Hierarchical sampling from populations, incipient and recognized varieties within Phacelia dubia and P. maculata has revealed high levels of intraspecific polymorphism in chloroplast DNA. Much of the variation is partitioned between populations as evidenced by population-specific variants at fixation in all three populations of P. dubia var. interior and in both populations of P. maculata. Nine of 16 populations were polymorphic for cpDNA haplotypes. A total of 16 haplotypes was found in a sample of 106 individuals; the most common occurred in eight of the 16 populations and in 31 per cent of the individuals in the entire sample. A phylogenetic analysis revealed four basic plastome types. The two major groups of plastomes were separated by four independent base-pair mutations which suggests an ancient split in the evolution of plastid genomes. Representatives from each major plastome division were found in each of five populations spanning two allopatric varieties of P. dubia. The geographical distribution of haplotypes and lack of evidence for recent admixture argue against migration as a source of the polymorphism. It is more likely that the current taxonomic varieties are descendants of a polymorphic common ancestor.

Arsenic contamination of the environment is a worldwide health hazard. This research project focused on four areas: development and testing of low cost, field portable devices capable of measuring levels of arsenic at 10 μg L-1 or less; specific chemical techniques for such testing; creation of educational tools and techniques to allow operators who lack advanced chemistry training to perform accurate testing; and the determination and use of a biomarker in DNA as a cancer predictor in individuals exposed to environmental arsenic. The analytical techniques explored include: (1) the Gutzeit method of arsenic determination though arsine gas production, which was investigated in three experiments: measuring arsenic levels in soil samples, using Gutzeit-based kits using silver nitrate as a reactant for arsine gas, and sensitivity comparison of three commercial test kits over varying time periods up to twenty-four hours. (2) The molybdenum blue method, technologically quantified through three different experiments: digital photographic analysis, spectroscopic analysis, and flow injection. (3) Filtration of arsenic contaminated water with wood-ash, sand, ferric oxide, and commercially available steel wool; and the construction of a filtering device constructed of recyclable discarded soda bottles. Further, single nucleotide polymorphisms in the DNA of arsenic exposed individuals were studied to determine what immune response genes might be implicated in arsenic susceptibility. The major conclusions of this research were: digital image analysis used with the Gutzeit method improves precision and accuracy; silver nitrate proved to be a better measurement tool at low concentrations of arsenic than mercuric bromide; and the Gutzeit method can be applied to soils in the Hach kit.

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

No description available.

No description available.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Cytokines mediate crucial functions in innate and adaptive immunity. They play valuable roles in immune cell growth and lineage specification, and are associated with various disease pathologies. A large number of low, medium and high throughput studies have implicated association of single nucleotide polymorphisms (SNPs) in cytokine genes with diseases. A preponderance of such experiments have not shown any causality of an identified SNP to the associated disease. Instead, they have identified statistically significant SNP-disease associations; hence, it is likely that some of these cytokine gene variants may directly or indirectly cause the disease phenotype(s). To fill this knowledge gap and derive study parameters for cytokine SNP-disease causality relationships, we have designed and developed the Disease Associated Cytokine SNP Database (DACS-DB). DACS-DB has data on 456 cytokine genes, approximately 61,000 SNPs, and 891 SNP-associated diseases. In DACS-DB, among other attributes, we present functional annotation, and heterozygosity allele frequency for the SNPs, and literature-validated SNP association for diseases. Users of the DB can run queries such as the ones to find disease-associated SNPs in a cytokine gene, and all the SNPs involved in a disease. We have developed a web front end (available at http://www.iupui.edu/~cytosnp) to disseminate this information for immunologists, biomedical researchers, and other interested biological researchers. Since there is no such comprehensive collection of disease associated cytokine SNPs, this DB will be vital to understanding the role of cytokine SNPs as markers in disease, and, more importantly, in causality to disease thus helping to identify drug targets for common inflammatory diseases. Due to the presence of rich annotations, the DACS-DB can be a good source for building a tool for the prediction of the "disease association potential (DAP)" of a given SNP. In a preliminary effort to devise such a methodology for DAP prediction, we have applied a support vector machine (SVM) to classify SNPs. Employing the SNP attributes of function class, heterozygosity value, and heterozygosity standard error, 864 SNPs were classified into two classes, "disease" and "non-disease". The SVM returned a classification of these SNPs into the disease and non-disease classes with an accuracy of 74%. By modifying various SNP and disease attributes in the training data sets, such a predictive algorithm can be extrapolated to identify potential disease associated SNPs among newly sequenced cytokine variations. In the long run, this approach can provide a means for future gene variation based therapeutic regimens.

