Autism is a severe childhood neuropsychiatric condition characterized by impairments in socialization and communication, and by restricted and repetitive behaviours. Autism spectrum disorder (ASD) is a complex and largely unknown disease with a strong genetic basis, multiple genes involved and environmental factors determining its phenotype. Interestingly, the DLX1/DLX2 and DLX5/DLX6 bigene clusters are located in autism susceptibility loci and Dlx genes are involved in GABAergic interneurons differentiation and migration to the cortex during forebrain development. Dlx gene expression is controlled by different cis-regulatory elements. Of these, 4 are active in the forebrain, URE2, I12b, I56ii and I56i. In order to determine the role of the DLX genes in ASD, variants were found in gene exons and in cis-regulatory elements in autistic individuals. A single nucleotide polymorphism (SNP), a change of an adenine for a guanine, was identified in I56i enhancer. Finding a SNP in I56i was very surprising considering that it is located in a Dlx binding motif highly conserved among >40 species. We showed, using in vitro approaches, that the presence of this SNP affects the affinity of Dlx for their binding site and reduces the transcriptional activation of the enhancer. The SNP also affects activity of the I56i enhancer in transgenic mice. In order to determine the real impact of the SNP in vivo, mutant mice harboring the SNP in their I56i enhancer were produced. That involved the insertion of the I56i enhancer with the SNP, using homologous recombination in mouse embryonic stem cells to replace the wild type version of the enhancer. With these mutant mice, we demonstrated that, in vivo, this SNP reduces Dlx5 and Dlx6 expression in the forebrain. Furthermore, this decrease in Dlx5/Dlx6 expression could affect the differentiation and/or migration of specific populations of inhibitory interneurons in the forebrain. No distinct iv behavioural phenotypes were observed between wild type mice and those carrying the SNP, during social interaction and anxiety tests. Therefore, these results suggest that even a subtle change in a regulatory element can have an impact in the development of the forebrain and may even contribute to disorders such as autism.

Improving feed efficiency has become a top priority in beef cattle production because of the rapidly increasing cost of feed provision. However, because of the expense associated with collecting individual animal feed intake data, only a relatively small number of animals have been tested, leading to low accuracies of estimated breeding values (EBV). Three studies were conducted to demonstrate the usefulness of including DNA marker information in RFI genetic evaluations. In the first study, the effect of period of testing on RFI was assessed. Beef cattle steers were tested for feed intake, with different cohorts tested in the fall-winter and winter-spring seasons. Seasonal differences were detected although these were confounded by differences in age and weight among the seasons. Additionally, mean EBV accuracy obtained was low, ranging between 0.47 and 0.51, implying that strategies to increase this accuracy are necessary. In the 2nd study, a suite of genetic markers predictive of RFI, DMI and ADG were pre-selected using single marker regression analysis and the top 100 SNPs analyzed further in 5 replicates of the training data to provide prediction equations for RFI, DMI and ADG. Cumulative marker phenotypes (CMP) were used to predict trait phenotypes and accuracy of prediction ranged between 0.007 and 0.414. Given that this prediction accuracy was lower than the polygenic EBV accuracy, the CMP would need to be combined with EBV for effective marker assisted selection. In study 3, genomic selection (GS) theory and methodology were used to derive genomic breeding values (GEBV) for RFI, DMI and ADG. The accuracy of prediction obtained with GEBV was low, ranging from 0.223 to 0.479 for marker panel with 200 SNPs, and 0.114 to 0.246 for a marker panel with 37,959 SNPs, depending on the GS method used. The results from these studies demonstrate that the utility of genetic markers for genomic prediction of RFI in beef cattle may be possible, but will likely be more effective if a tool that combines GEBV with traditional BLUP EBV is used for selection. / Animal Science

