Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2018-05-02T19:07:13Z No. of bitstreams: 1 2014 - Rafael Sanches Pacheco.pdf: 1387070 bytes, checksum: c73e833c8d495bf472f647befeedd57e (MD5) / Made available in DSpace on 2018-05-02T19:07:13Z (GMT). No. of bitstreams: 1 2014 - Rafael Sanches Pacheco.pdf: 1387070 bytes, checksum: c73e833c8d495bf472f647befeedd57e (MD5) Previous issue date: 2014-08-19 / CNPq / The 15N natural abundance technique to measure the biological nitrogen fixation (BNF) in legumes is based on the slight enrichment of 15N observed in soils to the atmosphere and does not require the application of labelled fertilizers. The objectives of this work were: to quantify the BNF contribution in common bean (Phaseolus vulgaris) cultivars under field conditions through the 15N natural abundance technique; to evaluate the agronomic performance of these cultivars under inoculation; to estimate the isotopic fractionation value during BNF (B value) of common bean cultivars grown in nutrient solution. Two experiments were conducted in greenhouse in nutrient solution without N, in the years 2012 and 2013 in Serop?dica, RJ. Three common bean cultivars were evaluated. Plant biomass was sampled at different growth stages, where the N natural abundance was analyzed. In 2012 experiment, the nodules showed positive ?15N, with deltas of 6.63? and 8.18? in Grafite and Radiante cultivars, respectively. The B value in shoot was ?0.919?, ?0.734? and ?0.724? for cultivars Grafite, Ouro Negro and Radiante, respectively. In 2013 experiment, the nodules showed great enrichment in 15N, with 9.11? and 8.36? deltas in Radiante and Grafite cultivars. The B value in shoot was -1.06? and -1.40? for cultivars Radiante and Grafite, respectively. The average B value in shoots of common bean cultivars was ?1.23?, and the B value of ?1.2 was used in the calculation of the BNF contribution in the field experiments of this study. Eight common bean cultivars were grown in field conditions in 2011 and 2012, with two N sources (inoculation with commercial inoculant with rizhobia or fertilization with 90 kg ha-1 of mineral N), with four replications, at Embrapa Rice and Bean in Santo Antonio de Goias, GO. Biomass of the shoot, root, nodules and grains were sampled, and total N and 15N natural abundance were analyzed in shoots and grains. In the experiment in 2011, the Ouro Negro cultivar produced higher nodule mass under inoculation, also at reproductive stages, and the Jalo Precoce and Radiante cultivars showed the lowest nodule mass. The grain yield of the eight cultivars was 1614 and 2942 kg ha-1 under inoculation or mineral N, respectively. Under inoculation, the Ouro Negro cultivar showed the highest N accumulation in shoots and grains and the highest grain yield, reaching 2200 kg ha-1, equivalent to 73% of yield under mineral N. In 2011, the N accumulation derived from BNF in grains of the eight cultivars ranged from 5.8 to 16.4 kg ha-1, which corresponded to a BNF contribution of 17% and 33% in Marfim and Ouro Negro cultivars, respectively. In 2012, the higher nodule mass occurred in Vereda and Estilo cultivars, in the first and second samples, respectively, whereas Radiante cultivar had the lowest nodule mass. The average grain yield of the eight cultivars was 3284 and 3919 kg ha-1 under inoculation and mineral N, respectively. The N accumulation derived from BNF in grains was 68.4, 38.7, and 21.6 kg ha-1 in Ouro Negro, Grafite and Radiante cultivars, respectively, corresponding to a BNF contribution of 49, 29 and 32%. The method of 15N natural abundance provided the identification of BNF potential for the supply of N in different common bean