Made available in DSpace on 2014-06-11T19:30:12Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-04-04Bitstream added on 2014-06-13T20:39:52Z : No. of bitstreams: 1 paes_adnva_me_botib.pdf: 667583 bytes, checksum: 1b9f159d6044dacd37b4d8cb58168da7 (MD5) / A hibridação interespecífica é considerada por diversos autores um método de melhoramento genético de difícil compreensão, uma vez que os produtos obtidos do acasalamento de diferentes espécies podem originar diversos produtos genéticos tais como Ginogenéticos e Androgenéticos Haplóides ou Diplóides, Híbridos Diplóides simples, Triplóides ou até indivíduos Tetraplóides. A aplicação da hibridação Interespecífica é utilizada no sistema de manejo nas grandes Pisciculturas que visa produzir animais que possuam melhor desempenho que as espécies parentais (vigor híbrido), como o aumento da taxa de crescimento, melhor qualidade da carne, resistência a doenças e capacidade de tolerar variações ambientais, além do aperfeiçoamento de diversas outras características a fim produzir peixes mais proveitosos para o cultivo. Dentre aproximadamente 40 espécies de peixes de interesse comercial no Brasil, destaca-se o gênero Brycon, de grande interesse nos centros de Pisciculturas devido à qualidade da carne, ao hábito alimentar, ao rápido crescimento e ganho de peso.Com o objetivo de compreender o mecanismo de hibridação, foram analisados 10 exemplares de cada espécie parental B.amazonicus (Matrinxã) e B.hilarii (Piraputanga) e 10 exemplares da respectiva geração Filial. Através da técnica de coloração com Giemsa, observou-se um conjunto diplóide 2n = 50 cromossomos em todos os indivíduos analisados das espécies parentais e da geração Filial. No entanto, houve diferenciação na formação cariotípica dos parentais e da geração filial.O parental Matrinxã (B.amazonicus) e sua geração Filial apresentaram uma constituição cariotíca de seis cromossomos do tipo metacêntrico, nove cromossomos submetacêntrico e dez cromossomos subtelocêntrico, enquanto que o parental Piraputanga (B.hilarii) apresentou uma constituição cariotípica de dez pares... / Interspecific hybridization is considered by many authors as a method of breeding that is difficult to understand, since the mating products obtained from different species can cause many genetic products, such as gynogenetic and androgenetic haploid or diploid, diploid hybrid simple, triploid or even individuals tetraploids. The application of Interspecific hybridization is used in the management system in major fisheries that aims to produce animals that have better performance than the parental species (hybrid vigor), as increased growth rate, meat quality, disease resistance and ability to tolerate environmental changes besides the improvement of several other characteristics in order to produce more profitable fish farming.Out of approximately 40 fish species of commercial interest in Brazil, there is the genus Brycon, of great interest in the centers of fish farms due to meat quality, the feeding habits of the rapid growth and weight gain. Aiming to understand the mechanism of hybridization, we analyzed 10 specimens of each parental species B.amazonicus (Matrinxã) and B.hilarii (Piraputanga) and 10 of filial generation. By staining with Giemsa, all specimens analyzed have a number diploid with 2n = 50 chromosomes. However, there was karyotypic differentiation in the formation of parental and filial generation. The parental Matrinxã (B.amazonicus) and his generation branch had a karyotype constitution of six chromosomes of metacentric type, nine submetacentric chromosomes and ten subtelocêntric chromosomes, while the parental Piraputanga (B.hilarii) showed a karyotype constitution of ten pairs of chromosomes metacentric, nine pairs of chromosomes submetacentric and six subtelocentric chromosomes pairs.The detection of Nucleolar Organizer Regions (RONs) was obtained by the technique of impregnation by silver nitrate (Ag-NORs) that showed markings on the telomeric region... (Complete abstract click electronic access below)

