UA Open Access Publishing Fund / Background: Current understanding of tuberculosis (TB) genotype clustering in the US is based on individual risk factors. This study sought to identify whether area-based socioeconomic status (SES) was associated with genotypic clustering among culture-confirmed TB cases. Methods: A retrospective cohort analysis was performed on data collected on persons with incident TB in King County, Washington, 2004–2008. Multilevel models were used to identify the relationship between area-level SES at the block group level and clustering utilizing a socioeconomic position index (SEP). Results: Of 519 patients with a known genotyping result and block group, 212 (41%) of isolates clustered genotypically. Analyses suggested an association between lower area-based SES and increased recent TB transmission, particularly among US-born populations. Models in which community characteristics were measured at the block group level demonstrated that lower area-based SEP was positively associated with genotypic clustering after controlling for individual covariates. However, the trend in higher clustering odds with lower SEP index quartile diminished when additional block-group covariates. Conclusions: Results stress the need for TB control interventions that take area-based measures into account, with particular focus on poor neighborhoods. Interventions based on area-based characteristics, such as improving case finding strategies, utilizing location-based screening and addressing social inequalities, could reduce recent rates of transmission.

Giardia lamblia is a eukaryotic parasite that causes diarrhea in humans worldwide. Diarrheal diseases cause stunting and mental retardation in children in developing nations, therefore it is important to understand the molecular epidemiology of G. lamblia. Compounding this, it is not clear if companion animals such as dogs contribute to infections in humans through zoonotic transmission. The genotypes of G. lamblia that have been found in humans are A1, A2 and B, while those in dogs have been on rare occasions all three human genotypes, but largely C and D, which have only been reported in dogs and appear to be species-specific. The molecular epidemiology of G. lamblia in humans and dogs was assessed in an endemic region of Lima, Peru. With one exception, dogs were found to harbor the C and D dog genotypes of G. lamblia. A single family dog was found to harbor a human genotype of G. lamblia. A2 and B genotypes of G. lamblia, but not A1, were found in humans in the endemic region. Previous literature reported that A2 and B typing within genotype tools were available, however the A2 samples from the endemic region could not be distinguished from one another through nucleotide polymorphism sequence analysis. A molecular typing technique was developed to type A2 samples. The extensive sequence analysis performed on two chromosomes of G. lamblia, yielded different phylogenetic tree groupings for the same samples. This lead to algorithmic analysis, which demonstrated a significantly high probability that meiotic recombination is occurring in the A2 samples of G. lamblia. As G. lamblia is largely believed to be asexual, the conclusion of doctoral research performed in this study yielded controversial, yet significant evidence that sex in G. lamblia A2 genotype samples is indeed occurring.

Single nucleotide polymorphisms (SNPs) and insertions/deletions (indels) constitute important sources of genetic variation which provide insight into disease aetiology and idiosyncratic differences in drug response. The analysis of such genetic variation relies upon the generation of allele-specific products, typically by enzymatic extension or the hybridization of allele-specific DNA probes. Herein, a distinct enzyme-free, dynamic chemistry-based method of producing allele-specific products for genotyping was developed. The approach was initially demonstrated in model systems using synthetic DNA, which was used as a template in a base-filling reductive amination reaction on a PNA backbone. The templated dynamic reaction between a free secondary amine at a ‘blank’ position on the PNA strand and four aldehyde-modified nucleobases drove selective formation of the ‘correct’ iminium intermediate according to Watson-Crick base-pairing rules. In a blind trial, the method was extended to genotype twelve cystic fibrosis patients for two mutations (one SNP and one indel) linked to this disease. Enzyme-free dynamic chemistry thus permitted successful genotyping in both singleplex and duplex formats, demonstrating the application of dynamic chemistry as a distinct method of allelediscrimination with certain advantages over those reported previously. The application of this method as a tool for the discovery of non-natural nucleobases with improved properties for antisense and genotyping applications was also investigated. Furthermore, progress was made towards the use of dynamic chemistry as a means of full nucleic acid sequence analysis, through the templated sequence-selective extension of PNA probes by reductive amination.

