Analysis of genetic diversity within Australian lucerne cultivars and implications for future genetic improvement

Lucerne (Medicago sativa L.) is autotetraploid, and predominantly allogamous. This complex breeding structure maximises the genetic diversity within lucerne populations making it difficult to genetically discriminate between populations. The objective of this study was to evaluate the level of random genetic diversity within and between a selection of Australian-grown lucerne cultivars, with tetraploid M. falcata included as a possible divergent control source. This diversity was evaluated using random amplified polymorphic DNA (RAPDs). Nineteen plants from each of 10 cultivars were analysed. Using 11 RAPD primers, 96 polymorphic bands were scored as present or absent across the 190 individuals. Genetic similarity estimates (GSEs) of all pair-wise comparisons were calculated from these data. Mean GSEs within cultivars ranged from 0.43 to 0.51. Cultivar Venus (0.43) had the highest level of intra-population genetic diversity and cultivar Sequel HR (0.51) had the lowest level of intra-population genetic diversity. Mean GSEs between cultivars ranged from 0.31 to 0.49, which overlapped with values obtained for within-cultivar GSE, thus not allowing separation of the cultivars. The high level of intra- and inter-population diversity that was detected is most likely due to the breeding of synthetic cultivars using parents derived from a number of diverse sources. Cultivar-specific polymorphisms were only identified in the M. falcata source, which like M. sativa, is outcrossing and autotetraploid. From a cluster analysis and a principal components analysis, it was clear that M. falcata was distinct from the other cultivars. The results indicate that the M. falcata accession tested has not been widely used in Australian lucerne breeding programs, and offers a means of introducing new genetic diversity into the lucerne gene pool. This provides a means of maximising heterozygosity, which is essential to maximising productivity in lucerne.

Development and characterization of polymorphic microsatellite markers in taro (Colocasia esculenta)

Microsatellite-containing sequences were isolated from enriched genomic libraries of taro (Colocasia esculenta (L.) Schott). The sequencing of 269 clones yielded 77 inserts containing repeat motifs. The majority of these (81.7%) were dinucleotide or trinucleotide repeats. The GT/CA repeat motif was the most common, accounting for 42% of all repeat types. From a total of 43 primer pairs designed, 41 produced markers within the expected size range. Sixteen (39%) were polymorphic when screened against a restricted set of taro genotypes from Southeast Asia and Oceania, with an average of 3.2 alleles detected on each locus. These markers represent a useful resource for taro germplasm management, genome mapping, and marker-assisted selection.

A rapid PCR protocol for marker assisted detection of heterozygotes in segregating generations involving 1BL/1RS translocation and normal wheat lines

A rapid and reliable polymerase chain reaction (PCR)-based protocol was developed for detecting zygosity of the 1BL/1RS translocation in hexaploid wheat. The protocol involved a multiplex PCR with 2 pairs of oligonucleotide primers, rye-specific Ris-1 primers, and consensus 5S intergenic spacer (IGS) primers, and digestion of the PCR products with the restriction enzyme, MseI. A small piece of alkali-treated intact leaf tissue is used as a template for the PCR, thereby eliminating the necessity for DNA extraction. The test is simple, highly sensitive, and rapid compared with the other detection systems of 1BS1RS heterozygotes in hexaploid wheat. PCR results were confirmed with AFLP analyses. Diagnostic tests for 1BL/1RS translocation based on Sec-1-specific ELISA, screening for chromosome arm 1RS controlled rust resistance locus Yr9, and the PCR test differed in their ability to detect heterozygotes. The PCR test and rust test detected more heterozygotes than the ELISA test. The PCR test is being used to facilitate S1 family recurrent selection in the Germplasm Enhancement Program of the Australian Northern Wheat Improvement Program. A combination of the PCR zygosity test with other markers currently being implemented in the breeding program makes this test economical for 1BL/1RS characterisation of S1 families.

