An siRNA-based functional genomics screen for the identification of regulators of ciliogenesis and ciliopathy genes

<p>Defects in primary cilium biogenesis underlie the ciliopathies, a growing group of genetic disorders. We describe a whole-genome siRNA-based reverse genetics screen for defects in biogenesis and/or maintenance of the primary cilium, obtaining a global resource. We identify 112 candidate ciliogenesis and ciliopathy genes, including 44 components of the ubiquitin-proteasome system, 12 G-protein-coupled receptors, and 3 pre-mRNA processing factors (PRPF6, PRPF8 and PRPF31) mutated in autosomal dominant retinitis pigmentosa. The PRPFs localize to the connecting cilium, and PRPF8- and PRPF31-mutated cells have ciliary defects. Combining the screen with exome sequencing data identified recessive mutations in PIBF1, also known as CEP90, and C21orf2, also known as LRRC76, as causes of the ciliopathies Joubert and Jeune syndromes. Biochemical approaches place C21orf2 within key ciliopathy-associated protein modules, offering an explanation for the skeletal and retinal involvement observed in individuals with C21orf2 variants. Our global, unbiased approaches provide insights into ciliogenesis complexity and identify roles for unanticipated pathways in human genetic disease.</p>

Application of SNPs for population genetics of nonmodel organisms:new opportunities and challenges

<p>Recent improvements in the speed, cost and accuracy of next generation sequencing are revolutionizing the discovery of single nucleotide polymorphisms (SNPs). SNPs are increasingly being used as an addition to the molecular ecology toolkit in nonmodel organisms, but their efficient use remains challenging. Here, we discuss common issues when employing SNP markers, including the high numbers of markers typically employed, the effects of ascertainment bias and the inclusion of nonneutral loci in a marker panel. We provide a critique of considerations specifically associated with the application and population genetic analysis of SNPs in nonmodel taxa, focusing specifically on some of the most commonly applied methods.</p>

Population genetics of C. Maenas : oceanography and larval dispersal

Decifrar a complexa interac����o entre os ciclos de vida de esp��cies marinhas e a oceanografia revela-se fundamental para a compreens��o do fluxo gen��tico e da conectividade no meio marinho. Nas esp��cies marinhas com desenvolvimento indirecto o fluxo de genes entre popula����es depende da dist��ncia que separa as popula����es, bem como da interac����o entre a dura����o do desenvolvimento larvar, do comportamento das larvas e dos padr��es de circula����o oce��nica. A conectividade larvar influencia uma variedade de processos como a din��mica de stocks e de popula����es, a distribui����o e limites geogr��ficos das esp��cies, a estrutura gen��tica das popula����es e a dispers��o de esp��cies invasivas e reveste-se consequentemente de uma import��ncia fundamental na identifica����o das unidades populacionais evolucionariamente relevantes e para a gest��o e conserva����o marinhas. Os marcadores gen��ticos e os Modelos Individuais Acoplados a Modelos F��sico-Biol��gicos (���ICPBMs���) s��o actualmente ferramentas fundamentais para o estudo dos padr��es de dispers��o larvar e para avaliar o n��vel de conectividade populacional. A presente tese respeita �� avalia����o das escalas espaciais de conectividade de popula����es de uma esp��cie costeira, o caranguejo Carcinus maenas, e utiliza conjuntamente informa����o de marcadores gen��ticos, an��lise de s��ries temporais de fornecimento de larvas e um modelo num��rico de circula����o oce��nica. O primeiro cap��tulo introduz a tem��tica da conectividade em esp��cies marinhas e inclui algumas refer��ncias aos m��todos moleculares, anal��ticos e de modela����o seguidos ao longo da tese. Atrav��s da utiliza����o de m��ltiplas ferramentas ��� avalia����o da estrutura gen��tica geogr��fica de C. maenas na sua distribui����o nativa com recurso a marcadores de DNA (microssat��lites) (Cap��tulo 2), avalia����o da estrutura gen��tica temporal das larvas que formam os eventos de fornecimento larvar �� Ria de Aveiro, NW Portugal (Cap��tulo 3), descri����o da variabilidade inter-anual do fornecimento larvar �� Ria de Aveiro, NW Portugal (Cap��tulo 4) e valida����o de um modelo ICPBM que descreve os padr��es observados de fornecimento (Cap��tulo 5) ��� esta tese espera poder contribuir para uma melhor compreens��o dos mecanismos que regulam o fluxo de genes e a conectividade entre popula����es de organismos marinhos. No Cap��tulo 6 s��o apresentadas as principais conclus��es da investiga����o. A an��lise gen��tica com recurso a microssat��lites indicou que as popula����es de C. maenas s��o geneticamente homog��neas ao longo de v��rias centenas de km, dentro da distribui����o nativa da esp��cie. Paralelamente, n��o foram encontrados ind��cios da exist��ncia de reprodu����o por ���sweepstakes��� em C. maenas de popula����es da costa oeste da Pen��nsula Ib��rica, visto que n��o se obtiveram diferen��as gen��ticas significativas entre os eventos larvares. Tamb��m n��o se encontrou qualquer estrutura familiar entre as larvas que formam cada epis��dio de fornecimento, e n��o houve nenhuma redu����o significativa da variabilidade gen��tica das larvas quando comparada com a de caranguejos adultos. A an��lise de s��ries temporais de suprimento de larvas na Ria de Aveiro em cinco anos estudados indica que este �� um fen��meno epis��dico e vari��vel, sendo os maiores epis��dios de fornecimento coincidentes com as mar��s vivas e acentuados por fortes ventos de sul. O modelo ICPBM foi validado com sucesso e parece fornecer uma estimativa real��stica das escalas espaciais e temporais de dispers��o larvar, de acordo com as observa����es da estrutura gen��tica e da aus��ncia de reprodu����o por ���sweepstake��� em C. maenas da costa oeste da Pen��nsula Ib��rica