Improving feed efficiency has become a top priority in beef cattle production because of the rapidly increasing cost of feed provision. However, because of the expense associated with collecting individual animal feed intake data, only a relatively small number of animals have been tested, leading to low accuracies of estimated breeding values (EBV). Three studies were conducted to demonstrate the usefulness of including DNA marker information in RFI genetic evaluations. In the first study, the effect of period of testing on RFI was assessed. Beef cattle steers were tested for feed intake, with different cohorts tested in the fall-winter and winter-spring seasons. Seasonal differences were detected although these were confounded by differences in age and weight among the seasons. Additionally, mean EBV accuracy obtained was low, ranging between 0.47 and 0.51, implying that strategies to increase this accuracy are necessary. In the 2nd study, a suite of genetic markers predictive of RFI, DMI and ADG were pre-selected using single marker regression analysis and the top 100 SNPs analyzed further in 5 replicates of the training data to provide prediction equations for RFI, DMI and ADG. Cumulative marker phenotypes (CMP) were used to predict trait phenotypes and accuracy of prediction ranged between 0.007 and 0.414. Given that this prediction accuracy was lower than the polygenic EBV accuracy, the CMP would need to be combined with EBV for effective marker assisted selection. In study 3, genomic selection (GS) theory and methodology were used to derive genomic breeding values (GEBV) for RFI, DMI and ADG. The accuracy of prediction obtained with GEBV was low, ranging from 0.223 to 0.479 for marker panel with 200 SNPs, and 0.114 to 0.246 for a marker panel with 37,959 SNPs, depending on the GS method used. The results from these studies demonstrate that the utility of genetic markers for genomic prediction of RFI in beef cattle may be possible, but will likely be more effective if a tool that combines GEBV with traditional BLUP EBV is used for selection. / Animal Science

<p>Double-stranded DNA breaks are potentially lethal lesions that can be repaired in mitotic cells by either homologous recombination (HR) or non-homologous end- joining (NHEJ) pathways. In the HR pathway, the broken DNA molecule is repaired using either the sister chromatid or the homolog as a template. Mitotic recombination events involving the homolog often result in loss of heterozygosity (LOH) of markers located distal to the crossover. In humans that are heterozygous for a mutation in a tumor suppressor gene, mitotic recombination leading to LOH can be an early step in cancer development.</p><p> In my thesis research, I analyzed mitotic recombination in the yeast Saccharomyces cerevisiae using oligonucleotide-containing microarrays to detect LOH of single-nucleotide polymorphisms (SNPs). In analyzing cells treated with ionizing radiation, I performed the first whole-genome analysis of LOH events done in any organism (Chapter 2). I showed that irradiated cells had between two and three unselected LOH events. I also showed that crossovers were often associated with non- reciprocal exchanges of genetic information (gene conversion events) and that these conversion events were more complex than predicted by standard models of homologous recombination.</p><p> In Chapter 3, I describe my mapping of spontaneous crossovers in a 1.1 Mb region of yeast chromosome IV. This analysis is the first high-resolution mitotic recombination map of a substantial fraction (about 10%) of a eukaryotic genome. I demonstrated the existence of recombination "hotspots" and showed that some of these hotspots were homolog-specific. Two of the strongest hotspots were formed by closely- spaced inverted repeats of retrotransposons. I demonstrated that the hotspot activity was a consequence of a secondary DNA structure formed by these repeats. Additionally, the majority of spontaneous LOH events reflect DNA lesions induced in unreplicated chromosomes during G1 of the cell cycle, indicating that G1-initiated lesions threaten genome stability more than G2-initiated lesions.</p><p> In Chapter 4, I describe mitotic crossovers associated with DNA replication stress induced by hydroxyurea (HU) treatment. Surprisingly, most HU-induced crossovers had conversion tracts indicative of DNA lesions initiated in G1. Additionally, HU- induced recombination events were very significantly associated with solo delta elements, a 330 bp sequence that is repeated several hundred times in the yeast genome.</p> / Dissertation

The evolutionary relationship between Salmonella enterica serovar Typhi, other typhoid-like enteric fever causing serovars and 10 non-Typhoid serovars from S. enterica subspecies I, could not be determined by comparative nucleotide sequences of six genes. Phylogenetic analyses of the dataset showed that the genes of interest underwent frequent recombination, suggesting a low level of clonality within subspecies I of S. enterica. To establish the evolutionary relationships within serovar Typhi, genome-wide Single Nucleotide Polymorphism (SNP) was explored as a marker for both typing purposes and phylogenetic analysis. Thirty eight SNPs were typed in 73 global Typhi isolates, including 18 isolates expressing the special flagellar antigen z66, using restriction enzyme digestion method. The isolates were differentiated into 23 SNP profiles and grouped into four distinct clusters. The z66 isolates were divided into four SNP profiles and were all grouped into one cluster, suggesting a single origin. An alternative SNP typing method using the hairpin real time PCR assay was investigated to type four additional SNPs, termed as biallelic polymorphisms (BiP). These BiPs were found to classify 481 global Typhi isolates into five major clusters (Roumagnac et al., 2006). Typing four BiPs resulted in the identification of four additional SNP profiles. We proposed nine SNPs were required to type Typhi isolates into 13 subclusters for global epidemiology. An enzymatic-based method using CelI nuclease was evaluated to discover more SNPs from other Typhi genomes. The efficiency of the CelI was shown to be unsatisfactory and we were unable to demonstrate the effectiveness of the proposed method. Nine Variable Number of Tandem Repeats (VNTRs) were typed in the 73 Typhi isolates using fluorescent-labelled universal primers, and analysed on an automated DNA sequencer. Five isolates were unable to give PCR products in one or more VNTR loci. Nine VNTRs could differentiate 68 Typhi isolates into 65 MLVA profiles, suggesting a higher discriminating power than SNP typing. SNPs were shown to be a more appropriate marker for phylogenetic tracing for Typhi while VNTRs were highly discriminating but could not be used to establish the evolutionary relationships of diverse Typhi isolates.