Identification of research to improve the efficiency of breeding strategies for white clover in Australia - a review

A major challenge faced by today's white clover breeder is how to manage resources within a breeding program. It is essential to utilise these resources with sufficient flexibility to build on past progress from conventional breeding strategies, but also take advantage of emerging opportunities from molecular breeding tools such as molecular markers and transformation. It is timely to review white clover breeding strategies. This background can then be used as a foundation for considering how to continue conventional plant improvement activities and complement them with molecular breeding opportunities. In this review, conventional white clover breeding strategies relevant to the Australian dryland target population environments are considered. Attention is given to: (i) availability of genetic variation, (ii) characterisation of germplasm collections, (iii) quantitative models for estimation of heritability, (iv) the role of multi-environment trials to accommodate genotype-by-environment interactions, (v) interdisciplinary research to understand adaptation to dryland environments, (vi) breeding and selection strategies, and (vii) cultivar structure. Current achievements in biotechnology with specific reference to white clover breeding in Australia are considered, and computer modelling of breeding programs is discussed as a useful integrative tool for the joint evaluation of conventional and molecular breeding strategies and optimisation of resource use in breeding programs. Four areas are identified as future research priorities: (i) capturing the potential genetic diversity among introduced accessions and ecotypes that are adapted to key constraints such as summer moisture stress and the use of molecular markers to assess the genetic diversity, (ii) understanding the underlying physiological/morphological root and shoot mechanisms involved in water use efficiency of white clover, with the objective of identifying appropriate selection criteria, (iii) estimation of quantitative genetic parameters of important morphological/physiological attributes to enable prediction of response to selection in target environments, and (iv) modelling white clover breeding strategies to evaluate the opportunities for integration of molecular breeding strategies with conventional breeding programs.

T cell regulation of the immune response to infection in periodontal diseases

Although T cells have been implicated in the pathogenesis and are considered to be central both in progression and control of the chronic inflammatory periodontal diseases, the precise contribution of T cells to the regulation of tissue destruction has not been fully elucidated. Current dogma suggests that immunity to infection is controlled by distinct T helper 1 (Th1) and T helper 2 (Th2) subsets of T cells classified on the basis of their cytokine profile. Further, a subset of T cells with immunosuppressive function and cytokine profile distinct from Th1 or Th2 has been described and designated as regulatory T cells. Although these regulatory T cells have been considered to maintain self-tolerance resulting in the suppression of auto-immune responses, recent data suggest that these cells may also play a role in preventing infection-induced immunopathology. In this review, the role of functional and regulatory T cells in chronic inflammatory periodontal diseases will be summarized. This should not only provide an insight into the relationship between the immune response to periodontopathic bacteria and disease but should also highlight areas of development for potentially new therapeutic modalities.

Quantitation of alternatively spliced NMDA receptor NR1 isoform mRNA transcripts in human brain by competitive RT-PCR

The N-methyl-D-aspartate (NMDA)-selective subtype of ionotropic glutamate receptor is of importance in neuronal differentiation and synapse consolidation, activity-dependent forms of synaptic plasticity, and excitatory amino acid-mediated neuronal toxicity [Neurosci. Res. Program, Bull. 19 (1981) 1; Lab. Invest. 68 (1993) 372]. NMDA receptors exist in vivo as tetrameric or pentameric complexes comprising proteins from two families of homologous subunits, designated NR1 and NR2(A-D) [Biochem. Biophys. Res. Commun. 185 (1992) 826]. The gene coding for the human NR1 subunit (hNR1) is composed of 21 exons, three of which (4, 20 and 21) can be differentially spliced to generate a total of eight distinct subunit variants. We detail here a competitive RT-PCR (cRT-PCR) protocol to quantify endogenous levels of hNR1 splice variants in autopsied human brain. Quantitation of each hNR1 splice variant is performed using standard curve methodology in which a known amount of synthetic ribonucleic acid competitor (internal standard) is co-amplified against total RNA. This method can be used for the quantitation of hNR1 mRNA levels in response to acute or chronic disease states, in particular in the glutamatergic-associated neuronal loss observed in Alzheimer's disease [J. Neurochem. 78 (2001) 175]. Furthermore, alterations in hNR1 mRNA expression may be reflected at the translational level, resulting in functional changes in the NMDA receptor. (C) 2003 Elsevier Science B.V. All rights reserved.

