Phylogeography and non-invasive landscape genetics of the European pine marten ("martes martes" L. 1758) : insights into ancient and contemporary processes shaping genetic variation

283 p. : graf., map.

New genetic markers for treatment personalization in pediatric acute lymphoblastic leukemia

La Leucemia Linfoblástica Aguda (LLA) es el cáncer pediátrico más común. Es un desorden de las células linfoblásticas, que son las precursoras de las células linfáticas, y se caracteriza por la acumulación en médula ósea y sangre de pequeñas células blásticas con poco citoplasma y cromatina dispersa. En las últimas décadas, se ha conseguido aumentar la supervivencia del 10% al 80% pero todavía hay un 20% de pacientes que no responden al tratamiento. Esta mejoría se ha conseguido mediante la implantación de terapias combinadas y la adecuación de la terapia a grupos de riesgo. Los pacientes se separan en tres grupos de riesgo, Riesgo Estándar (RE), Alto Riesgo (AR) y Muy Alto Riesgo (MAR), en base a marcadores pronósticos, entre los que se incluyen alteraciones citogenéticas. Sin embargo, a lo largo del tratamiento, nos encontramos con dos problemas:1) Por un lado, algunos de los pacientes incluidos en el grupo de RE y AR no responden bien al tratamiento y pasan AR y MAR respectivamente. Esto quiere decir que los grupos de riesgo no están bien definidos. Por lo tanto, sería de interés poder caracterizar los pacientes que realmente son RE y AR y aquéllos que desde un principio deberían haber sido considerados como de mayor riesgo.2) Por otro lado, un alto porcentaje de pacientes experimenta toxicidad, que puede llegar a ser muy grave en algunos casos, siendo necesario parar el tratamiento. Por este motivo, sería altamente beneficioso poder reconocer a los pacientes que van a ser más sensibles al tratamiento para, de ese modo, poder ajustar la dosis.Por todo esto, creemos que una mejor asignación de los pacientes de LLA a grupos de riesgo y la personalización de la dosis, mediante nuevos marcadores genéticos, permitiría mejorar la respuesta al tratamiento.En este estudio nos planteamos, por lo tanto, dos objetivos: 1) Llevar a cabo la identificación de nuevas alteraciones genéticas presentes en el tumor para una mejor caracterización del riesgo y 2) Realizar una caracterización genética del individuo que permita predecir la respuesta al tratamiento.

Design,optimization and in vivo evaluation of non-viral vectors for gene therapy. Applications in ocular disorders

227 págs.

Multi-drug resistance profiles and the genetic features of Acinetobacter baumannii isolates from Bolivia

Introduction: Acinetobacter baumannii is opportunistic in debilitated hospitalised patients. Because information from some South American countries was previously lacking, this study examined the emergence of multi-resistant A. baumannii in three hospitals in Cochabamba, Bolivia, from 2008 to 2009. Methodology: Multiplex PCR was used to identify the main resistance genes in 15 multi-resistant A. baumannii isolates. RT-PCR was used to measure gene expression. The genetic environment of these genes was also analysed by PCR amplification and sequencing. Minimum inhibitory concentrations were determined for key antibiotics and some were determined in the presence of an efflux pump inhibitor, 1-(1-napthylmethyl) piperazine. Results: Fourteen strains were found to be multi-resistant. Each strain was found to have the bla(OXA-58) gene with the ISAba3-like element upstream, responsible for over-expression of the latter and subsequent carbapenem resistance. Similarly, ISAba1, upstream of the bla(ADC) gene caused over-expression of the latter and cephalosporin resistance; mutations in the gyrA(Ser83 to Leu) and parC (Ser-80 to Phe) genes were commensurate with fluoroquinolone resistance. In addition, the adeA, adeB efflux genes were over-expressed. All 15 isolates were positive for at least two aminoglycoside resistance genes. Conclusion: This is one of the first reports analyzing the multi-drug resistance profile of A. baumannii strains isolated in Bolivia and shows that the over-expression of thebla(OXA-58), bla(ADC) and efflux genes together with aminoglycoside modifying enzymes and mutations in DNA topoisomerases are responsible for the multi-resistance of the bacteria and the subsequent difficulty in treating infections caused by them.