genotypes, arising as a tool for providing subsidies to guide breeding programs in order to increase BNF efficiency. / A t?cnica da abund?ncia natural de 15N para mensurar a fixa??o biol?gica de nitrog?nio (FBN) em leguminosas baseia-se no ligeiro enriquecimento de 15N observado nos solos em rela??o ? atmosfera e n?o exige a aplica??o de fertilizantes marcados. Os objetivos deste trabalho foram: quantificar, atrav?s da t?cnica da abund?ncia natural de 15N, a contribui??o da FBN em cultivares de feijoeirocomum (Phaseolus vulgaris) em condi??es de campo; avaliar o desempenho agron?mico dessas cultivares sob inocula??o; estimar o valor do fracionamento isot?pico durante a FBN (valor B) de cultivares de feijoeiro crescidas em solu??o nutritiva. Foram conduzidos dois experimentos em casa de vegeta??o, em solu??o nutritiva isenta de N, nos anos de 2012 e 2013, em Serop?dica, RJ. Foram avaliadas tr?s cultivares de feijoeiro, efetuando-se coletas de biomassa em diferentes est?dios e analisando-se a abund?ncia natural de 15N do material vegetal. No experimento em 2012, os n?dulos apresentaram ?15N positivo, com deltas de 6,63? e 8,18? nas cultivares Grafite e Radiante, respectivamente. O valor B da parte a?rea foi de ?0,92?, ?0,73? e ?0,72?, nas cultivares Grafite, Ouro Negro e Radiante, respectivamente. No experimento em 2013, os n?dulos apresentaram deltas de 9,11? e 8,36? nas cultivares Radiante e Grafite. O valor B da parte a?rea foi de ?1,06? e ?1,40? nas cultivares Radiantee Grafite, respectivamente. O valor B m?dio da parte a?rea das cultivares foi de ?1,23?, sendo o valor B de ?1,2 utilizado nos c?lculos da contribui??o da FBN nos experimentos de campo desse estudo. Oito cultivares de feijoeiro foram crescidas em condi??es de campo em 2011 e 2012, sob duas fontes de N (inocula??o com inoculante comercial com riz?bio ou aduba??o com 90 kg ha-1 de N mineral), com quatro repeti??es, na Embrapa Arroz e Feij?o, em Santo Antonio de Goi?s, GO. Foram efetuadas amostragens de biomassa de parte a?rea, raiz, n?dulos e gr?os e an?lises de N total e abund?ncia natural de 15N na parte a?rea e gr?os. No experimento em 2011, a cultivar Ouro Negro produziu maior massa de n?dulos sob inocula??o, inclusive nos est?dios reprodutivos, e as cultivares Jalo Precoce e Radiante apresentaram a menor massa de n?dulos. O rendimento m?dio de gr?os das oito cultivares foi de 1614 e 2942 kg ha-1 sob inocula??o ou N mineral, respectivamente. Sob inocula??o, a cultivar Ouro Negro apresentou os maiores ac?mulos de N na parte a?rea e nos gr?os e a maior produtividade, atingindo 2200 kg ha-1 de gr?os, equivalente a 73% da produ??o sob N mineral. Em 2011, o ac?mulo de N derivado da FBN nos gr?os das oito cultivares variou de 5,8 a 16,4 kg ha-1, que correspondeu a 17% e 33% de contribui??o da FBN nas cultivares Marfim e Ouro Negro, respectivamente. Em 2012, a maior massa de n?dulos ocorreu nas cultivares Vereda e Estilo, na primeira e segunda coletas, respectivamente, e a cultivar Radiante apresentou a menor massa de n?dulos. O rendimento m?dio de gr?os das oito cultivares foi de 3284 e 3919 kg ha-1 sobinocula??o e N mineral, respectivamente. O ac?mulo de N derivado da FBN nos gr?os foi de 68,4 kg ha-1 na cultivar Ouro Negro, 38,7 kg ha-1 na cultivar Grafite e 21,6 kg ha-1 na cultivar Radiante, que correspondeu a 49, 29 e 32% de contribui??o da FBN, respectivamente. A t?cnica da abund?ncia natural de 15N possibilita a identifica??o do potencial de FBN para o fornecimento de N em diferentes gen?tipos de feijoeiro, e mostra-se como uma ferramenta capaz de fornecer subs?dios para orientar os programas de melhoramento da esp?cie, visando aumentar a efici?ncia da FBN.