This PhD Thesis includes five main parts on diverse topics. The first two parts deal with the trophic ecology of wolves in Italy consequently to a recent increase of wild ungulates abundance. Data on wolf diet across time highlighted how wild ungulates are important food resource for wolves in Italy. Increasing wolf population, increasing numbers of wild ungulates and decreasing livestock consume are mitigating wolf-man conflicts in Italy in the near future. In the third part, non-invasive genetic sampling techniques were used to obtain genotypes and genders of about 400 wolves. Thus, wolf packs were genetically reconstructed using diverse population genetic and parentage software. Combining the results on pack structure and genetic relatedness with sampling locations, home ranges of wolf packs and dispersal patterns were identified. These results, particularly important for the conservation management of wolves in Italy, illustrated detailed information that can be retrieved from genetic identification of individuals. In the fourth part, wolf locations were combined with environmental information obtained as GIS-layers. Modern species distribution models (niche models) were applied to infer potential wolf distribution and predation risk. From the resulting distribution maps, information pastures with the highest risk of depredation were derived. This is particularly relevant as it allows identifying those areas under danger of carnivore attack on livestock. Finally, in the fifth part, habitat suitability models were combined with landscape genetic analysis. On one side landscape genetic analyses on the Italian wolves provided new information on the dynamics and connectivity of the population and, on the other side, a profound analysis of the effects that habitat suitability methods had on the parameterization of landscape genetic analyses was carried out to contributed significantly to landscape genetic theory.

Root-yield-1.06 is a major QTL affecting root system architecture (RSA) and other agronomic traits in maize. The effect of this QTL has been evaluated with the development of near isogenic lines (NILs) differing at the QTL position. The objective of this study was to fine map qroot-yield-1.06 by marker-assisted searching for chromosome recombinants in the QTL interval and concurrent root phenotyping in both controlled and field conditions, through successive generations. Complementary approaches such as QTL meta-analysis and RNA-seq were deployed in order to help prioritizing candidate genes within the QTL target region. Using a selected group of genotypes, field based root analysis by ‘shovelomics’ enabled to accurately collect RSA information of adult maize plants. Shovelomics combined with software-assisted root imaging analysis proved to be an informative and relatively highly automated phenotyping protocol. A QTL interval mapping was conducted using a segregating population at the seedling stage grown in controlled environment. Results enabled to narrow down the QTL interval and to identify new polymorphic markers for MAS in field experiments. A collection of homozygous recombinant NILs was developed by screening segregating populations with markers flanking qroot-yield-1.06. A first set of lines from this collection was phenotyped based on the adapted shovelomics protocol. QTL analysis based on these data highlighted an interval of 1.3 Mb as completely linked with the target QTL but, a larger safer interval of 4.1 Mb was selected for further investigations. QTL meta-analysis allows to synthetize information on root QTLs and two mQTLs were identified in the qroot-yield-1.06 interval. Trascriptomics analysis based on RNA-seq data of the two contrasting QTL-NILs, confirmed alternative haplotypes at chromosome bin 1.06. qroot-yield-1.06 has now been delimited to a 4.1-Mb interval, and thanks to the availability of additional untested homozygous recombinant NILs, the potentially achievable mapping resolution at qroot-yield-1.06 is c. 50 kb.

Durum wheat is the second most important wheat species worldwide and the most important crop in several Mediterranean countries including Italy. Durum wheat is primarily grown under rainfed conditions where episodes of drought and heat stress are major factors limiting grain yield. The research presented in this thesis aimed at the identification of traits and genes that underlie root system architecture (RSA) and tolerance to heat stress in durum wheat, in order to eventually contribute to the genetic improvement of this species. In the first two experiments we aimed at the identification of QTLs for root trait architecture at the seedling level by studying a bi-parental population of 176 recombinant inbred lines (from the cross Meridiano x Claudio) and a collection of 183 durum elite accessions. Forty-eight novel QTLs for RSA traits were identified in each of the two experiments, by means of linkage- and association mapping-based QTL analysis, respectively. Important QTLs controlling the angle of root growth in the seedling were identified. In a third experiment, we investigated the phenotypic variation of root anatomical traits by means of microscope-based analysis of root cross sections in 10 elite durum cultivars. The results showed the presence of sizeable genetic variation in aerenchyma-related traits, prompting for additional studies aimed at mapping the QTLs governing such variation and to test the role of aerenchyma in the adaptive response to abiotic stresses. In the fourth experiment, an association mapping experiment for cell membrane stability at the seedling stage (as a proxy trait for heat tolerance) was carried out by means of association mapping. A total of 34 QTLs (including five major ones), were detected. Our study provides information on QTLs for root architecture and heat tolerance which could potentially be considered in durum wheat breeding programs.

