Andreia Amaral

Prospects and challenges for the conservation of farm animal genomic resources, 2015-2025

Livestock conservation practice is changing rapidly in light of policy developments, climate change and diversifying market demands. The last decade has seen a step change in technology and analytical approaches available to define, manage and conserve Farm Animal Genomic Resources (FAnGR). However, these rapid changes pose challenges for FAnGR conservation in terms of technological continuity, analytical capacity and integrative methodologies needed to fully exploit new, multidimensional data. The final conference of the ESF Genomic Resources program aimed to address these interdisciplinary problems in an attempt to contribute to the agenda for research and policy development directions during the coming decade. By 2020, according to the Convention on Biodiversitys Aichi Target 13, signatories should ensure that “…the genetic diversity of …farmed and domesticated animals and of wild relatives …is maintained, and strategies have been developed and implemented for minimizing genetic erosion and safeguarding their genetic diversity.” However, the real extent of genetic erosion is very difficult to measure using current data. Therefore, this challenging target demands better coverage, understanding and utilization of genomic and environmental data, the development of optimized ways to integrate these data with social and other sciences and policy analysis to enable more flexible, evidence-based models to underpin FAnGR conservation. At the conference, we attempted to identify the most important problems for effective livestock genomic resource conservation during the next decade. Twenty priority questions were identified that could be broadly categorized into challenges related to methodology, analytical approaches, data management and conservation. It should be acknowledged here that while the focus of our meeting was predominantly around genetics, genomics and animal science, many of the practical challenges facing conservation of genomic resources are societal in origin and are predicated on the value (e.g., socio-economic and cultural) of these resources to farmers, rural communities and society as a whole. The overall conclusion is that despite the fact that the livestock sector has been relatively well-organized in the application of genetic methodologies to date, there is still a large gap between the current state-of-the-art in the use of tools to characterize genomic resources and its application to many non-commercial and local breeds, hampering the consistent utilization of genetic and genomic data as indicators of genetic erosion and diversity. The livestock genomic sector therefore needs to make a concerted effort in the coming decade to enable to the democratization of the powerful tools that are now at its disposal, and to ensure that they are applied in the context of breed conservation as well as development.

Expression Profile of microRNAs Regulating Proliferation and Differentiation in Mouse Adult Cardiac Stem Cells

The identification of cardiac cells with stem cell properties changed the paradigm of the heart as a post mitotic organ. These cells proliferate and differentiate into cardiomyocytes, endothelial and vascular smooth muscle cells, providing for cardiac cell homeostasis and regeneration. microRNAs are master switches controlling proliferation and differentiation, in particular regulating stem cell biology and cardiac development. Modulation of microRNAs -regulated gene expression networks holds the potential to control cell fate and proliferation, with predictable biotechnologic and therapeutic applications. To obtain insights into the regulatory networks active in cardiac stem cells, we characterized the expression profile of 95 microRNAs with reported functions in stem cell and tissue differentiation in mouse cardiac stem cells, and compared it to that of mouse embryonic heart and mesenchymal stem cells. The most highly expressed microRNAs identified in cardiac stem cells are known to target key genes involved in the control of cell proliferation and adhesion, vascular function and cardiomyocyte differentiation. We report a subset of differentially expressed microRNAs that are proposed to act as regulators of differentiation and proliferation of adult cardiac stem cells, providing novel insights into active gene expression networks regulating their biological properties.

Advances in Modeling of Water Quality in Estuaries

Water quality models are in great demand to complement studies about the status of estuarine waters. However, local models do not perform well when boundary conditions are not properly defined and when biogeochemical processes are not described with adequate detail. This chapter presents advanced modeling applications to perform water quality studies in Portuguese estuaries. Boundary conditions for hydrodynamics and biogeochemistry are provided by the Portuguese Coast Operational Model, downscaled by using nested domains with increasing resolution from the regional to the local scale. The nested models of the estuaries are described, and case studies are presented for specific estuaries to compute sediment transport (Tagus estuary), to calculate residence time of water (Mondego estuary), to forecast quality of bathing waters (Estoril Coast), and to quantify nutrient fluxes between estuaries and the open ocean (Ria de Aveiro). The level of detail used to represent biological processes in water quality models is also addressed, including the description of a case study about modeling of species vulnerable to water quality, such as Zostera noltii in Ria de Aveiro. The need for high level of detail to represent microbial loop and carbon cycle in estuaries is discussed with the application of a complex biological model to the Tagus estuary.

A long journey to effective obesity treatments: is there light at the end of the tunnel?

As the obesity epidemic continues, more Americans are getting fatter, having more weight-related problems such as cardiovascular disease, and are experiencing new metabolic dysfunctions. For over 50 years, the adipose tissue (AT), commonly referred to as fat, has been of interest to academic and clinical scientists, public health officials and individuals interested in body composition and image including much of the average public, athletes, parents, etc. On one hand, efforts to alter body shape, weight and body fat percentage still include bizarre and scientifically unfounded methods. On the other hand, significant new scientific strides have been made in understanding the growth, function and regulation of anatomical and systemic AT. Markers of transition/conversion of precursor cells that mature to form lipid assimilating adipocytes have been identified. Molecular ‘master’ regulators such as peroxisome proliferator-activated receptor gamma and CCAAT-enhancer-binding proteins were uncovered and regulatory mechanisms behind variables of adiposity defined and refined. Interventions including pharmaceutical compounds, surgical, psychosocial interventions have also been tested. Has all of the preceding research helped alleviate the adverse physiologies of overweight and/or obese people? Does research to date point to new modalities that should be the focus of efforts to rid the world of obesity-related problems in the 21st century? This review provides a general overview of scientific efforts to date and a provocative view of the future for adiposity.