Doctor of Philosophy / Department of Agronomy / Tesfaye Tesso / The approach used to identify inbred lines that can produce superior hybrids is costly and time-consuming. It requires creation of all possible crosses and evaluation of the crosses to estimate combining abilities for the desired traits. Predicting heterosis or hybrid performance in any way possible may help to reduce the number of crosses to be made and evaluated. In this study, four sets of experiments were conducted to determine whether heterosis can be predicted based on inbred line performance, genetic distance between parents and genomic prediction model. The first experiment was aimed at assessing the levels of genetic diversity, population structure and linkage disequilibrium (LD) in 279 public sorghum inbred lines, based on 66,265 SNPs generated using the genotyping-by-sequencing (GBS) platform. The inbred lines were developed at different times over the last two decades and harbor robust diversity in pedigree and agronomic characteristics. Some of the inbreds are resistant to Acetolactate synthase (ALS) and Acetyl co-enzyme-A carboxylase (ACC) inhibitor herbicides. The mean polymorphic information content (PIC) and gene diversity across the entire inbreds were 0.35 and 0.46, respectively with non-herbicide resistant inbreds harboring more diversity than the herbicide resistant ones. The population structure analysis clustered the inbred lines into three major subgroups according to pedigree and fertility-reaction with the maintainer lines (B-lines) distinctly forming a separate cluster. Analysis of molecular variance (AMOVA) revealed more variation within subgroups than among subgroups. Substantial linkage disequilibrium (LD) was detected between the markers in the population with marked variation between chromosomes. This information may facilitate the use of the inbreds in sorghum breeding programs and provide perspectives for optimizing marker density for gene mapping and marker-assisted breeding. The second experiment, based on 102 F1 hybrids developed by intercrossing closely and distantly related inbreds, was conducted to investigate the relationship of genetic distance between parents with hybrid vigor or heterosis. The F1 hybrids alongside their parents were evaluated at two environments in a randomized complete block design with three replications. The results show that correlations of genetic distance between parents with hybrid performance and heterosis were variable and dependent on the trait. Though most were statistically non-significant and not strong to be used as predictor for heterosis, the results tend to show that certain level of genetic distance between parents is needed to capture maximum heterosis and hybrid performance. The objective of the third research study was to determine whether traits measured on parents can be used to predict hybrid performance in sorghum and to assess the combining ability of selected inbreds. Forty-six parental inbred lines and 75 F1 hybrids generated from intercrossing the inbreds were evaluated in four environments in a randomized complete block design with three replications. The average performance of the parents (mid-parent) was significantly correlated with hybrid performance for thousand kernel weight, days to flowering and plant height. Significant general (GCA) and specific (SCA) combining abilities were observed for most traits, with highly significant GCA effects observed for most traits as compared to SCA indicating that additive genetic effects are more important in affecting the inheritance of the traits measured. Results show that studying parental inbred line performance could generate important information for predicting hybrid performance in sorghum. The fourth experiment was aimed at assessing the efficacy of genomic prediction of hybrid performance in sorghum. Genomic prediction was performed with five-fold cross-validation procedure on 204 F1 hybrids developed using 102 inbred lines. A total of 66,265 SNP markers generated using genotyping-by-sequencing were used in this study. Results showed that increasing training population size increased prediction accuracies for all traits with the effect being different for different traits. Also, considering additive effects alone versus additive and dominance effects in the model showed similar trend of prediction accuracy but the full model (considering both additive and dominance effects of the markers) provided better prediction at least for some of the traits. The results suggest that genomic prediction could become an effective tool for predicting the performance of untested sorghum hybrids thus adding efficiency to hybrid selection.