The role of melanocortin-1 receptor polymorphism in skin cancer risk phenotypes

We have examined melanocortin-1 receptor (MC1R) variant allele frequencies in the general population and in a collection of adolescent dizygotic and monozygotic twins to determine statistical associations of pigmentation phenotypes with increased skin cancer risk. This included hair and skin color, freckling, mole count and sun exposed skin reflectance. Nine variants were studied and designated as either strong R (OR = 63; 95% CI 32-140) or weak r (OR = 5; 95% CI 3-11) red hair alleles. Penetrance of each MC1R variant allele was consistent with an allelic model where effects were multiplicative for red hair but additive for skin reflectance. To assess the interaction of the brown eye color gene BEY2/OCA2 on the phenotypic effects of variant MC1R alleles we imputed OCA2 genotype in the twin collection. A modifying effect of OCA2 on MC1R variant alleles was seen on constitutive skin color, freckling and mole count. In order to study the individual effects of these variants on pigmentation phenotype we have established a series of human primary melanocyte strains genotyped for the MC1R receptor. These include strains which are MC1R wild-type consensus, variant heterozygotes, and homozygotes for strong R alleles Arg151Cys and Arg160Trp. Ultrastructural analysis demonstrated that only consensus strains contained stage III and IV melanosomes in their terminal dendrites whereas Arg151Cys and Arg160Trp homozygous strains contained only immature stage I and II melanosomes. Such genetic association studies combined with the functional analysis of MC1R variant alleles in melanocytic cells should provide a link in understanding the association between pigmentary phototypes and skin cancer risk.

Identification of a novel protein kinase mediating Akt survival signaling to the ATM protein

We identified a novel human AMP-activated protein kinase (AMPK) family member, designated ARK5, encoding 661 amino acids with an estimated molecular mass of 74 kDa. The putative amino acid sequence reveals 47, 45.8, 42.4, and 55% homology to AMPK-alpha1, AMPK-alpha2, MELK and SNARE respectively, suggesting that it is a new member of the AMPK family. It has a putative Akt phosphorylation motif at amino acids 595600, and Ser(600) was found to be phosphorylated by active Akt resulting in the activation of kinase activity toward the SAMS peptide, a consensus AMPK substrate. During nutrient starvation, ARK5 supported the survival of cells in an Akt-dependent manner. In addition, we also demonstrated that ARK5, when activated by Akt, phosphorylated the ATM protein that is mutated in the human genetic disorder ataxia-telangiectasia and also induced the phosphorylation of p53. On the basis of our current findings, we propose that a novel AMPK family member, ARK5, is the tumor cell survival factor activated by Akt and acts as an ATM kinase under the conditions of nutrient starvation.

GRIP domain-mediated targeting of two new coiled-coil proteins, GCC88 and GCC185, to subcompartments of the trans-Golgi network

The GRIP domain is a targeting sequence found in a family of coiled-coil peripheral Golgi proteins. Previously we demonstrated that the GRIP domain of p230/golgin245 is specifically recruited to tubulovesicular structures of the traps-Golgi network (TGN). Here we have characterized two novel Golgi proteins with functional GRIP domains, designated GCC88 and GCC185. GCC88 cDNA encodes a protein of 88 kDa, and GCC185 cDNA encodes a protein of 185 kDa. Both molecules are brefeldin A-sensitive peripheral membrane proteins and are predicted to have extensive coiled-coil regions with the GRIP domain at the C terminus. By immunofluorescence and immunoelectron microscopy GCC88 and GCC185, and the GRIP protein golgin97, are all localized to the TGN of Hela cells. Overexpression of full-length GCC88 leads to the formation of large electron dense structures that extend from the traps-Golgi. These de novo structures contain GCC88 and co-stain for the TGN markers syntaxin 6 and TGN38 but not for alpha2,6-sialyltransferase, beta-COP, or cis-Golgi GM130. The formation of these abnormal structures requires the N-terminal domain of GCC88. TGN38, which recycles between the TGN and plasma membrane, was transported into and out of the GCC88 decorated structures. These data introduce two new GRIP domain proteins and implicate a role for GCC88 in the organization of a specific TGN subcompartment involved with membrane transport.

Integrated control of nitrate recirculation and external carbon addition in a predenitrification system

The integrated control of nitrate recirculation and external carbon addition in a predenitrification biological wastewater treatment system is studied. The proposed control structure consists of four feedback control loops, which manipulate the nitrate recirculation and the carbon dosage flows in a highly coordinated manner such that the consumption of external carbon is minimised while the nitrate discharge limits (based on both grab and composite samples) are met. The control system requires the measurement of the nitrate concentrations at the end of both the anoxic and the aerobic zones. Distinct from ordinary control systems, which typically minimise the variation in the controlled variables, the proposed control system essentially maximises the diurnal variation of the effluent nitrate concentration and through this maximises the use of influent COD for denitrification, thus minimising the requirement for external carbon source. Simulation studies using a commonly accepted simulation benchmark show that the controlled system consistently achieves the designated effluent quality with minimum costs.