O gênero Yersinia pertence à família Enterobacteriaceae e compreende 17 espécies. Y. pestis, Y. pseudotuberculosis e Y. enterocolitica são reconhecidamente patógenos de humanos e animais. Y. pestis cause a peste. Y. pseudotuberculosis e Y. enterocolitica são agentes causadores, sobretudo, de gastroenterites transmitidas por água e alimentos. As demais 14 espécies são, usualmente, consideradas não-patogênicas, com exceção de Y. ruckeri sorogrupo O:1 que causa infecções em peixes. Nas últimas décadas, a tipagem molecular tornou-se uma importante ferramenta nos estudos filogenéticos de numerosos micro-organismos e o desenvolvimento de sistemas de tipagem rápidos e baratos pode facilitar os estudos epidemiológicos de infecções bacterianas. No presente estudo objetivou-se desenvolver um método de genotipagem de Yersinia spp. baseado em high-resolution melting analysis (HRMA) para diferenciar os single-nucleotide polymorphisms (SNPs) presentes nas sequências dos genes 16S rRNA, glnA, gyrB, hsp60 e recA e aplicá-lo na tipagem de 40 linhagens de Y. pseudotuberculosis e 50 linhagens de Y. enterocolitica, bem como separar por HRMA as espécies Y. pseudotuberculosis e Y. enterocolitica. Os SNPs foram determinados nas sequências dos loci acima citados a partir de um conjunto de 119 linhagens de Yersinia spp. depositadas no GenBank/EMBL/DDBJ. Foram encontrados nas sequências dos genes analisados de Y. pseudotuberculosis, Y. enterocolitica, Y. bercovieri, Y. rohdei, Y. intermedia, Y. mollaretii e Y. ruckeri 10, 10, 9, 6, 4, 1 e 1 SNPs, respectivamente. Nenhum SNP foi encontrado nas sequências analisadas de Y. pestis e um grande número de SNPs foi encontrado nas sequências analisadas de Y. frederiksenii, Y. kristensenii e Y. massiliensis, o que impossibilitou a genotipagem dessas espécies por HRMA. As demais espécies não foram analisadas. Foram desenhados pares de primers para flanquear os SNPs encontrados em cada espécie de Yersinia testada. Usando um conjunto de primers espécie-específicos, a diversidade genética de cada espécie de Yersinia foi determinada por HRMA e a análise filogenética foi baseada na sequência concatenada composta pelos nucleotídeos identificados em cada fragmento analisado. O agrupamento foi realizado com o software BioNumerics usando o método UPGMA com 1.000 replicatas de bootstrap. A árvore filogenética ii construída para Y. pseudotuberculosis agrupou as linhagens em clusters bio-sorogrupo específicos. As linhagens do bio-sorogrupo 1/O:1 foram agrupadas em um cluster e as linhagens do bio-sorogrupo 2/O:3 em outro. A árvore filogenética construída para Y. enterocolitica agrupou as linhagens em três grupos. As linhagens altamente patogênicas, do biotipo 1B, foram agrupadas em um cluster, as linhagens de média patogenicidade, dos biotipos 2, 3, 4 e 5, foram agrupadas em um segundo cluster e as linhagens consideradas nãopatogênicas, do biotipo 1A, foram agrupadas em um terceiro cluster. O agrupamento encontrado em Y. pseudotuberculosis e Y. enterocolitica foi consistente com o perfil patogênico característico dessas duas espécies. Nenhuma correlação epidemiológica significativa foi encontrada no agrupamento de Y. bercovieri, Y. rohdei, Y. intermedia, Y. mollaretii e Y. ruckeri de acordo com os resultados de HRMA. Ademais, o método de HRMA aqui desenvolvido foi capaz de separar as espécies Y. pseudotuberculosis e Y. enterocolitica. O método de HRMA desenvolvido nesse estudo pode ser usado como