Autoimmunity and allergy control in adults submitted to complete thymectomy early in infancy

The contribution of the decline in thymic activity for the emergence of autoimmunity is still debatable. Immune-competent adults submitted to complete thymectomy early in life provide a unique model to address this question. We applied here strict criteria to identify adults lacking thymic activity based on sjTREC levels, to exclude thymic rebound and/or ectopic thymuses. In agreement, they featured severe naïve CD4 T-cell depletion and contraction of T-cell receptor diversity. Notwithstanding this, there was neither increased incidence of autoimmune disease in comparison with age-matched controls nor significant changes in their IgG/IgA/IgM/IgE autoreactivity profiles, as assessed through extensive arrays. We reasoned that the observed relative preservation of the regulatory T-cell compartment, including maintenance of naïve regulatory CD4 T-cells, may contribute to limit the emergence of autoimmunity upon thymectomy. Our findings have implications in other clinical settings with impaired thymic activity, and are particularly relevant to studies of autoimmunity in ageing.

miRNA profiling of human naive CD4 T cells links miR-34c-5p to cell activation and HIV replication

Cell activation is a vital step for T‐cell memory/effector differentiation as well as for productive HIV infection. To identify novel regulators of this process, we used next‐generation sequencing to profile changes in microRNA expression occurring in purified human naive CD4 T cells in response to TCR stimulation and/or HIV infection. Our results demonstrate, for the first time, the transcriptional up‐regulation of miR‐34c‐5p in response to TCR stimulation in naive CD4 T cells. The induction of this miR was further consistently found to be reduced by both HIV‐1 and HIV‐2 infections. Overexpression of miR‐34c‐5p led to changes in the expression of several genes involved in TCR signaling and cell activation, confirming its role as a novel regulator of naive CD4 T‐cell activation. We additionally show that miR‐34c‐5p promotes HIV‐1 replication, suggesting that its down‐regulation during HIV infection may be part of an anti‐viral host response.

Quality assessment and control of tissue specific RNA-seq libraries of Drosophila transgenic RNAi models

RNA-sequencing (RNA-seq) is rapidly emerging as the technology of choice for whole-transcriptome studies. However, RNA-seq is not a bias free technique. It requires large amounts of RNA and library preparation can introduce multiple artifacts, compounded by problems from later stages in the process. Nevertheless, RNA-seq is increasingly used in multiple studies, including the characterization of tissue-specific transcriptomes from invertebrate models of human disease. The generation of samples in this context is complex, involving the establishment of mutant strains and the delicate contamination prone process of dissecting the target tissue. Moreover, in order to achieve the required amount of RNA, multiple samples need to be pooled. Such datasets pose extra challenges due to the large variability that may occur between similar pools, mostly due to the presence of cells from surrounding tissues. Therefore, in addition to standard quality control of RNA-seq data, analytical procedures for control of “biological quality” are critical for successful comparison of gene expression profiles. In this study, the transcriptome of the central nervous system (CNS) of a Drosophila transgenic strain with neuronal-specific RNAi of an ubiquitous gene was profiled using RNA-seq. After observing the existence of an unusual variance in our dataset, we showed that the expression profile of a small panel of marker genes, including GAL4 under control of a tissue specific driver, can identify libraries with low levels of contamination from neighboring tissues, enabling the selection of a robust dataset for differential expression analysis. We further analyzed the potential of profiling a complex tissue to identify cell-type specific changes in response to target gene down-regulation. Finally, we showed that trimming 5′ ends of reads decreases nucleotide frequency biases, increasing the coverage of protein coding genes with a potential positive impact in the incurrence of systematic technical errors.

76PStimulation of RAC1/PAK1 signalling upregulates DNA damage repair genes via the BCL6/STAT5-switch

Center grant UID/MULTI/04046/2013 to BioISI, IF2012 Development grant and PTDC/SAU/GMG/119586/2010 to PM, FCT fellowship SFRH/BPD/94322/2013 to PB and EMBO long-term fellowship ALT-33-2010 and FCT fellowship SFRH/BPD/65976/2009 to AJA.

Large-Scale Transcriptomic Approaches for Characterization of Post-Transcriptional Control of Gene Expression

MicroRNAs are critical regulators of gene expression programs. It has been demonstrated that during the maturation of miRNAs some changes can happen leading to the production of isoforms called isomiRs. During our study, a PERL pipeline was developed to find all the miRs (miRNAs) and isomiRs in two sets of Next Generation Sequencing (NGS) of small RNA libraries derived from naive and activated CD4+ T cells. Then, a differential expression analysis was performed using Bioconductor package DESeq. Our pipeline allowed us to find all the different types of isomiRs in both of conditions. Also, we found that the isoforms coming from changes on 3’ are more frequent that in 5’ ends. Tailing isoforms are described as the less frequent isomiRs. The use of DESeq on the read count dataset of these miRs and isomiRs identified a total of 5 miRs and 22 isomiRs which were differentially expressed. So, in addition to creating a new tool for isomiR analysis, we have been able to obtain evidence that upon activation miRs and isomiRs in T-cells are differentially expressed.