Hereditary hearing loss is the most common sensory disorder, affecting 1 in 500 newborns. There are more than 538 million individuals with genetic hearing loss worldwide and this number is expected to grow to 1 billion over the next three decades. Currently, the only option for individuals with hearing loss is mechanical intervention such as hearing aids or cochlear implants. In the past decade, many studies have highlighted the need for personalized gene therapy or molecular intervention to treat genetic deafness. However, in order to fulfill this vision a comprehensive understanding of the intricate mutation-gene-phenotype nuances and relationships is required. Toward this goal, we unraveled novel mutation-gene-phenotype associations and mechanisms in four deafness-causing genes (CIB2, COL11A1, CEACAM16 and DFNA5), by using a combination of in-depth phenotyping, human genetics, cutting edge genomic technologies, murine mutant models, and functional assays. These novel insights revealed mutations in CIB2 do not cause Usher Syndrome, mutations in COL11A1 can cause either non-syndromic or syndromic hearing loss, CEACAM16-related deafness is due to two distinct mechanisms, loss of function and gain of function, and coding variants can influence mRNA assembly and cause DFNA5-related hearing loss. Elucidating these novel mutation-gene-phenotype relationships has improved our knowledge of the pathogenic mechanisms underlying hearing loss and provided much needed answers to individuals seeking a diagnosis for their deafness. Recognizing the complexities associated with genetic hearing loss and the challenges in interpreting the clinical significance of genetic variants, we established the first deafness-specific variant database, the Deafness Variation Database (DVD), which classifies over 876,000 variants across 152 deafness-associated genes. This breadth of data provided us with a unique opportunity to explore the molecular landscape of deafness. We show that over 96% of coding variants are rare and novel and that mutational signatures are unique to each gene and are driven by minor allele frequency thresholds, variant effect, and protein domain. The mutational landscape we define shows complex gene-specific variability, making an understanding of these nuances foundational for improved accuracy in variant interpretation. Overall the work presented in this thesis improves our understanding of deafness biology, identifies novel targets for therapeutics and enhances clinical decision-making.

The purpose of this investigation was to explore the phenotypic and performance outcomes associated with ACTN3 and ACE polymorphisms. Ten trained men (age = 25.8 ± 3.0 years, height = 183.3 ± 4.1 cm, body mass = 92.3 ± 9.3 kg, and back squat to body mass ratio = 1.8 ± 0.3) participated. Blood samples were analyzed to determine ACTN3 and ACE polymorphisms. Standing ultrasonography images of the vastus lateralis (VL) were collected to determine whole muscle cross-sectional area (CSA-M), and a percutaneous muscle biopsy of the VL was collected to determine type I–specific CSA (CSA-T1), type II–specific CSA (CSA-T2), and type II to type I CSA ratio (CSA-R). Isometric squats were performed on force platforms with data used to determine peak force (IPF), allometrically scaled peak force (IPFa), and rate of force development (RFD) at various timepoints. One repetition maximum back squats were performed, whereby allometrically scaled dynamic strength (DSa) was determined. Cohen's d effect sizes revealed ACTN3 RR and ACE DD tended to result in greater CSA-M but differ in how they contribute to performance. ACTN3 RR's influence seems to be in the type II fibers, altering maximal strength, and ACE DD may influence RFD capabilities through a favorable CSA-R. Although the findings of the current investigation are limited by the sample size, the findings demonstrate the potential influence of ACTN3 and ACE polymorphisms on isometric and dynamic strength testing. This study may serve as a framework to generate hypotheses regarding the effect of genetics on performance.

