia����es fenol��gicas foram realizadas em intervalos mensais, no per��odo de novembro de 2012 a outubro de 2013. Realizou-se a medi����o da altura das ��rvores, di��metro �� altura do peito (DAP), ��ndice de enfolhamento, taxa de sobreviv��ncia e c��lculo do ICC (��ndice Combinado de Crescimento), bem como a determina����o das esp��cies mais adaptadas. Das 109 esp��cies estudadas, 64,22% apresentaram adapta����o funcional e estrutural ��s condi����es de solo e clima da regi��o experimental, 42,22 % floresceram e frutificaram e 90% apresentaram senesc��ncia e brota����o acompanhando a sazonalidade clim��tica. Vinte e nove esp��cies apresentaram ICC maior do que o ICC m��dio. A maioria das esp��cies destacou-se como alternativa para recupera����o da cobertura vegetal local, com destaque para Inga uruguensis e Schizolobium amazonicum. As an��lises de fluoresc��ncia da clorofila revelaram que o aparato fotossint��tico da Schizolobium amazonicum foi capaz de proteg��-la da fotoinibi����o e promover boa convers��o da energia luminosa em fotoqu��mica.

Caracteriza����o molecular de tr��s esp��cies de Trachycephalus (Anura: Hylidae) : investigando potenciais h��bridos interespec��ficos

Animais h��bridos representam um desafio �� taxonomia e sistem��tica, pois correspondem a unidades evolutivas geralmente sem clara delimita����o morfol��gica, comportamental e gen��tica. H��bridos podem ser morfologicamente intermedi��rios aos parentais ou, devido �� introgress��o e retrocruzamentos, suas caracter��sticas podem se misturar tornando dif��cil sua identifica����o. Uma das formas de identifica����o de h��bridos �� por meio de ferramentas de biologia molecular, que ao utilizarem marcadores de DNA mitocondrial (heran��a exclusiva materna) e DNA nuclear (heran��a materna e paterna), permitem a compara����o entre informa����es gen��ticas. Al��m da hibridiza����o existem outras fontes de conflito entre dados moleculares provenientes do DNA mitocondrial e DNA nuclear, como por exemplo a reten����o de polimorfismos ancentrais. Em localidades do Esp��rito Santo, Brasil, foram coletados indiv��duos de morfologia distinta de Trachycephalus mesophaeus e T. nigromaculatus, que s��o as ��nicas esp��cies do g��nero conhecidas nesse estado. Por��m, estudos piloto usando o gene mitocondrial Citocromo Oxidase subunidade I (COI) agruparam esses esp��cimes com amostras de T. typhonius. Devido a estas incongru��ncias, foram sequenciados fragmentos de dois genes mitocondriais - COI e Nicotinamida Desidrogenase subunidade 2 (ND2) e um exon nuclear (tirosinase) de 173 indiv��duos de Trachycephalus, de forma a esclarecer as identifica����es taxon��micas e investigar a correspond��ncia entre caracteres morfol��gicos e gen��ticos nesta linhagem, na sua ��rea de ocorr��ncia As filogenias moleculares, diverg��ncias gen��ticas, redes de hapl��tipos e polimorfismos de nucleot��deos ��nicos (SNPs) confirmaram as tr��s esp��cies acima mencionadas como linhagens evolutivas distintas e revelaram mais sete indiv��duos potencialmente h��bridos, mas morfologicamente assinalados a T. mesophaeus, T. nigromaculatus ou T. typhonius.. Devido �� taxa de evolu����o lenta da tirosinase, as esp��cies mais recentes T. typhonius e T. nigromaculatus parecem n��o terem sido sorteadas completamente nesse gene. J�� T. mesophaeus, que �� a esp��cie mais antiga das tr��s, foi recuperada inequivocamente em todas as an��lises. De forma in��dita, as an��lises moleculares evidenciaram a ocorr��ncia de introgress��o bidirecional entre T. nigromaculatus e T. typhonius e entre T. nigromaculatus e T. mesophaeus, sendo que h�� ind��cios de indiv��duos F1 (cruzamentos entre esp��cies parentais puras gerando h��bridos). A utiliza����o do gene ND2 mostrou-se mais eficiente do que o gene COI nas filogenias e, apesar da tirosinase ser um gene nuclear de evolu����o lenta, contribuiu para a identifica����o de incongru��ncias citonucleares. Nossos resultados mostram que a hist��ria filogen��tica de Trachycephalus �� complexa e que o uso de marcadores nucleares de evolu����o mais r��pida e amplia����o dessas an��lises para outras esp��cies do g��nero podem revelar mais eventos de hibridiza����o.

Program slicing by calculation

Program slicing is a well known family of techniques used to identify code fragments which depend on or are depended upon specific program entities. They are particularly useful in the areas of reverse engineering, program understanding, testing and software maintenance. Most slicing methods, usually oriented towards the imperative or object paradigms, are based on some sort of graph structure representing program dependencies. Slicing techniques amount, therefore, to (sophisticated) graph transversal algorithms. This paper proposes a completely different approach to the slicing problem for functional programs. Instead of extracting program information to build an underlying dependencies��� structure, we resort to standard program calculation strategies, based on the so-called Bird-Meertens formalism. The slicing criterion is specified either as a projection or a hiding function which, once composed with the original program, leads to the identification of the intended slice. Going through a number of examples, the paper suggests this approach may be an interesting, even if not completely general, alternative to slicing functional programs

Higher-order lazy functional slicing

Program slicing is a well known family of techniques intended to identify and isolate code fragments which depend on, or are depended upon, specific program entities. This is particularly useful in the areas of reverse engineering, program understanding, testing and software maintenance. Most slicing methods, and corresponding tools, target either the imperative or the object oriented paradigms, where program slices are computed with respect to a variable or a program statement. Taking a complementary point of view, this paper focuses on the slicing of higher-order functional programs under a lazy evaluation strategy. A prototype of a Haskell slicer, built as proof-of-concept for these ideas, is also introduced

Slicing for architectural analysis

Current software development often relies on non-trivial coordination logic for combining autonomous services, eventually running on different platforms. As a rule, however, such a coordination layer is strongly woven within the application at source code level. Therefore, its precise identification becomes a major methodological (and technical) problem and a challenge to any program understanding or refactoring process. The approach introduced in this paper resorts to slicing techniques to extract coordination data from source code. Such data are captured in a specific dependency graph structure from which a coordination model can be recovered either in the form of an Orc specification or as a collection of code fragments corresponding to the identification of typical coordination patterns in the system. Tool support is also discussed

Slicing functional programs by calculation

Program slicing is a well known family of techniques used to identify code fragments which depend on or are depended upon specific program entities. They are particularly useful in the areas of reverse engineering, program understanding, testing and software maintenance. Most slicing methods, usually targeting either the imperative or the object oriented paradigms, are based on some sort of graph structure representing program dependencies. Slicing techniques amount, therefore, to (sophisticated) graph transversal algorithms. This paper proposes a completely different approach to the slicing problem for functional programs. Instead of extracting program information to build an underlying dependencies��� structure, we resort to standard program calculation strategies, based on the so-called Bird- Meertens formalism. The slicing criterion is specified either as a projection or a hiding function which, once composed with the original program, leads to the identification of the intended slice. Going through a number of examples, the paper suggests this approach may be an interesting, even if not completely general alternative to slicing functional programs