Framework for the Shape Optimization of Aerodynamic Profiles Using Genetic Algorithms

This study developed a framework for the shape optimization of aerodynamics profiles using computational fluid dynamics (CFD) and genetic algorithms. Agenetic algorithm code and a commercial CFD code were integrated to develop a CFD shape optimization tool. The results obtained demonstrated the effectiveness of the developed tool. The shape optimization of airfoils was studied using different strategies to demonstrate the capacity of this tool with different GA parameter combinations.

The Expanded mtDNA Phylogeny of the Franco-Cantabrian Region Upholds the Pre-Neolithic Genetic Substrate of Basques

9 p.

Epitope mapping of antibodies to alpha-synuclein in LRRK2 mutation carriers, idiopathic Parkinson disease patients, and healthy controls

Alpha-synuclein (Snca) plays a major role in Parkinson disease (PD). Circulating anti-Snca antibodies has been described in PD patients and healthy controls, but they have been poorly characterized. This study was designed to assess the prevalence of anti-Snca reactivity in human subjects carrying the LRRK2 mutation, idiopathic PD (iPD) patients, and healthy controls and to map the epitopes of the anti-Snca antibodies. Antibodies to Snca were detected by ELISA and immunoblotting using purified recombinant Snca in plasma from individuals carrying LRRK2 mutations (104), iPD patients (59), and healthy controls (83). Epitopes of antibodies were mapped using recombinant protein constructs comprising different regions of Snca. Clear positive anti-Snca reactivity showed no correlation with age, sex, years of evolution, or the disability scores for PD patients and anti-Snca reactivity was not prevalent in human patients with other neurological or autoimmune diseases. Thirteen of the positive individuals were carriers of LRRK2 mutations either non-manifesting (8 out 49 screened) or manifesting (5 positive out 55), three positive (out of 59) were iPD patients, and five positive (out of 83) were healthy controls. Epitope mapping showed that antibodies against the N-terminal (a.a. 1-60) or C-terminal (a.a. 109-140) regions of Snca predominate in LRRK2 mutation carriers and iPD patients, being N122 a critical amino acid for recognition by the anti-C-terminal directed antibodies. Anti-Snca circulating antibodies seem to cluster within families carrying the LRRK2 mutation indicating possible genetic or common environmental factors in the generation of anti-Snca antibodies. These results suggest that case-controls' studies are insufficient and further studies in family cohorts of patients and healthy controls should be undertaken, to progress in the understanding of the possible relationship of anti-Snca antibodies and PD patholog

The Carpathian range represents a weak genetic barrier in South-East Europe

Background: In the present study we have assessed whether the Carpathian Mountains represent a genetic barrier in East Europe. Therefore, we have analyzed the mtDNA of 128 native individuals of Romania: 62 of them from the North of Romania, and 66 from South Romania. Results: We have analyzed their mtDNA variability in the context of other European and Near Eastern populations through multivariate analyses. The results show that regarding the mtDNA haplogroup and haplotype distributions the Romanian groups living outside the Carpathian range (South Romania) displayed some degree of genetic differentiation compared to those living within the Carpahian range (North Romania). Conclusion: The main differentiation between the mtDNA variability of the groups from North and South Romania can be attributed to the demographic movements from East to West (prehistoric or historic) that differently affected in these regions, suggesting that the Carpathian mountain range represents a weak genetic barrier in South-East Europe.

Analysis and evaluation of public health policy in Cuba : progress on millennium development goals

[EN] The main objective of this project is to analyze Cuban public health policy and the Millennium Development Goals, especially those linked to the issue of health, presenting their potential and strengths with a well-defined time horizon (2000-2015). The Millennium Development Goals are the international consensus on development and was signed as an international minimum agreement, with which began the century. The MDGs promote various goals and targets, with the corresponding monitoring indicators, which should be achieved by all countries for the present year. Health is an area that is at the center of the Millennium Development Goals, which reinforce each other to get a true human development itself. The research was done through theoretical frameworks of social production of health and disease, social justice and the power structure. A retrospective analysis of Cuban economic and social context is performed in order to study whether health-related MDGs are likely to be completed by the deadline on the island and likewise, the main parameters related to health compared with those of the neighboring countries in the Americas.

