Definição dos mecanismos de polaridade celular durante a citocinese

Dissertação de Mestrado, Oncobiologia – Mecanismos Moleculares do Cancro, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2016

Geographical variation in genetic structure of an Atlantic Coastal Forest frog reveals regional differences in habitat stability

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Stability Predicts Genetic Diversity in the Brazilian Atlantic Forest Hotspot

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Genetic parameters and simultaneous selection for root yield, adaptability and stability of cassava genotypes

O objetivo deste trabalho foi estimar parâmetros genéticos e avaliar a seleção simultânea quanto à produtividade de raízes e à adaptabilidade e estabilidade de genótipos de mandioca. Os efeitos dos genótipos foram considerados como fixos e aleatórios, e a metodologia de modelos mistos (REML/Blup) foi utilizada para estimar os parâmetros genéticos e a média harmônica do desempenho relativo dos valores genotípicos (MHPRVG), para seleção simultânea. Dez genótipos foram avaliados em delineamento de blocos ao acaso, com quatro repetições. O experimento foi realizado nos municípios de Altamira, Santarém e Santa Luzia do Pará, PA, nos anos agrícolas de 2009/2010, 2010/2011 e 2011/2012. As raízes foram colhidas 12 meses após o plantio, em todos os locais testados. A produtividade de raízes apresentou baixo coeficiente de variação genotípica (4,25%) e herdabilidade de parcelas individuais no sentido amplo (0,0424), o que resultou em baixo ganho genético. Em razão da baixa correlação genotípica (0,15), a classificação dos genótipos quanto à produtividade de raízes variou de acordo com o ambiente. Os genótipos CPATU 060, CPATU 229 e CPATU 404 destacaram-se quanto à produtividade, adaptabilidade e estabilidade.

XIPHOPHORUS ENZYME GENE EXPRESSION: TISSUE SPECIFICITY, GENETIC CONTROL, AND STABILITY IN CULTURED CELLS

Five permanent cell lines were developed from Xiphophorus maculatus, X. helleri, and their hybrids using three tissue sources, including adults and embryos of different stages. To evaluate cell line gene expression for retention of either tissue-of-origin-specific or ontogenetic stage-specific characters, the activity distribution of 44 enzyme loci was determined in 11 X. maculatus tissues, and the developmental genetics of 17 enzyme loci was charted in X. helleri and in helleri x maculatus hybrids using starch gel electrophoresis. In the process, eight new loci were discovered and characterized for Xiphophorus.^ No Xiphophorus cell line showed retention of tissue-of-origin-specific or ontogenetic stage-specific enzyme gene expressional traits. Instead, gene expression was similar among the cell lines. One enzyme, adenosine deaminase (ADA) was an exception. Two adult-origin cell lines expressed ADA, whereas, three cell lines derived independently from embryos did not. ADA('-) expression of Xiphophorus embryo-derived cell lines may represent retention of an embryonic gene expressional trait. In one cell line (T(,3)) derived from 13 pooled interspecific hybrid (F(,2)) embryos, shifts with time were observed at enzyme loci polymorphic between the two species. This suggested shifts in ratios of cells of different genotypes in the population rather than unstable gene expression in one dominant cell type.^ Verification of this hypothesis was attempted by cloning the culture--seeking clones having different genetic signatures. The large number of loci electrophoretically polymorphic between the two species and whose alleles segregated independently into the 13 progeny from which this culture originated almost guaranteed the presence of different genetic signatures (lineages) in T(,3).^ Seven lineages of cells were found within T(,3), each expressing genotypes at some loci not characteristic of the expression of the culture-as-a-whole, supporting the hypothesis tested. Quantitative studies of ADA expression in the whole culture (ADA('-)) and in clones of these seven lineages suggested the predominance in T(,3) of ADA deficient cell lineages, although moderate to high ADA output clones also occurred. Thus, T(,3) has the potential to shift phenotypes from ADA('-) to ADA('+) by simply changing proportions of its constituent cell types, demonstrating that such shifts can occur in any cell culture containing cells of mixed expressional characteristics.^