Crayfish plague is an emerging disease caused by the oomycete Aphanomyces astaci, a pathogen listed among the 100 World's Worst Invasive Alien Species. It was introduced into Europe in the second half of 19th century from North America and caused collapses of European native crayfish populations. Nowadays, A. astaci is widespread in Europe and has spread also to other parts of the world, threatening all susceptible crayfish of non-North American origin. The aims of this MSc thesis were 1) to provide information about crayfish plague outbreaks from recent years, and by using microsatellite and mtDNA markers reveal A. astaci genotypes involved; 2) to test healthy-looking indigenous crayfish for potential occurrence of chronic infections by A. astaci in Czechia. Six new crayfish plague outbreaks were confirmed from 2016 to 2018, involving at least five distinct pathogen strains. My results provide first evidence of the A. astaci genotype group D causing Astacus astacus and Austropotamobius torrentium mass mortalities in Czechia. MtDNA sequencing revealed two haplotypes of the D haplogroup, indicating two independent sources of infection presumably either from ornamental crayfish or spreading from neighbouring countries. The genotype group A was recorded in two A. astacus mortalities and genotype group...

Because tuberculosis (TB) is a public health threat that continues to elude elimination in the United States, there is a need to identify contributing factors that may have implications for targeted control measures. Molecular studies of genetic clustering are crucial for pinpointing these contributing factors. It is for this reason this study was conducted. This was a non-experimental, cross-sectional population-based molecular epidemiological study of TB in SC from 2005 to 2011. Its purpose was to estimate the proportion of TB that may be due to recently acquired infection and to determine the risk factors associated with the genetic clustering of identical M. tuberculosis isolates from TB patients in South Carolina from 2005-2011. The analysis sample included 627 confirmed pulmonary and/or pleural cases of TB, for which complete data on all covariates and a valid genotype were available. The results strongly suggested that about 50% of TB in South Carolina is recently transmitted. The study also revealed that being born in the United States and Black race were independently and significantly associated with being part of a TB genotype cluster. The key messages of this study were as follows: a substantial portion of TB in South Carolina is due to recent transmission, not reactivation or importation, and transmission of TB in South Carolina occurs in groups often defined by American birth and Black race. These important findings indicate that most TB in South Carolina is preventable and that enhanced TB control efforts should be explored. The implication for positive social change is that employing targeted contact investigation informed by these findings could lead to decreased disease transmission. Future studies should explore pilot programs that investigate alternatives to the traditional TB contact investigation.

Thesis (M.Sc. (Chemical Pathology)) --University of Limpopo, 2013 / Objectives The present study investigated how the serum lipids responded to a high-fat meal in black South African subjects with different APOE genotypes, a population that until recently was reported to be consuming a traditional diet of low fat and high carbohydrates. Methods Sixty students (males and females) of the University of Limpopo, Turfloop Campus were successfully genotyped using Restriction Fragment Length Polymorphism (RFLP) and grouped into four APOE genotype groups; ε2, ε2/ε4, ε3 and ε4. Only thirty-three subjects volunteered to participate in the oral fat-tolerance test (OFTT), but two were excluded for having abnormal total cholesterol (6.05 mmol/l) and LDL cholesterol (3.12 mmol/l) so only 31 subjects were left. The numbers per group were ε2=5, ε2/ε4=8, ε3=9 and ε4=9. After an overnight fast blood was drawn for measurements of baseline serum parameters. Subjects were administered a high fat meal 30 minutes after the baseline blood sample was drawn. Blood was drawn at intervals of 20, 40, 60, 120, 180, 240, 300 and 360 minutes for measurements of postprandial serum parameter levels. Serum parameters measured were triglyceride, total cholesterol, low density lipoprotein cholesterol, high density lipoprotein cholesterol, glucose and insulin. Results Mean levels of serum lipids at baseline in mmol/l were as follows; group 1[TG=0.69(0.55-0.81), TCHOL=3.10±0.29, HDL-C=1.12±0.32, LDLC= 1.67±0.28]; group 2 [TG=0.61(0.53-1.00), TCHOL=2.98±0.53, HDLC= 1.20±0.37, LDL-C=1.43±0.37]; group 3 [TG=0.67(0.28-0.86), TCHOL=2.96±0.54, HDL-C=1.22±0.30, LDL-C=1.46±0.47]; group 4 [TG=0.76(0.51-1.16), TCHOL=3.27±0.51, HDL-C=1.12±0.17, LDLC= 1.79±0.47]. There was no significant difference in the mean levels of baseline triglyceride, total cholesterol, low density lipoprotein cholesterol, and ix high density lipoprotein cholesterol between the APOE groups hence no significant difference in the response to a fatty meal. Conclusions There was no significant change in serum lipid concentrations after a fatty meal in individuals with different APOE genotypes in a population that consume a traditional diet of low fat and high carbohydrates. Due to the small sample size, the results should be interpreted with caution. A larger study is recommended to ascertain the role of APOE genotypes on serum lipid response to a fatty meal in Black South African population.