The MLH1 c.1852_1853delinsGC (p.K618A) Variant in Colorectal Cancer: Genetic Association Study in 18,723 Individuals

Colorectal cancer is one of the most frequent neoplasms and an important cause of mortality in the developed world. Mendelian syndromes account for about 5% of the total burden of CRC, being Lynch syndrome and familial adenomatous polyposis the most common forms. Lynch syndrome tumors develop mainly as a consequence of defective DNA mismatch repair associated with germline mutations in MLH1, MSH2, MSH6 and PMS2. A significant proportion of variants identified by screening these genes correspond to missense or noncoding changes without a clear pathogenic consequence, and they are designated as "variants of uncertain significance'', being the c.1852_1853delinsGC (p.K618A) variant in the MLH1 gene a clear example. The implication of this variant as a low-penetrance risk variant for CRC was assessed in the present study by performing a case-control study within a large cohort from the COGENT consortium-COST Action BM1206 including 18,723 individuals (8,055 colorectal cancer cases and 10,668 controls) and a case-only genotype-phenotype correlation with several clinical and pathological characteristics restricted to the Epicolon cohort. Our results showed no involvement of this variant as a low-penetrance variant for colorectal cancer genetic susceptibility and no association with any clinical and pathological characteristics including family history for this neoplasm or Lynch syndrome.

Characterisation of patients with brachydactyly type E associated or not with hormonal resistance: clinical, genetic and radiological approach

334 p.

Inferring Population Genetic Structure in Widely and Continuously Distributed Carnivores: The Stone Marten (Martes foina) as a Case Study

The stone marten is a widely distributed mustelid in the Palaearctic region that exhibits variable habitat preferences in different parts of its range. The species is a Holocene immigrant from southwest Asia which, according to fossil remains, followed the expansion of the Neolithic farming cultures into Europe and possibly colonized the Iberian Peninsula during the Early Neolithic (ca. 7,000 years BP). However, the population genetic structure and historical biogeography of this generalist carnivore remains essentially unknown. In this study we have combined mitochondrial DNA (mtDNA) sequencing (621 bp) and microsatellite genotyping (23 polymorphic markers) to infer the population genetic structure of the stone marten within the Iberian Peninsula. The mtDNA data revealed low haplotype and nucleotide diversities and a lack of phylogeographic structure, most likely due to a recent colonization of the Iberian Peninsula by a few mtDNA lineages during the Early Neolithic. The microsatellite data set was analysed with a) spatial and non-spatial Bayesian individual-based clustering (IBC) approaches (STRUCTURE, TESS, BAPS and GENELAND), and b) multivariate methods [discriminant analysis of principal components (DAPC) and spatial principal component analysis (sPCA)]. Additionally, because isolation by distance (IBD) is a common spatial genetic pattern in mobile and continuously distributed species and it may represent a challenge to the performance of the above methods, the microsatellite data set was tested for its presence. Overall, the genetic structure of the stone marten in the Iberian Peninsula was characterized by a NE-SW spatial pattern of IBD, and this may explain the observed disagreement between clustering solutions obtained by the different IBC methods. However, there was significant indication for contemporary genetic structuring, albeit weak, into at least three different subpopulations. The detected subdivision could be attributed to the influence of the rivers Ebro, Tagus and Guadiana, suggesting that main watercourses in the Iberian Peninsula may act as semi-permeable barriers to gene flow in stone martens. To our knowledge, this is the first phylogeographic and population genetic study of the species at a broad regional scale. We also wanted to make the case for the importance and benefits of using and comparing multiple different clustering and multivariate methods in spatial genetic analyses of mobile and continuously distributed species.