Doctor of Philosophy / Genetics Interdepartmental Program / Jesse Poland / The Green Revolution is credited with saving billions of lives by effectively harnessing new genetic resources and breeding strategies to create high-yielding varieties for countries lacking adequate food security. To keep the next billion people in a state of food security, plant breeders will need to rapidly incorporate novel approaches and technologies into their breeding programs. The work presented here describes new genomic and phenomic strategies and tools aimed at accelerating genetic gain in plant breeding. Plant breeders have long relied on regional testing networks to evaluate new breeding lines across many locations. These are an attractive resource for both retrospective and contemporary analysis due to the vast amount of data available. To characterize genetic progress of plant breeding programs in the Central Plains, entries from the Southern Regional Performance Nursery dating back to 1992 were evaluated in field trials. The trend for annual improvement was 1.1% yr⁻¹, matching similar reports for genetic gain. During the same time period, growth of on-farm yields stagnated. Genomic selection, a promising method to increase genetic gain, was tested using historical data from the SRPN. A temporal-based model showed that, on average, yield predictions outperformed a year-to-year phenotypic correlation. A program-based model found that the predictability of a breeding program was similar when using either data from a single program or from the entire regional collection. Modern DNA marker platforms either characterize a small number of loci or profile an entire genome. Spiked genotyping-by-sequencing (sGBS) was developed to address the need in breeding programs for both targeted loci and whole-genome selection. sGBS uses a low-cost, integrated approach that combines targeted amplicons with reduced representation genotyping-by-sequencing. This approach was validated using converted and newly-designed markers targeting known polymorphisms in the leaf rust resistance gene Lr34. Plant breeding programs generate vast quantities of data during evaluation and selection of superior genotypes. Many programs still rely on manual, error-prone methods to collect data. To make this process more robust, we have developed several open-source phenotyping apps with simple, intuitive interfaces. A contemporary Green Revolution will rely on integrating many of these innovative technologies into modern breeding programs.

Scrapie ou paraplexia enzoótica dos ovinos é uma doença neurodegenerativa fatal que acomete ovinos e raramente caprinos. A doença é influenciada por polimorfismos nos códons 136, 154 e 171 do gene prnp que codifica a proteína priônica. Os animais podem ser susceptíveis ou resistentes, de acordo com as sequências alélicas observadas nos referidos códons. No Brasil ocorreram apenas casos de animais que foram importados, sendo o país considerado livre da doença. Neste trabalho foi realizada a genotipagem dos diferentes polimorfismos associados ao desenvolvimento do scrapie e a categorização em animais susceptíveis e resistentes. Foram sequenciadas 118 amostras provenientes de ovinos da raça Santa Inês criados em propriedades localizadas no Estado de São Paulo. Destas amostras foram identificados 6 alelos e 11 genótipos (ARQ/ARQ, ARR/ARQ, ARQ/AHQ, ARQ/VRQ, AHQ/AHQ, ARR/ARR, ARR/AHQ, VRQ/VRQ, ARQ/TRQ, TRR/TRR, TRQ/TRQ), dentre os quais o genótipo ARQ/ARQ teve ocorrência de 56,7%. Em nosso estudo foi detectada a presença da tirosina no códon 136, observação rara na medida em quenão existem relatos nacionais e relatos envolvendo a raça Santa Inês descrevendo este polimorfismo. Com os resultados obtidos, foi possível determinar a existência de grande variabilidade genética relacionada à raça Santa Inês no Estado de São Paulo, apesar da variabilidade, apenas 1,69% dos genótipos observados são extremamente resistentes ao scrapie. Estes dados demonstram que a raça nativa Santa Inês pode ser considerada potencialmente susceptível ao scrapie. / Enzootic paraplexia or scrapie is a fatal neurodegenerative disease affecting mainly sheep and rarely goats. The disease is influenced by polymorphisms at codons 136, 154 and 171 of prnp gene that encodes the prion protein. The animals may be susceptible or resistant to the development of the disease according to the allelic sequences observed in these codons. In Brazil there were only cases of scrapie in imported animals, therefore the country is considered free of the disease. This study performed the genotyping of different polymorphisms associated to the development of scrapie. Then, based on these findings the animals were categorized in resistant and susceptible. A total of 118 samples were sequenced from the Santa Ines sheep raised on properties located in the State of Sao Paulo. From these samples, 6 alleles and 11 genotypes were identified (ARQ / ARQ, ARR / ARQ, ARQ / AHQ, ARQ / VRQ, AHQ / AHQ, ARR / ARR, ARR / AHQ, VRQ / VRQ, ARQ / TRQ, TRR / TRR, TRQ / TRQ), the genotype ARQ / ARQ presented a frequency of 56.7%. It was also detected the presence of tyrosine at codon 136, which may be considered a rare observation, since there is no report regarding Santa Ines breeding presenting this polymorphism. These results showed the great genetic variability in Santa Ines in Sao Paulo and only 1,69% of the genotypes observed are extremely resistant to scrapie. These data demonstrate that the Santa Ines sheep can be considered potentially susceptible to scrapie.