Thesis (M.Sc. Agriculture (Crop Science)) -- University of Limpopo, 2011 / The nature and magnitude of the genotype by environment interactions is important to identify superior and stable genotypes under the target environments. This will assist to maximize specific adaptation and to speed up the transfer of new cultivars to growers. The objective of this study was to determine the stability of selected soybean genotypes with regards to the agronomic traits, high yield and nodule formation. Field experiments were conducted under dryland conditions during the 2007/2008 and 2008/2009 growing seasons at the University of Limpopo’s experimental farm (Syferkuil) and at a farmer’s field at Gabaza community, Mopani District near Tzaneen. Ten selected soybean cultivars were evaluated under a randomised complete block design with three replications. Stability was assessed via joint regression and superiority analyses. Significant differences were found for genotypes, environments and genotype by environment interactions. Stability analysis after Eberhart and Russell’s model suggested that the genotypes showed marked differences to environmental changes. The cultivar superiority measure for seed yield indicated that variety Clark was the most stable genotype with an average yield of 5235 kg/ha, followed by L81-4858 and Barc-2 that provided average yield of 4839 kg/ha and 4582 kg/ha, respectively. In terms of number of nodules Magoye was observed to be stable with average of five nodules per plant. Cultivar Barc-2 was found stable for number of active nodules with an average of 3.17 active nodules per plant. Most of the genotypes performed better at Syferkuil than at Gabaza. In general Barc-2 was found stable for yield and other agronomic traits considered in this study. This variety could be suitable for large scale production in these or other similar environments in Limpopo Province.

Thesis (M.Sc. (Chemical Pathology)) --University of Limpopo, 2013 / Objectives: The present study investigated how the serum lipids responded to a high-fat meal in black South African subjects with different APOE genotypes, a population that until recently was reported to be consuming a traditional diet of low fat and high carbohydrates. Methods: Sixty students (males and females) of the University of Limpopo, Turfloop Campus were successfully genotyped using Restriction Fragment Length Polymorphism (RFLP) and grouped into four APOE genotype groups; ε2, ε2/ε4, ε3 and ε4. Only thirty-three subjects volunteered to participate in the oral fat-tolerance test (OFTT), but two were excluded for having abnormal total cholesterol (6.05 mmol/l) and LDL cholesterol (3.12 mmol/l) so only 31 subjects were left. The numbers per group were ε2=5, ε2/ε4=8, ε3=9 and ε4=9. After an overnight fast blood was drawn for measurements of baseline serum parameters. Subjects were administered a high fat meal 30 minutes after the baseline blood sample was drawn. Blood was drawn at intervals of 20, 40, 60, 120, 180, 240, 300 and 360 minutes for measurements of postprandial serum parameter levels. Serum parameters measured were triglyceride, total cholesterol, low density lipoprotein cholesterol, high density lipoprotein cholesterol, glucose and insulin. Results Mean levels of serum lipids at baseline in mmol/l were as follows; group 1[TG=0.69(0.55-0.81), TCHOL=3.10±0.29, HDL-C=1.12±0.32, LDLC= 1.67±0.28]; group 2 [TG=0.61(0.53-1.00), TCHOL=2.98±0.53, HDLC= 1.20±0.37, LDL-C=1.43±0.37]; group 3 [TG=0.67(0.28-0.86), TCHOL=2.96±0.54, HDL-C=1.22±0.30, LDL-C=1.46±0.47]; group 4 [TG=0.76(0.51-1.16), TCHOL=3.27±0.51, HDL-C=1.12±0.17, LDLC= 1.79±0.47]. There was no significant difference in the mean levels of baseline triglyceride, total cholesterol, low density lipoprotein cholesterol, and high density lipoprotein cholesterol between the APOE groups hence no significant difference in the response to a fatty meal. Conclusions There was no significant change in serum lipid concentrations after a fatty meal in individuals with different APOE genotypes in a population that consume a traditional diet of low fat and high carbohydrates. Due to the small sample size, the results should be interpreted with caution. A larger study is recommended to ascertain the role of APOE genotypes on serum lipid response to a fatty meal in Black South African population.