Childhood neuroblastoma is the most common solid tumour of infancy and highly refractory to therapy. One of the most powerful prognostic indicators for this disease is the N-Myc gene amplification, which occurs in approximately 25% of all neuroblastomas. N-Myc is a member of transcription factors belonging to a subclass of the larger group of proteins sharing Basic-Region/Helix–Loop–Helix/Leucin-Zipper (BR/HLH/LZ) motif. N-Myc oncoproteins may determine activation or repression of several genes thanks to different protein-protein interactions that may modulate its transcriptional regulatory ability and therefore its potential for oncogenicity. Chromatin modifications, including histone methylation, have a crucial role in transcription de-regulation of many cancer-related genes. Here, it was investigated whether N-Myc can functionally and/or physically interact with two different factors involved in methyl histone modification: WDR5 (core member of the MLL/Set1 methyltransferase complex) and the de- methylase LSD1. Co-IP assays have demonstrated the presence of both N-Myc-WDR5 and N-Myc-LSD1 complexes in two neuroblastoma cell lines. Human N-Myc amplified cell lines were used as a model system to investigate on transcription activation and/or repression mechanisms carried out by N-Myc-LSD1 and N-Myc-WDR5 protein complexes. qRT-PCR and immunoblot assays underlined the ability of both complexes to positively (N-Myc-WDR5) and negatively (N-Myc-LSD1) influence transcriptional regulation of crititical neuroblastoma N-Myc-related genes, MDM2, p21 and Clusterin. Ch-IP experiments have revealed the binding of the N-Myc complexes above mentioned to the gene promoters analysed. Finally, pharmacological treatment pointed to abolish N-Myc and LSD1 activity were performed to test cellular alterations, such as cell viability and cell cycle progression. Overall, the results presented in this work suggest that N-Myc can interact with two distinct histone methyl modifiers to positively and negatively affect gene transcription in neuroblastoma.

In Drosophila the steroid hormone ecdysone regulates a wide range of developmental and physiological responses, including reproduction, embryogenesis, postembryonic development and metamorphosis. Drosophila provides an excellent system to address some fundamental questions linked to hormone actions. In fact, the apparent relative simplicity of its hormone signaling pathways taken together with well-established genetic and genomic tools developed to this purpose, defines this insect as an ideal model system for studying the molecular mechanisms through which steroid hormones act. During my PhD research program I’ve analyzed the role of ecdysone signaling to gain insight into the molecular mechanisms through which the hormone fulfills its pleiotropic functions in two different developmental stages: the oogenesis and the imaginal wing disc morphogenesis. To this purpose, I performed a reverse genetic analysis to silence the function of two different genes involved in ecdysone signaling pathway, EcR and ecd.