Diss. (sammanfattning) Uppsala : Univ., 2007. / Härtill 4 uppsatser.

The variability of the marker TG5 TG gene and its association with the intramuscular fat and marbling of meat in cattle was studied. Randomly selected population, composed of 237 individuals (bulls) of the Czech Pied cattle breed from four farms in the Czech Republic, waschosen for testing polymorphisms. Polymerase chain reaction was used to amplify a fragment of TG 545 bp in size. After amplification, the fragments digestion was performed using the restriction endonuclease BstYI. To verify the presence of the PCR product and to identify the sizes of digested frag-ments the horizontal agarose electrophoresis was performed. The population's absolute and relative frequencies of alleles and genotypes were calculated. The calculations reve-aled a high frequency of the T allele (25.98 %). In conclusion, statisticalanalysis was performed. As a result, the influence of C422T polymorphism in the 5'promoter region of the gene on the TG and on the IMT marblinghave not been proven, but on the contra-ry there was detected significant effect (p<0.05) of polymorphism content on the region of Lauric acid (C12:0). The results also reveal some evidence of polymorphism having influence on Timnodonic acid and meat pH.

IBSP gene for bone sialoprotein plays an important role in bone mineralization. The aim of the study was to find out if there is an association between bone mineral composition and A211G polymorphism in gene for bone sialoprotein. PCR-RFLP method was used for polymorphism genotyping. Calcium and phosphorus content of bone tissue were determined photometrically. The three-point bending test was applied to measure bone strength. Results was evaluated by one-way ANOVA with Fisher LSD post-hoc testing. No statistically significant differences between genotypes were found. This findings shows no support for association between bone content and polymorphism. So A211G polymorphism cannot been recommended for MAS.

The purpose of this project was to develop inexpensive, standardized, and high throughput Single Nucleotide Polymorphism (SNP) markers that discriminate between pallid (Scaphirhynchus albus) and shovelnose (S. platorynchus) sturgeon for use as a larval identification tool. A total of 67 polymorphic sites was identified in DNA sequences from three genes: Recombination Activating Gene-1, Beta Actin, and Beta-2-Microglobulin. Allele frequencies from the 10 most variable SNPs were characterized for both pallid and shovelnose sturgeon in three geographically separated populations throughout the range of the pallid sturgeon. To create a standardized method of genotyping SNPs for larval pallid and shovelnose sturgeon, 5' nuclease allelic discrimination (TaqMan) assays were designed for two unlinked SNPs that exhibited the greatest allele frequency differences between species. A power analysis compared these SNP loci and their diagnostic power for species discrimination compared to sixteen microsatellite loci currently used for species discrimination (Schrey et al. 2007) One SNP locus was the most powerful marker for species identification in the upper and middle Missouri River. This study provides practical genetic tools for species discrimination between pallid and shovelnose that will facilitate understanding addressing questions that were previously too costly, labor intensive or technically challenging to answer.