Scrapie ou paraplexia enzoótica dos ovinos é uma doença neurodegenerativa fatal que acomete ovinos e raramente caprinos. A doença é influenciada por polimorfismos nos códons 136, 154 e 171 do gene prnp que codifica a proteína priônica. Os animais podem ser susceptíveis ou resistentes, de acordo com as sequências alélicas observadas nos referidos códons. No Brasil ocorreram apenas casos de animais que foram importados, sendo o país considerado livre da doença. Neste trabalho foi realizada a genotipagem dos diferentes polimorfismos associados ao desenvolvimento do scrapie e a categorização em animais susceptíveis e resistentes. Foram sequenciadas 118 amostras provenientes de ovinos da raça Santa Inês criados em propriedades localizadas no Estado de São Paulo. Destas amostras foram identificados 6 alelos e 11 genótipos (ARQ/ARQ, ARR/ARQ, ARQ/AHQ, ARQ/VRQ, AHQ/AHQ, ARR/ARR, ARR/AHQ, VRQ/VRQ, ARQ/TRQ, TRR/TRR, TRQ/TRQ), dentre os quais o genótipo ARQ/ARQ teve ocorrência de 56,7%. Em nosso estudo foi detectada a presença da tirosina no códon 136, observação rara na medida em quenão existem relatos nacionais e relatos envolvendo a raça Santa Inês descrevendo este polimorfismo. Com os resultados obtidos, foi possível determinar a existência de grande variabilidade genética relacionada à raça Santa Inês no Estado de São Paulo, apesar da variabilidade, apenas 1,69% dos genótipos observados são extremamente resistentes ao scrapie. Estes dados demonstram que a raça nativa Santa Inês pode ser considerada potencialmente susceptível ao scrapie. / Enzootic paraplexia or scrapie is a fatal neurodegenerative disease affecting mainly sheep and rarely goats. The disease is influenced by polymorphisms at codons 136, 154 and 171 of prnp gene that encodes the prion protein. The animals may be susceptible or resistant to the development of the disease according to the allelic sequences observed in these codons. In Brazil there were only cases of scrapie in imported animals, therefore the country is considered free of the disease. This study performed the genotyping of different polymorphisms associated to the development of scrapie. Then, based on these findings the animals were categorized in resistant and susceptible. A total of 118 samples were sequenced from the Santa Ines sheep raised on properties located in the State of Sao Paulo. From these samples, 6 alleles and 11 genotypes were identified (ARQ / ARQ, ARR / ARQ, ARQ / AHQ, ARQ / VRQ, AHQ / AHQ, ARR / ARR, ARR / AHQ, VRQ / VRQ, ARQ / TRQ, TRR / TRR, TRQ / TRQ), the genotype ARQ / ARQ presented a frequency of 56.7%. It was also detected the presence of tyrosine at codon 136, which may be considered a rare observation, since there is no report regarding Santa Ines breeding presenting this polymorphism. These results showed the great genetic variability in Santa Ines in Sao Paulo and only 1,69% of the genotypes observed are extremely resistant to scrapie. These data demonstrate that the Santa Ines sheep can be considered potentially susceptible to scrapie.