A genetic algorithm for slope stability analyses with concave slip surfaces using custom operators

Heuristic methods are popular tools to find critical slip surfaces in slope stability analyses. A new genetic algorithm (GA) is proposed in this work that has a standard structure but a novel encoding and generation of individuals with custom-designed operators for mutation and crossover that produce kinematically feasible slip surfaces with a high probability. In addition, new indices to assess the efficiency of operators in their search for the minimum factor of safety (FS) are proposed. The proposed GA is applied to traditional benchmark examples from the literature, as well as to a new practical example. Results show that the proposed GA is reliable, flexible and robust: it provides good minimum FS estimates that are not very sensitive to the number of nodes and that are very similar for different replications

Genetic interactions between ATM and the nonhomologous end-joining factors in genomic stability and development

DNA ligase IV (Lig4) and the DNA-dependent protein kinase (DNA-PK) function in nonhomologous end joining (NHEJ). However, although Lig4 deficiency causes late embryonic lethality, deficiency in DNA-PK subunits (Ku70, Ku80, and DNA-PKcs) does not. Here we demonstrate that, similar to p53 deficiency, ataxia-telangiectasia-mutated (ATM) gene deficiency rescues the embryonic lethality and neuronal apoptosis, but not impaired lymphocyte development, associated with Lig4 deficiency. However, in contrast to p53 deficiency, ATM deficiency enhances deleterious effects of Lig4 deficiency on growth potential of embryonic fibroblasts (MEFs) and genomic instability in both MEFs and cultured progenitor lymphocytes, demonstrating significant differences in the interplay of p53 vs. ATM with respect to NHEJ. Finally, in dramatic contrast to effects on Lig4 deficiency, ATM deficiency causes early embryonic lethality in Ku- or DNA-PKcs-deficient mice, providing evidence for an NHEJ-independent role for the DNA-PK holoenzyme.

Sociometric stability and the behavioral correlates of peer acceptance in early childhood

This paper presents findings from an Australian study examining the behavioral correlates and stability of social status for preschool-aged children. The social status of an initial sample of 187 (94 boys and 93 girls) preschool children (mean age 62.4 months, SD = 4.22) was determined through sociometric assessment. Children classified as rejected, neglected and popular (n = 70) were selected for observation. Children were observed for a total of 25 minutes over a three-month period engaging in free play within their preschool centers. Results indicated that children classified as popular were more likely than rejected or neglected children to engage in cooperative play, ongoing connected conversation and to display positive affect. Popular children were less likely than rejected or neglected children to engage in parallel play, onlooker behavior or alone directed behavior. Six months after initial sociometric classification, sociometric interviews were repeated to test for stability and change. Results indicated that preschool-aged children’s social status classifications showed a moderate to high rate of stability for those children classified as popular, rejected and neglected.

Spatial population genetic structure reveals strong natal site fidelity in Echinocladius martini (Diptera: Chironomidae) in northeast Queensland, Australia

1. A diverse array of patterns has been reported regarding the spatial extent of population genetic structure and effective dispersal in freshwater macroinvertebrates. In river systems, the movements of many taxa can be restricted to varying degrees by the natural stream channel hierarchy. 2. In this study, we sampled populations of the non-biting freshwater midge Echinocladius martini in the Paluma bioregion of tropical northeast Queensland to investigate fine scale patterns of within- and among-stream dispersal and gene flow within a purported historical refuge. We amplified a 639 bp fragment of mitochondrial COI and analysed genetic structure using pairwise ΦST, hierarchical AMOVA, Mantel tests and a parsimony network. Genetic variation was partitioned among stream sections using Streamtree to investigate the effect of potential instream dispersal barriers. 3. The data revealed strong natal site fidelity and significant differentiation among neighbouring, geographically proximate streams. We found evidence for only episodic adult flight among sites on separate stream reaches. Overall, however, our data suggested that both larval and adult dispersal was largely limited to within a stream channel. 4. This may arise from a combination of the high density of riparian vegetation physically restricting dispersal and from the joint effects of habitat stability and large population sizes. Together these may mitigate the requirement for movement among streams to avoid inbreeding and local extinction due to habitat change and may thus enable persistence of upstream populations in the absence of regular compensatory upstream flight. Taken together, these data suggest that dispersal of E. martini is highly restricted, to the scale of only a few kilometres, and hence occurs predominantly within the natal stream.