Patterns of genetic variation in the endangered European mink (Mustela lutreola L., 1761)

Background: The European mink (Mustela lutreola, L. 1761) is a critically endangered mustelid, which inhabits several main river drainages in Europe. Here, we assess the genetic variation of existing populations of this species, including new sampling sites and additional molecular markers (newly developed microsatellite loci specific to European mink) as compared to previous studies. Probabilistic analyses were used to examine genetic structure within and between existing populations, and to infer phylogeographic processes and past demography. Results: According to both mitochondrial and nuclear microsatellite markers, Northeastern (Russia, Estonia and Belarus) and Southeastern (Romania) European populations showed the highest intraspecific diversity. In contrast, Western European (France and Spain) populations were the least polymorphic, featuring a unique mitochondrial DNA haplotype. The high differentiation values detected between Eastern and Western European populations could be the result of genetic drift in the latter due to population isolation and reduction. Genetic differences among populations were further supported by Bayesian clustering and two main groups were confirmed (Eastern vs. Western Europe) along with two contained subgroups at a more local scale (Northeastern vs. Southeastern Europe; France vs. Spain). Conclusions: Genetic data and performed analyses support a historical scenario of stable European mink populations, not affected by Quaternary climate oscillations in the Late Pleistocene, and posterior expansion events following river connections in both North-and Southeastern European populations. This suggests an eastern refuge during glacial maxima (as already proposed for boreal and continental species). In contrast, Western Europe was colonised more recently following either natural expansions or putative human introductions. Low levels of genetic diversity observed within each studied population suggest recent bottleneck events and stress the urgent need for conservation measures to counteract the demographic decline experienced by the European mink.

Exploring Genetic Factors Involved in Huntington Disease Age of Onset: E2F2 as a New Potential Modifier Gene

Age of onset (AO) of Huntington disease (HD) is mainly determined by the length of the CAG repeat expansion (CAGexp) in exon 1 of the HTT gene. Additional genetic variation has been suggested to contribute to AO, although the mechanism by which it could affect AO is presently unknown. The aim of this study is to explore the contribution of candidate genetic factors to HD AO in order to gain insight into the pathogenic mechanisms underlying this disorder. For that purpose, two AO definitions were used: the earliest age with unequivocal signs of HD (earliest AO or eAO), and the first motor symptoms age (motor AO or mAO). Multiple linear regression analyses were performed between genetic variation within 20 candidate genes and eAO or mAO, using DNA and clinical information of 253 HD patients from REGISTRY project. Gene expression analyses were carried out by RT-qPCR with an independent sample of 35 HD patients from Basque Country Hospitals. We found suggestive association signals between HD eAO and/or mAO and genetic variation within the E2F2, ATF7IP, GRIN2A, GRIN2B, LINC01559, HIP1 and GRIK2 genes. Among them, the most significant was the association between eAO and rs2742976, mapping to the promoter region of E2F2 transcription factor. Furthermore, rs2742976 T allele patient carriers exhibited significantly lower lymphocyte E2F2 gene expression, suggesting a possible implication of E2F2-dependent transcriptional activity in HD pathogenesis. Thus, E2F2 emerges as a new potential HD AO modifier factor.

New Nuclear SNP Markers Unravel the Genetic Structure and Effective Population Size of Albacore Tuna (Thunnus alalunga)

In the present study we have investigated the population genetic structure of albacore (Thunnus alalunga, Bonnaterre 1788) and assessed the loss of genetic diversity, likely due to overfishing, of albacore population in the North Atlantic Ocean. For this purpose, 1,331 individuals from 26 worldwide locations were analyzed by genotyping 75 novel nuclear SNPs. Our results indicated the existence of four genetically homogeneous populations delimited within the Mediterranean Sea, the Atlantic Ocean, the Indian Ocean and the Pacific Ocean. Current definition of stocks allows the sustainable management of albacore since no stock includes more than one genetic entity. In addition, short-and long-term effective population sizes were estimated for the North Atlantic Ocean albacore population, and results showed no historical decline for this population. Therefore, the genetic diversity and, consequently, the adaptive potential of this population have not been significantly affected by overfishing.