Genetic characterization of pilin glycosylation and phase variation in Neisseria meningitidis

Pili of Neisseria meningitidis are a key virulence factor, being the major adhesin of this capsulate organism and contributing to specificity for the human host. Pili are post-translationally modified by addition of either an O-linked trisaccharide, Gal (beta1-4) Gal (alpha1-3) 2,4-diacetamido-2,4,6-trideoxyhexose or an O-linked disaccharide Gal (alpha1,3) GlcNAc. The role of these structures in meningococcal pathogenesis has not been resolved. In previous studies we identified two separate genetic loci, pglA and pglBCD, involved in pilin glycosylation. Putative functions have been allocated to these genes; however, there are not enough genes to account for the complete biosynthesis of the described structures, suggesting additional genes remain to be identified. In addition, it is not known why some strains express the trisaccharide structure and some the disaccharide structure. In order to find additional genes involved in the biosynthesis. of these structures, we used the recently published group A strain Z2491 and group B strain MC58 Neisseria meningitidis genomes and the unfinished Neisseria meningitidis group C strain FAM18 and Neisseria gonorrhoeae strain FA1090 genomes to identify novel genes involved in pilin glycosylation, based on homology to known oligosaccharide biosynthetic genes. We identified a new gene involved in pilin glycosylation designated pglE and examined four additional genes pgIB/B2, pglF, pglG and pglH. A strain survey revealed that pglE and pglF were present in each strain examined. The pglG, pglH and pgIB2 polymorphisms were not found in strain C311#3 but were present in a large number of clinical isolates. Insertional mutations were constructed in pglE and pglF in N. meningitidis strain C311#3, a strain with well-defined lipopolysaccharide (LPS) and pilin-linked glycan structures. Increased gel migration of the pilin subunit molecules of pglE and pglF mutants was observed by Western analysis, indicating truncation of the trisaccharide structure. Antisera specific for the C311#3 trisaccharide failed to react with pilin from these pglE and pglF mutants. GC-MS analysis of the sugar composition of the pglE mutant showed a reduction in galactose compared with C311#3 wild type. Analysis of amino acid sequence homologies has suggested specific roles for pglE and pglF in the biosynthesis of the trisaccharide structure. Further, we present evidence that pglE, which contains heptanucleotide repeats, is responsible for the phase variation between trisaccharide and disaccharide structures in strain C311#3 and other strains. We also present evidence that pglG, pglH and pgIB2 are potentially phase variable.

Characterization of the types of the Neotropical Pseudisobrachium (Hymenoptera: Bethylidae), with a key to species

Atualmente é difícil reconhecer a identidade de muitas espécies neotropicais de Pseudisobrachium Kieffer, 1904, principalmente por que as descrições e ilustrações disponíveis não são suficientes para permitir identificações precisas. Para resolver este problema, foram examinadas 115 espécies válidas, além de seus sinônimos juniores. Foram realizados doze atos nomenclaturais, e reconhecidas 110 espécies válidas para a região Neotropical. Foram designados dois lectótipos: Pristocera crassicornis Westwood and Pristocera haemorrhoidalis Westwood. Foram propostas sete sinonímias novas para espécies: Pseudisobrachium retusum Evans syn. nov. de P. pauxillum Evans; P. cunco Perez syn. nov. de P. erythrocephalum Evans; P. navajo Evans, P. rectangulatum Evans, P. emarginatum Evans e P. foutsi Evans syn. nov. de P. flavinervis Fouts; P. acuminatum Waichert & Azevedo syn. nov. de P. latum Waichert & Azevedo. Foi proposta a seguinte sinonímia nova para gênero: Parisobrachium Kieffer syn. nov. de Dissomphalus Ashmead. Foi estabelecida a seguinte combinação nova e revalidado o nome: Dissomphalus albipes (Kieffer) comb. nov. e nom. rev. de Pseudisobrachium paraguayense Kieffer.