Submitted by Rosivalda Pereira (mrs.pereira@ufma.br) on 2017-09-12T19:35:08Z No. of bitstreams: 1 WalnaRamos.pdf: 1885249 bytes, checksum: a716015a5d512aaba735cf4af13d0e13 (MD5) / Made available in DSpace on 2017-09-12T19:35:08Z (GMT). No. of bitstreams: 1 WalnaRamos.pdf: 1885249 bytes, checksum: a716015a5d512aaba735cf4af13d0e13 (MD5) Previous issue date: 2017-04-28 / Human papillomavirus (HPV) is the etiologic agent of one of the most common sexually transmitted infections (STIs). According to the oncogenic potential, it is classified in low risk and high risk; high risk subtypes cause lesions that may progress to invasive carcinomas and has been recognized as a possible etiologic agent in penile carcinoma. Brazil has one of the highest incidences of penile cancer in the world. The general objective of this work is associate HPV infection with the histopathological profile of squamous cell carcinomas in patients from the State of Maranhão. This is a prospective analytical study of 29 samples of patients who underwent penectomy surgery at the Aldenora Bello Institute of Oncology of Maranhão. Sociodemographic data were collected through a questionnaire applied to the patients, clinical and histopathological data were collected from the medical records. Tumor samples were collected during penectomy surgeries, and the following steps were performed at the laboratory: DNA extraction and quantification, Nested PCR with PGMY and GP + primers for HPV detection, visualization of amplified products, purification of the PCR product and automated sequencing. The sequencing products were decoded through the BLAST program. Data were analyzed through the SPSS statistical program, with a 5% level of significance. It was observed that the majority of the men were over 60 years of age (69%), had stable marital union (75.9%), family income of up to one minimum wage (75.9%), were illiterate (55. 2%), lived in countryside towns (79.3%), reported using tobacco at some moment in their lives (62%). Regarding sexual habits, 62.1% reported not having performed circumcision, 41.4% had a previous STD, 72.4% had never used a condom, 48.3% had the first intercourse before age 18. The presence of HPV was detected in 69% of the cases, among the viral types, HPV 16 was the most prevalent (55.5%). Regarding to lesion, 51.7% of the cases presented more than one affected region, and the glans was affected in 93.1% of the cases. The most prevalent type of lesion was ulceration (51.7%). Among the histopathological classifications, 41.4% of the samples were classified in degree I of the Broders scale, 27.7% in stage T1 of the TNM staging and according to the Jackson classification, both stages I and II presented the same prevalence, 27.6% of samples. There was no statistically significant difference between the variables evaluated and the presence of HPV. Low education, low income and no circumcision were highly observed in the study, being possible to correlate such variables with the increase of the risk of penile cancer development. The presence of HPV in 69% of the samples and the prevalence of high risk subtypes suggest the need for more actions to prevent the spread of HPV over the population / O papilomavírus humano (HPV) é o agente etiológico de umas das infecções sexualmente transmissíveis (ISTs) mais comuns. É classificado de acordo com o potencial oncogênico em baixo risco e alto risco, os subtipos de alto risco causam lesões que podem progredir para carcinomas invasivos e vem sendo reconhecido como possível agente etiológico no carcinoma peniano. O Brasil tem uma das maiores incidências de câncer de pênis do mundo. O objetivo geral deste trabalho é associar a infecção do HPV com o perfil histopatológico de carcinomas epidermóides penianos em pacientes do Estado do Maranhão. Trata-se de um estudo analítico prospectivo de 29 amostras de pacientes que foram submetidos a cirurgia de penectomia no Instituto Maranhense de Oncologia Aldenora Bello. Dados sociodemográficos foram coletados através de um questionário aplicado aos paciente, os dados clínicos e histopatológicos foram coletados dos prontuários. As amostras tumorais foram coletadas durante as cirurgias de penectomia, e em laboratório foram