Animal neocentromeres are defined as ectopic centromeres that have formed in non-centromeric locations and avoid some of the features, like the DNA satellite sequence, that normally characterize canonical centromeres. Despite this, they are stable functional centromeres inherited through generations. The only existence of neocentromeres provide convincing evidence that centromere specification is determined by epigenetic rather than sequence-specific mechanisms. For all this reasons, we used them as simplified models to investigate the molecular mechanisms that underlay the formation and the maintenance of functional centromeres. We collected human cell lines carrying neocentromeres in different positions. To investigate the region involved in the process at the DNA sequence level we applied a recent technology that integrates Chromatin Immuno-Precipitation and DNA microarrays (ChIP-on-chip) using rabbit polyclonal antibodies directed against CENP-A or CENP-C human centromeric proteins. These DNA binding-proteins are required for kinetochore function and are exclusively targeted to functional centromeres. Thus, the immunoprecipitation of DNA bound by these proteins allows the isolation of centromeric sequences, including those of the neocentromeres. Neocentromeres arise even in protein-coding genes region. We further analyzed if the increased scaffold attachment sites and the corresponding tighter chromatin of the region involved in the neocentromerization process still were permissive or not to transcription of within encoded genes. Centromere repositioning is a phenomenon in which a neocentromere arisen without altering the gene order, followed by the inactivation of the canonical centromere, becomes fixed in population. It is a process of chromosome rearrangement fundamental in evolution, at the bases of speciation. The repeat-free region where the neocentromere initially forms, progressively acquires extended arrays of satellite tandem repeats that may contribute to its functional stability. In this view our attention focalized to the repositioned horse ECA11 centromere. ChIP-on-chip analysis was used to define the region involved and SNPs studies, mapping within the region involved into neocentromerization, were carried on. We have been able to describe the structural polymorphism of the chromosome 11 centromeric domain of Caballus population. That polymorphism was seen even between homologues chromosome of the same cells. That discovery was the first described ever. Genomic plasticity had a fundamental role in evolution. Centromeres are not static packaged region of genomes. The key question that fascinates biologists is to understand how that centromere plasticity could be combined to the stability and maintenance of centromeric function. Starting from the epigenetic point of view that underlies centromere formation, we decided to analyze the RNA content of centromeric chromatin. RNA, as well as secondary chemically modifications that involve both histones and DNA, represents a good candidate to guide somehow the centromere formation and maintenance. Many observations suggest that transcription of centromeric DNA or of other non-coding RNAs could affect centromere formation. To date has been no thorough investigation addressing the identity of the chromatin-associated RNAs (CARs) on a global scale. This prompted us to develop techniques to identify CARs in a genome-wide approach using high-throughput genomic platforms. The future goal of this study will be to focalize the attention on what strictly happens specifically inside centromere chromatin.

Doctor en Ciencias con mención en Biología

Magister en Ciencias Biológicas con mención en Botánica / Las nuevas herramientas biotecnológicas ofrecen una amplia gama de posibilidades de desarrollo en todas las áreas donde el hombre se desenvuelve. La agricultura no esta ajena a esta posibilidad. El trigo de pan ( Triticum aestivum L), uno de los alimentos más importantes del mundo, puede beneficiarse significativamente al incorporar el uso de esta nueva tecnología dentro del proceso de creación de nuevas variedades. Los microsatélites o SSR (Simple Sequence Repeats), marcadores genéticos hipervariables, han demostrado ser una de las mejores alternativas para este cultivo. Por este motivo fueron los seleccionados para llevar a cabo el objetivo de la presente investigación, en la cual se busco asociar microsatélites a loci genéticos de proteínas de reserva y de dureza del grano, factores determinantes de la calidad panadera de este cereal. Para llevar a cabo está investigación se seleccionaron 80 genotipos de trigo de pan, a los cuales se les determinó: porcentaje de proteína del grano, volumen de sedimentación, gluten seco, gluten húmedo, gluten índex e índice de dureza. Esta información fue utilizada como base para seleccionar aquellos marcadores genéticos que estuvieran asociados con estos parámetros de calidad. De los seis microsatélites analizados cuatro estuvieron asociados con diferentes pruebas de calidad. El marcador Xglu A3 presentó correlaciones significativas con volumen de sedimentación (0,385), gluten seco (0,394) y gluten índex (0,377). Xgwm 164 se asoció significativamente con sedimentación (0,235) y gluten índex (0,233), el partidor Xgwm 135 presento correlaciones significativas con gluten seco (0,172) y gluten índex (0,221) y el SSR Xgdm 19 fue el marcador con mayor número de pruebas asociadas: porcentaje de proteínas (0,447), sedimentación (0,408), gluten seco (0,487), gluten índex (0,391) e índice de dureza (0,483). De estos cuatro microsatélites, Xglu A3 y Xgdm 19, son los más informativos con índices de polimorfismos (PIC) de 0,721 y 0,758 respectivamente. Ninguno de los SSR estudiados presentó asociación con gluten húmedo. Los marcadores Xgwm 498 y Xgdm 98, no se correlacionaron con ninguno de los parámetros de calidad. Al analizar la distribución de las frecuencia genotipica de los marcadores seleccionados, en los grupos de genotipos de buena, regular y mala calidad panadera, se seleccionó haplotipos promisorios para buena calidad. A todo el germoplasma analizado se le determinó la presencia de secalina, proteína de centeno, la cual existe en algunas líneas de trigo por efecto de translocaciones cromosomales con este cereal. Treinta y tres genotipos dieron positivo para este análisis. La mayoría de este grupo presentó bajos niveles de volumen de sedimentación. Este resultado indica la necesidad de tener presente este factor cuando se están determinando criterios de selección para calidad panadera. Paralelamente, en esta investigación se caracterizó las gluteninas de alto peso molecular, de las 80 líneas y variedades en ensayo. Las subunidades permitieron determinar el índice GLU-1, el que demostró ser un buen indicador de calidad panadera, por su buena asociación con volumen de sedimentación.