Human single-stranded DNA binding proteins are essential for maintaining genomic stability

The double-stranded conformation of cellular DNA is a central aspect of DNA stabilisation and protection. The helix preserves the genetic code against chemical and enzymatic degradation, metabolic activation, and formation of secondary structures. However, there are various instances where single-stranded DNA is exposed, such as during replication or transcription, in the synthesis of chromosome ends, and following DNA damage. In these instances, single-stranded DNA binding proteins are essential for the sequestration and processing of single-stranded DNA. In order to bind single-stranded DNA, these proteins utilise a characteristic and evolutionary conserved single-stranded DNA-binding domain, the oligonucleotide/oligosaccharide-binding (OB)-fold. In the current review we discuss a subset of these proteins involved in the direct maintenance of genomic stability, an important cellular process in the conservation of cellular viability and prevention of malignant transformation. We discuss the central roles of single-stranded DNA binding proteins from the OB-fold domain family in DNA replication, the restart of stalled replication forks, DNA damage repair, cell cycle-checkpoint activation, and telomere maintenance.

Reduced excision repair cross-complementing 1 expression associates with enhanced papilloma formation and fibroblast transformation after genetic disruption of secreted protein acidic and rich in cysteine

SPARC (secreted protein acidic and rich in cysteine)/ osteonectin/BM-40 is a matricellular protein implicated in development, cell transformation and tumorigenesis. We have examined the role of SPARC in cell transformation induced chemically with 7,12-dimethylbenz[a]anthracene (DMBA) and 12- tetradecanoylphorbol-13-acetate (TPA) in embryonic fibroblasts and in the skin of mice. Embryonic fibroblasts from SPARCnull mice showed increases in cell proliferation, enhanced sensitivity to DMBA and a higher number of DMBA/TPA-induced transformation foci. The number of DMBA-DNA adducts was 9 times higher in SPARCnull fibroblasts and their stability was lower than wild-type fibroblasts, consistent with a reduction in excision repair cross-complementing 1 the nucleotide excision repair enzyme in these cells. The SPARCnull mice showed an increase in both the speed and number of papillomas forming after topical administration of DMBA/TPA to the skin. These papillomas showed reduced growth and reduced progression to a more malignant phenotype, indicating that the effect of SPARC on tumorigenesis depends upon the transformation stage and/or tissue context. These data reinforce a growing number of observations in which SPARC has shown opposite effects on different tumor types/stages.

Diffusion imaging protocol effects on genetic associations

Large multi-site image-analysis studies have successfully discovered genetic variants that affect brain structure in tens of thousands of subjects scanned worldwide. Candidate genes have also associated with brain integrity, measured using fractional anisotropy in diffusion tensor images (DTI). To evaluate the heritability and robustness of DTI measures as a target for genetic analysis, we compared 417 twins and siblings scanned on the same day on the same high field scanner (4-Tesla) with two protocols: (1) 94-directions; 2mm-thick slices, (2) 27-directions; 5mm-thickness. Using mean FA in white matter ROIs and FA skeletons derived using FSL, we (1) examined differences in voxelwise means, variances, and correlations among the measures; and (2) assessed heritability with structural equation models, using the classical twin design. FA measures from the genu of the corpus callosum were highly heritable, regardless of protocol. Genome-wide analysis of the genu mean FA revealed differences across protocols in the top associations.