realizadas as etapas de: extração e quantificação do DNA, PCR Nested com os primers PGMY e GP+ para detecção do HPV, visualização dos produtos amplificados, purificação do produto da PCR e sequenciamento automatizado. Os produtos do sequenciamento foram decodificados através do programa BLAST. Os dados foram analisados através do programa estatístico SPSS, o nível de significância foi de 5%. Foi observado que a maioria dos homens estava na faixa etária acima de 60 anos (69%), apresentavam união conjugal estável (75,9%), renda familiar de até um salário mínimo (75,9%), eram analfabetos (55,2%), residiam em municípios do interior do Estado (79,3%), relataram o uso de tabaco em algum momento da vida (62%). Em relação aos hábitos sexuais, 62,1% relataram não ter realizado circuncisão, 41,4 % a ocorrência de DST prévia, 72,4% nunca fizeram uso de preservativo, 48,3% tiveram o 1º coito antes dos 18 anos. A presença de HPV foi detectada em 69% dos casos, dentre os tipos virais, o HPV 16 foi o mais prevalente (55,5%). Em relação a lesão, 51,7% dos casos apresentou mais de uma região afetada, estando a glande afetada em 93,1% dos casos. O tipo de lesão mais prevalente foi a do tipo ulceração (51,7%). Dentre as classificações histopatológicas, 41,4% das amostras foram classificadas no grau I da escala de Broders, 27,7% no estágio T1 do estadiamento TNM e na classificação de Jackson, os estágios I e II apresentam a mesma prevalência, 27,6% das amostras. Não houve diferença estatisticamente significante entre as variáveis avaliadas e a presença de HPV. A baixa escolaridade, a baixa renda e a não circuncisão foram observados no estudo com um percentual elevado, sendo possível correlacionar tais variáveis com o aumento no risco de desenvolvimento do câncer de pênis. A presença de HPV em 69% das amostras e a prevalências dos subtipos de alto risco sugerem a necessidade de mais ações de prevenção da disseminação desse vírus na população.

<p>About 30 % of all infertile couples suffer from infertility of an unexplained cause. Leukemia inhibitory factor (LIF) is a glycoprotein produced by the endometrium and is an important cytokine in the implantation process. LIF exerts its biological functions through heterodimerization of its two receptors: LIF receptor (LIFR) and gp130. Point mutations in the LIF gene have been associated with female infertility. The aim of this study was to investigate whether single nucleotide polymorphisms (SNPs) in the genes of LIFR and gp130 could cause reduced fertility in women. To this end, 115 samples from women diagnosed with unexplained infertility and 191 samples from fertile women were studied. Three SNPs in the gp130 gene and two SNPs in the LIFR gene were analyzed using real-time PCR. One significant difference and a tendency to difference were detected in the gp130 gene for women with unexplained infertility. There were no differences in the LIFR gene variations. In conclusion, polymorphisms in gp130, and thereby disturbances in the LIF pathway, could be one cause for infertility in women diagnosed with unexplained infertility.</p>

About 30 % of all infertile couples suffer from infertility of an unexplained cause. Leukemia inhibitory factor (LIF) is a glycoprotein produced by the endometrium and is an important cytokine in the implantation process. LIF exerts its biological functions through heterodimerization of its two receptors: LIF receptor (LIFR) and gp130. Point mutations in the LIF gene have been associated with female infertility. The aim of this study was to investigate whether single nucleotide polymorphisms (SNPs) in the genes of LIFR and gp130 could cause reduced fertility in women. To this end, 115 samples from women diagnosed with unexplained infertility and 191 samples from fertile women were studied. Three SNPs in the gp130 gene and two SNPs in the LIFR gene were analyzed using real-time PCR. One significant difference and a tendency to difference were detected in the gp130 gene for women with unexplained infertility. There were no differences in the LIFR gene variations. In conclusion, polymorphisms in gp130, and thereby disturbances in the LIF pathway, could be one cause for infertility in women diagnosed with unexplained infertility.