Made available in DSpace on 2014-06-11T19:30:57Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-11-28Bitstream added on 2014-06-13T19:40:34Z : No. of bitstreams: 1 ferraz_jaml_dr_arafcf.pdf: 1985252 bytes, checksum: 0ba79a8945f377afa95366862b572c08 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Universidade Estadual Paulista (UNESP) / Os marcadores polimórficos short tandem repeats do cromossomo X (X.STR) são de utilização recente na prática forense, sendo aplicados, principalmente, com a finalidade de complementar os dados obtidos com marcadores genéticos autossômicos. Tendo em vista a necessidade de ampliação dos dados da população brasileira em relação aos X.STRs, este projeto teve o objetivo de determinar as frequências alélicas e os parâmetros estatísticos de interesse forense para 10 X.STRs na população de São Paulo.SP, Rio de Janeiro.RJ, Belo Horizonte.BH e Vitória.ES (Sudeste do Brasil). Posteriormente, os X.STRs foram genotipados em casos deficientes de relação biológica e, com o objetivo de compilar os dados genéticos populacionais brasileiros publicados para X.STRs, este projeto desenvolveu o Banco Genético Brasileiro do Cromossomo X (BGBX). Para isso, foram analisados 1001 indivíduos não relacionados e residentes nas cidades descritas acima e 3 casos deficientes de relação biológica. Os marcadores X.STRs foram amplificados em sistema decaplex e submetidos à eletroforese em analisador genético ABI377 e ABI3500. Os programas GeneScan e GeneMapper ID.X foram utilizados para a determinação alélica e, a planilha Excel e o programa Arlequin, para a determinação das frequências alélicas, parâmetros estatísticos e análise de distância genética entre diferentes populações. Em relação aos X.STRs analisados, os marcadores DXS6809 e GATA172D05 foram os mais discriminativos, enquanto os marcadores DXS8378, DXS7133 e DXS7423 apresentaram os menores poder de discriminação. Diferenças significativas de frequências alélicas foram obtidas dentre as populações brasileiras e entre estas e demais populações estrangeiras, sendo que uma maior distância genética foi obtida em relação à população africana de Uganda... / The short tandem repeat polymorphic markers of X chromosome (X.STR) are of recent use in forensic practice, been applied mainly in order to complement the data obtained with autosomal genetic markers. Given the needs of expansion of the Brazilian population data in relation to X.STRs, this project aimed to determine the allele frequencies and the statistical parameters of forensic interest to 10 X.STRs in the population from São Paulo.SP, Rio de Janeiro.RJ, Belo Horizonte.BH e Vitória.ES (Southeast of Brazil). Subsequently, the X.STRs were genotyped in deficient kinship cases and, with the aim of compiling the Brazilian genetic population data published for X.STRs, this project developed the Brazilian Genetic Database of Chromosome X (BGBX). For this, we have analyzed 1001 unrelated individuals, residents in the cities previously described, and three deficient kinship cases. The X.STR markers were amplified in decaplex system and submitted to electrophoresis on ABI377 and ABI3500 genetic analyzers. GeneScan and GeneMapper ID.X softwares were used to determine the allele and, Excel spreadsheet and Arlequin software, for the determination of allele frequencies, statistical parameters and genetic distance analysis between different populations. Regarding the X.STRs analyzed, markers DXS6809 and GATA172D05 were the most discriminative, while the markers DXS8378, DXS7133 and DXS7423 showed the lowest discrimination power. Significant differences in allele frequencies were obtained within Brazilian populations and between these and other foreign populations, and a greater genetic distance was... (Complete abstract click electronic access below)