Super DNAging - New insights into DNA integrity, genome stability and telomeres in the oldest old

Reductions in DNA integrity, genome stability, and telomere length are strongly associated with the aging process, age-related diseases as well as the age-related loss of muscle mass. However, in people reaching an age far beyond their statistical life expectancy the prevalence of diseases, such as cancer, cardiovascular disease, diabetes or dementia, is much lower compared to “averagely” aged humans. These inverse observations in nonagenarians (90–99 years), centenarians (100–109 years) and super-centenarians (110 years and older) require a closer look into dynamics underlying DNA damage within the oldest old of our society. Available data indicate improved DNA repair and antioxidant defense mechanisms in “super old” humans, which are comparable with much younger cohorts. Partly as a result of these enhanced endogenous repair and protective mechanisms, the oldest old humans appear to cope better with risk factors for DNA damage over their lifetime compared to subjects whose lifespan coincides with the statistical life expectancy. This model is supported by study results demonstrating superior chromosomal stability, telomere dynamics and DNA integrity in “successful agers”. There is also compelling evidence suggesting that life-style related factors including regular physical activity, a well-balanced diet and minimized psycho-social stress can reduce DNA damage and improve chromosomal stability. The most conclusive picture that emerges from reviewing the literature is that reaching “super old” age appears to be primarily determined by hereditary/genetic factors, while a healthy lifestyle additionally contributes to achieving the individual maximum lifespan in humans. More research is required in this rapidly growing population of super old people. In particular, there is need for more comprehensive investigations including short- and long-term lifestyle interventions as well as investigations focusing on the mechanisms causing DNA damage, mutations, and telomere shortening.

Genetic, functional, and histopathological evaluation of two C-terminal BRCA1 missense variants

Background: The vast majority of BRCA1 missense sequence variants remain uncharacterised for their possible effect on protein expression and function, and therefore are unclassified in terms of their pathogenicity. BRCA1 plays diverse cellular roles and it is unlikely that any single functional assay will accurately reflect the total cellular implications of missense mutations in this gene. Objective: To elucidate the effect of two BRCA1 variants, 5236G>C (G1706A) and 5242C>A (A1708E) on BRCA1 function, and to survey the relative usefulness of several assays to direct the characterisation of other unclassified variants in BRCA genes. Methods and Results: Data from a range of bioinformatic, genetic, and histopathological analyses, and in vitro functional assays indicated that the 1708E variant was associated with the disruption of different cellular functions of BRCA1. In transient transfection experiments in T47D and 293T cells, the 1708E product was mislocalised to the cytoplasm and induced centrosome amplification in 293T cells. The 1708E variant also failed to transactivate transcription of reporter constructs in mammalian transcriptional transactivation assays. In contrast, the 1706A variant displayed a phenotype comparable to wildtype BRCA1 in these assays. Consistent with functional data, tumours from 1708E carriers showed typical BRCA1 pathology, while tumour material from 1706A carriers displayed few histopathological features associated with BRCA1 related tumours. Conclusions: A comprehensive range of genetic, bioinformatic, and functional analyses have been combined for the characterisation of BRCA1 unclassified sequence variants. Consistent with the functional analyses, the combined odds of causality calculated for the 1706A variant after multifactorial likelihood analysis (1:142) indicates a definitive classification of this variant as "benign". In contrast, functional assays of the 1708E variant indicate that it is pathogenic, possibly through subcellular mislocalisation. However, the combined odds of 262:1 in favour of causality of this variant does not meet the minimal ratio of 1000:1 for classification as pathogenic, and A1708E remains formally designated as unclassified. Our findings highlight the importance of comprehensive genetic information, together with detailed functional analysis for the definitive categorisation of unclassified sequence variants. This combination of analyses may have direct application to the characterisation of other unclassified variants in BRCA1 and BRCA2.