Seasonal variations in gross biochemical composition, percent edibility, and condition index of the clam Ruditapes Decussatus cultivated in the Ria Formosa (South Portugal)

The grooved carpet shell clam, Ruditapes decussatus (L. 1758), is one of the most popular and profitable molluscs exploited in rearing plots in the Mediterranean. However, annual catch has been declining steadily since the early nineties. In order to understand the seasonality of its nutritional value, thus providing an improved basis for economical valuation of the resource, gross biochemical composition, percentage edibility and condition index were investigated during a year with monthly periodicity in a commercially exploited population of the clam Ruditapes decussatus in the Ria Formosa, a temperate mesotidal coastal lagoon located in the south of Portugal. Our results show that total and non-protein nitrogen co-varied during the year, resulting in a protein content that peaked in the warmest months. Although complementary in summer, carbohydrate and lipid contents showed irregular annual trends. The observed seasonality was comparable to that shown by studies elsewhere at similar latitudes, and are underpinned by the reproductive cycle of the species. Our results show the clams to be at their prime nutritional value at the beginning of summer, when protein content peaks.

Ecological genetics in the North Atlantic: environmental gradients and adaptation at specific loci

The North Atlantic intertidal community provides a rich set of organismal and environmental material for the study of ecological genetics. Clearly defined environmental gradients exist at multiple spatial scales: there are broad latitudinal trends in temperature, meso-scale changes in salinity along estuaries, and smaller scale gradients in desiccation and temperature spanning the intertidal range. The geology and geography of the American and European coasts provide natural replication of these gradients, allowing for population genetic analyses of parallel adaptation to environmental stress and heterogeneity. Statistical methods have been developed that provide genomic neutrality tests of population differentiation and aid in the process of candidate gene identification. In this paper, we review studies of marine organisms that illustrate associations between an environmental gradient and specific genetic markers. Such highly differentiated markers become candidate genes for adaptation to the environmental factors in question, but the functional significance of genetic variants must be comprehensively evaluated. We present a set of predictions about locus-specific selection across latitudinal, estuarine, and intertidal gradients that are likely to exist in the North Atlantic. We further present new data and analyses that support and contradict these simple selection models. Some taxa show pronounced clinal variation at certain loci against a background of mild clinal variation at many loci. These cases illustrate the procedures necessary for distinguishing selection driven by internal genomic vs. external environmental factors. We suggest that the North Atlantic intertidal community provides a model system for identifying genes that matter in ecology due to the clarity of the environmental stresses and an extensive experimental literature on ecological function. While these organisms are typically poor genetic and genomic models, advances in comparative genomics have provided access to molecular tools that can now be applied to taxa with well-defined ecologies. As many of the organisms we discuss have tight physiological limits driven by climatic factors, this synthesis of molecular population genetics with marine ecology could provide a sensitive means of assessing evolutionary responses to climate change.

Biochemical indices and life traits of loggerhead turtles (Caretta caretta) from Cape Verde Islands

The loggerhead turtle (Caretta caretta) is an endangered marine reptile for whom assessing population health requires knowledge of demographic parameters such as individual growth rate. In Cape Verde, as within several populations, adult female loggerhead sea turtles show a size-related behavioral and trophic dichotomy. While smaller females are associated with oceanic habitats, larger females tend to feed in neritic habitats, which is reflected in their physiological condition and in their offspring. The ratio of RNA/DNA provides a measure of cellular protein synthesis capacity, which varies depending on changes in environmental conditions such as temperature and food availability. The purpose of this study was to evaluate the combined use of morphometric data and biochemical indices as predictors of the physiological condition of the females of distinct sizes and hatchlings during their nesting season and how temperature may influence the physiological condition on the offspring. Here we employed biochemical indices based on nucleic acid derived indices (standardized RNA/DNA ratio-sRD, RNA concentration and DNA concentration) in skin tissue as a potential predictor of recent growth rate in nesting females and hatchling loggerhead turtles. Our major findings were that the physiological condition of all nesting females (sRD) decreased during the nesting season, but that females associated with neritic habitats had a higher physiological condition than females associated with oceanic habitats. In addition, the amount of time required for a hatchling to right itself was negatively correlated with its physiological condition (sRD) and shaded nests produced hatchlings with lower sRD. Overall, our results showed that nucleic acid concentrations and ratios of RNA to DNA are an important tool as potential biomarkers of recent growth in marine turtles. Hence, as biochemical indices of instantaneous growth are likely temperature-, size- and age-dependent, the utility and validation of these indices on marine turtles stocks deserves further study.

Landscape genetics of a seagrass species in a tidal mudflat lagoon

Tese de doutoramento, Ci��ncias do Mar, da Terra e do Ambiente, Faculdade de Ci��ncias e Tecnologia, Universidade do Algarve, 2015

Population and landscape genetics of the stone marten and red fox in Portugal : implications for conservation management of common carnivores

Tese de doutoramento, Biologia (Biologia da Conserva����o), Universidade de Lisboa, Faculdade de Ci��ncias, 2015