O patrimônio arquitetônico da saúde : discussões sobre a história da arquitetura hospitalar do século XIX

O Patrimônio Cultural da Saúde consiste nos bens materiais e imateriais que expressam o processo da saúde individual e coletiva nas suas dimensões científica, histórica e cultural. Com a inserção do Brasil, através da COC-Fiocruz e do Ministério da Saúde, na Rede Latino-americana de Patrimônio Cultural da Saúde, iniciou-se o incentivo ao estudo da história da medicina e da arquitetura hospitalar, buscando também a proteção e a salvaguarda da memória das edificações hospitalares históricas. O século XIX foi marcado pela construção de várias edificações voltadas para o controle e reclusão dos pobres, essas instituições eram: a Casa de Correção, a Santa Casa da Misericórdia, o Hospício de Pedro II, o Asilo da Mendicidade e as Instituições de acolhimento de Menores. Dessas edificações destacam-se a Santa Casa da Misericórdia, o Hospício de Pedro II e o Asilo da Mendicidade que formam o Patrimônio Arquitetônico da Saúde tombado em nível federal. O Hospital da Santa Casa da Misericórdia foi construído em 1840-1852 sob os modernos preceitos da medicina do século XIX. A edificação até hoje mantém o uso hospitalar e apresenta um estado de conservação bom em seu exterior. Porém as condições internas foram consideradas ruins devido à falta de salubridade e higiene nos ambientes. O Hospital da Santa Casa é um Hospital de Referência, realiza atendimentos ambulatoriais, cirúrgicos e de internação. O Hospício de Pedro II foi criado para atender exclusivamente os alienados do Império. O estilo neoclássico e a monumentalidade da edificação o fizeram ser reconhecido como Palácio dos Loucos. O hospício funcionou até 1944 e quatro anos depois a edificação foi cedida à Universidade do Brasil, que adaptou sua arquitetura ao uso educacional. A edificação apresenta estado de conservação regular, com exceção da área central composta pela Capela que está ruim, devido ao incêndio de 2011. O Palácio dos Loucos tornou-se Palácio Universitário, modificando sua identidade através das mudanças que foram feitas em sua arquitetura. O Asilo da Mendicidade foi criado em 1876 para fechar o pentágono asilar. A edificação panóptica buscava a efetiva observação e controle dos internos. A edificação funcionou como Asilo para mendigos até 1920, quando transformou-se em Hospital de São Francisco de Assis. Posteriormente o hospital seria transferido para a Universidade do Brasil, que funcionou como hospital escola até 1978. O Hospital foi desativado e ficou sem uso por dez anos, quando enfim voltou a funcionar como um estabelecimento destinado aos mais pobres. O conjunto da edificação é o que apresenta o pior estado de conservação, considerado de ruim a péssimo. Comprovou-se com essa pesquisa que o mais importante para a preservação das características arquitetônicas e artísticas do bem é a manutenção do uso, seja ele qual for. Os novos usos devem ser adequados também às características e à capacidade da arquitetura em questão. Através de reformas e planos adequados, os hospitais oitocentistas, que hoje se apresentam como Patrimônio Arquitetônico da Saúde, podem manter um uso similar para o qual foi construído, como uma edificação voltada à promoção da saúde da população.

A formação de educadores e a travessia de cercas invisíveis de acesso/produção de conhecimentos : experiências do MST nas inter-relações com universidades brasileiras /