Submitted by Tatiana Silva (tsilva@icict.fiocruz.br) on 2013-02-08T13:08:41Z No. of bitstreams: 1 simone_t_b_santos_ioc_bcm_0006_2010.pdf: 2551865 bytes, checksum: 53317a265fddefc98babc5e7a0074acb (MD5) / Made available in DSpace on 2013-02-08T13:08:41Z (GMT). No. of bitstreams: 1 simone_t_b_santos_ioc_bcm_0006_2010.pdf: 2551865 bytes, checksum: 53317a265fddefc98babc5e7a0074acb (MD5) Previous issue date: 2010 / Fundação Oswaldo Cruz.Instituto Oswaldo Cruz. Rio de janeiro, RJ, Brasil / A população brasileira é considerada miscigenada, derivada de um processo relativamente recorrente e recente. Aqui viviam milhões de indígenas quando começou o processo colonizatório envolvendo integrantes europeus, principalmente portugueses do sexo masculino, tornando comum o acasalamento entre homens europeus e mulheres indígenas, começando assim a heterogeneidade étnica encontrada em nossa população. Posteriormente com a chegada dos escravos, durante o ciclo econômico da cana-de-açúcar, começou a ocorrer relacionamentos entre europeus e africanas. Basicamente, trata-se de uma população tri-híbrida que atualmente apresenta em sua composição outros grupos, entre eles: italianos, espanhóis, sírios, libaneses e japoneses. Para o melhor entendimento das raízes filogenéticas brasileiras, foram utilizados neste estudo marcadores bi-alélicos da região não recombinante do cromossomo Y. O objetivo foi analisar como esses grupos heterogêneos contribuíram para o pool genético de origem paterna encontrado na população masculina do Rio de Janeiro, e assim enriquecer os conhecimentos acerca dos movimentos migratórios no processo de estruturação desta população. Foram analisados, através do minissequenciamento multiplex, 13 polimorfismos de base única (SNPs) e foi possível a identificação de nove haplogrupos e quatro sub-haplogrupos, em uma amostra de 200 indivíduos não aparentados e residentes do Estado do Rio de Janeiro, escolhidos aleatoriamente entre participantes de estudos de paternidade da Defensoria Pública do Rio de Janeiro. Dos haplogrupos analisados, somente o R1a, não foi observado em nossa população. O haplogrupo mais representativo foi o de origem européia, o R1b1, com 51%, enquanto o menos representativo, com 1% foi o Q1a3a, encontrado entre os nativos americanos. Cerca de 85% dos cromossomos Y analisados são de origem européia; 10,5% de africanos e 1% de ameríndios, e o restante são de origem indefinida. Ao comparamos com dados da literatura nossa população mostrou-se semelhante a população branca de Porto Alegre e nenhuma diferença significativa foi encontrada entre o pool gênico da população masculina do Rio de Janeiro com a portuguesa. Os resultados aqui encontrados corroboram os dados históricos da fundação da população do Rio de Janeiro durante o século XVI, período onde foi observada uma significante redução da população ameríndia, com importante contribuição demográfica vinda da região Subsaariana da África e Europa, principalmente de portugueses. Tendo em vista o alto grau de miscigenação da nossa população e os avanços na medicina personalizada, estudos sobre a estrutura genética humana têm fundamental implicação no entendimento na evolução e no impacto em doenças humanas, uma vez que para esta abordagem, a coloração da pele é um preditor não confiável de ancestralidade étnica do indivíduo. / The Brazilian population is highly admixture, a relatively recurrent and recent process. Millions of indigenous people had been living here when the colonization process began, initially involving mainly Portuguese men. The immigration of European women during the first centuries was insignificant, making common the marriage between European men with indigenous women; hence, starting the ethnic heterogeneity found in our population nowadays. Subsequently, with the arrival of slaves during the economic cycle of sugarcane, began the relationships between Europeans and Africans. Basically, it is a tri-hybrid population with contributions from other groups, such as Italians, Germans, Syrians, Lebanese and Japanese. For a better understanding of Brazilian phylogenetic roots, biallelic markers of nonrecombining region of the Y chromosome were used in this study. The goal was to analyze how these heterogeneous groups contributed to the genetic pool found present-day in the population of Rio de Janeiro, and thus contribute to the understanding of migratory movements in the process of structuring this population. We analyzed, through minisequencing multiplex, 13 single nucleotide polymorphisms (SNPs) and through those it was able to identify nine haplogrupos and four subhaplogrupos, in a sample of 200 unrelated individuals, residents of the State of Rio de Janeiro, chosen randomly between participants from studies of fatherhood. Of the haplogrupos examined, only the R1a, has not been observed in our population. The more representative haplogroup was from European origin, the R1b1, with 51%, while the less representative, with 1% was the Q1a3a, found among Native Amerindians. 85% of Y chromosomes analyzed were from Europeans; 10.5% from Africans and 1% of Native Amerindians, and the rest have not had their origin defined. In this study sample, the vast majority of Y-chromosomes proved to be of European origin. Indeed, there were no significant differences when the haplogroup frequencies in Brazil and Portugal were compared by means of an exact test of population differentiation. These results corroborate historical data of the foundation of the population of Rio de Janeiro during the 16th century, a period where it was observed a significant reduction of Amerindian population was observed with important contribution from the Sub-Saharan region of Africa and Europe, particularly the Portuguese. In view of the high degree of admixture of oBrazilian population and advances in medicine, customized research on human genetic structure have fundamental implication in understanding the evolution and impact on human diseases, since for this approach, the skin color is an unreliable ancestry predictor of individual ethnic