This thesis principally addresses some problems in genetic programming (GP) and grammar-guided genetic programming (GGGP) arising from the lack of operators able to make small and bounded changes on both genotype and phenotype space. It proposes a new and flexible representation for genetic programming, using a state-of-the-art formalism from natural language processing, Tree Adjoining Grammars (TAGs). It demonstrates that the new TAG-based representation possesses two important properties: non-fixed arity and locality. The former facilitates the design of new operators, including some which are bio-inspired, and others able to make small and bounded changes. The latter ensures that bounded changes in genotype space are reflected in bounded changes in phenotype space. With these two properties, the thesis shows how some well-known difficulties in standard GP and GGGP tree-based representations can be solved in the new representation. These difficulties have been previously attributed to the treebased nature of the representations; since TAG representation is also tree-based, it has enabled a more precise delineation of the causes of the difficulties. Building on the new representation, a new grammar guided GP system known as TAG3P has been developed, and shown to be competitive with other GP and GGGP systems. A new schema theorem, explaining the behaviour of TAG3P on syntactically constrained domains, is derived. Finally, the thesis proposes a new method for understanding performance differences between GP representations requiring different ways to bound the search space, eliminating the effects of the bounds through multi-objective approaches.

Variations in gene expression have long been hypothesised to be the major cause of individual differences. An initial focus of this research thesis is to elucidate the genetic regulatory architecture of gene expression. Expression quantitative trait locus (eQTL) mapping analyses have been performed on expression levels of over 22,000 mRNAs from three tissues of a panel of recombinant inbred mice. These analyses are "single-locus" where "linkage" (i.e. significant correlation) between an expression trait and a putative eQTL is considered independently of other loci. Major conclusions from these analyses are: 1. Gene expression is mainly influenced by genetic (sequence) variations that act in trans rather than in cis; 2. Subsets of genes are controlled by master regulators that influence multiple genes; 3. Gene expression is a polygenic trait with multiple regulators. Single-locus mapping analyses are not designed for detecting multiple regulators of gene expression, and so observation of multiple-linkages (i.e. one expression trait mapped to multiple eQTLs) formed the basis of the second objective of this research project: to investigate the relationship between multiple-linkages and genotype pattern-association. A locus-pair is said to have associated genotype patterns if they have similar inheritance pattern across a panel of individuals, and these are attributed to one of fours sources: 1. linkage disequilibrium between loci located on the same chromosome; 2. non-syntenic association; 3. random association; 4. un-associated. To understand the validity of multiple-linkages observed in single-locus mapping studies, a newly developed method, bqtl.twolocus, is applied to confirm two-locus effects for a total of 898 out of 1,233 multiple-linkages identified from the three studies mentioned above as well as from seven publicly available eQTL-mapping studies. Combining these results with information of genotype pattern-association, a subset of 478 multiple-linkages has been deduced for which there is high confidence to be real.