Este estudo sistematiza e aborda a temática da formação de educadores desenvolvida nas inter-relações entre universidades brasileiras e o Movimento dos Trabalhadores Rurais Sem Terra, especificamente nos cursos de graduação em História na Universidade Federal da Paraíba e de Engenharia Agronômica na Universidade Federal de Sergipe, no período de 2004 a 2008. Tal processo desenvolve-se em um contexto difícil e complexo da luta pela reforma agrária no Brasil, principalmente pelas transformações ocorridas nos últimos anos oriundas da ampliação das lógicas de produção do agronegócio. Esta condição leva o MST a discutir e propor uma nova concepção de reforma agrária que a designa de popular em substituição à proposta de reforma agrária clássica. Por outro lado, apresenta uma visão das universidades brasileiras e dos projetos que são construídos e implementados nos últimos anos, inclusive, observando coincidências com as políticas econômicas gerais para a sociedade. Apresenta uma concepção de formação que vem sendo construída no interior das práticas do MST que também procura as universidades para firmar convênios e desenvolver processos de escolarização/formação de seus militantes educadores. Dentre as dimensões desse processo educativo/formativo, destacam-se: vínculo permanente com os processos orgânicos; a formação como um processo ético, estético, místico que trata das atitudes/comportamentos e como um processo dialógico, crítico e articulado que contempla saberes, experiências, em uma interação que busca superar as monoculturas. Captura por intermédio da pesquisa de campo: estranhamentos, entraves, sentidos da ocupação pedagógica e coletiva, alternativas, legados que permanecem tanto no MST como na universidade e apresenta o resultado do envolvimento e atuação dos egressos de ambos os cursos na atualidade. Aponta também para desafios, possibilidades outras de enfrentar a difícil mas necessária tarefa de formar educadores, militantes capazes de coletivamente levar adiante a luta por um mundo mais justo, solidário e democrático, em que a terra e o conhecimento, juntamente com os demais bens econômicos e culturais sejam profundamente democratizados. Pretende ser uma contribuição para o debate acerca da relevância dessas inter-relações entre universidade e movimentos sociais, em que essas experiências demarcam novas possibilidades de abertura e avanços democráticos e menos elitista da universidade e novos patamares de escolarização/formação para integrantes do Movimento dos Sem Terra.

Fenômeno da governança corporativa : um estudo de caso em uma pequena empresa familiar capixaba

Este estudo tem como objetivo compreender a forma com que os mecanismos de governança corporativa interferem na gestão de uma pequena empresa familiar. Para isso, adotaram-se a perspectiva de Hart (1995), que discorre sobre governança corporativa, e de Leone (2005) para empresas familiares. Para alcançar este objetivo, adotou-se, em relação à condução da pesquisa, uma abordagem qualitativa, por meio do método do estudo de caso. A triangulação de dados foi utilizada como instrumento de coleta de dados por meio de pesquisa documental, observação assistemática e entrevista semiestruturada, e a análise de dados foi realizada através da análise de conteúdo. Como contribuição teórica, este estudo amplia o Modelo de Quatro Círculos com Contexto e Sistema de Valores com a introdução de proprietários formais e informais, gerando o Modelo de Cinco Círculos. A presença da governança corporativa foi identificada através dos fatores de diferenciação e da implantação de onze mecanismos de governança que gerou mudanças no controle da empresa, no processo sucessório, na profissionalização e na captação de recursos. Os mecanismos encontrados foram denominados como: “empresa controladora”; “respeito fraternal”; “projetos pessoais”; “pró-labore dos gestores familiares”; “ausência de remuneração dos familiares não gestores”; “aconselhamento profissional”; “prestação de contas”; “proteção do empreendimento familiar”; “alinhamento de interesses na gestão”; “atribuições e responsabilidades”; e “atenção aos interesses dos stakeholders”. Tais mecanismos não possuem ordem cronológica, pois o respeito fraternal e projetos pessoas já existiam antes da criação da empresa familiar. Quanto ao controle, destaca-se o mecanismo prestação de contas, que possibilitou uma ligação entre a família e a empresa; permitiu clareza, transparência, igualdade entre todos os irmãos, diminuindo a assimetria informacional; facilitou uma comunicação aberta e honesta entre todos os proprietários, transmitindo uma sensação de segurança e previsibilidade; e contribuiu para eliminar e/ou minimizar conflitos entre os proprietários (formais e informais). Quanto à sucessão, os mecanismos proteção do empreendimento familiar, aconselhamento profissional, respeito fraternal estão possibilitando planejar o processo sucessório da empresa; facilitaram a comunicação entre os familiares, e assim, diminuiu a assimetria informacional; e realizaram a manutenção e administração dos bens mobiliários da família empresária. Quanto à profissionalização, os mecanismos proteção do empreendimento familiar e respeito fraternal viabilizaram a participação de todos para decidir sobre a profissionalização da empresa. Com relação à captação de recursos, os mecanismos atribuições e responsabilidades e atenção aos interesses dos stakeholders possibilitaram a empresa, durante todo seu ciclo de vida, a buscar recursos financeiros sem dificuldade, gerando um maior investimento, crescimento e geração de empregos