uma alternativa para a genotipagem e para a diferenciação de Y. pseudotuberculosis de Y. enterocolitica. Esse método também pode complementar os métodos baseados em sequências e facilitar os estudos epidemiológicos dessas duas espécies de Yersinia. / The genus Yersinia belongs to the family Enterobacteriaceae and comprises 17 species. Y. pestis, Y. pseudotuberculosis and Y. enterocolitica are well recognized human and animal pathogens. Y. pestis causes plague. Y. pseudotuberculosis and Y. enterocolitica are, usually, causative agents of food-waterborne gastroenteritis. The other 14 Yersinia species are considered to be non-pathogenic, with the exception of Y. ruckeri serogroup O:1 which causes infections in fishes. In the last few decades, molecular typing has become an important tool in phylogenetic studies of several microorganisms and the development of fast and inexpensive typing systems can facilitate epidemiological studies of bacterial infections. The present study aimed to develop a method of Yersinia spp. genotyping based on high-resolution melting analysis (HRMA) in order to differentiate the single-nucleotide polymorphisms (SNPs) present in the 16S rRNA, glnA, gyrB, hsp60 and recA sequences and apply it in the typing of 40 Y. pseudotuberculosis strains and 50 Y. enterocolitica strains, as well as, to separate by HRMA the Y. pseudotuberculosis and Y. enterocolitica species. The SNPs were determined in the sequences of the aforementioned loci using a set of 119 Yersinia strains deposited in the GenBank/EMBL/DDBJ database. It were found in the gene sequences analyzed of Y. pseudotuberculosis, Y. enterocolitica, Y. bercovieri, Y. rohdei, Y. intermedia, Y. mollaretii and Y. ruckeri 10, 10, 9, 6, 4, 1 and 1 SNPs, respectively. No SNPs was found in the analyzed sequences of Y. pestis and a large number of SNPs were found in the analyzed sequences of Y. frederiksenii, Y. kristensenii and Y. massiliensis what prevented their genotyping by HRMA. The remaining Yersinia species were not analyzed. It was designed primer pairs to flank the SNPs found in each Yersinia species tested. Using a specie-specific set of primers, the genetic diversity of each Yersinia species used was determined by HRMA and the phylogenetic analysis was based on the concatenated sequence composed by the nucleotides identified in each fragment analyzed. Clustering was performed with the software package BioNumerics using UPGMA method and 1,000 bootstrap replicates. The phylogenetic tree constructed for Y. pseudotuberculosis grouped the strains into bio-serogroups specific clusters. The strains of 1/O:1 bio-serogroup were grouped into one cluster and the strains of 2/O:3 bio-serogroup into iv other cluster. The phylogenetic tree constructed for Y. enterocolitica grouped the strains in three clusters. The highly pathogenic strains, of biotype 1B, were grouped into one cluster, the moderate pathogenic strains, of biotypes 2, 3, 4 and 5, were grouped into a second cluster and, the non-pathogenic strains, of biotype 1A, were grouped into a third cluster. The clusterization of Y. pseudotuberculosis and Y. enterocolitica were consistent with the pathogenic profile characteristic of these two Yersinia species. No significant epidemiological correlation was found in the grouping of Y. bercovieri, Y. rohdei, Y. intermedia Y. mollaretii and Y. ruckeri according to HRMA results. Moreover, the HRMA-based method develop here was able to separate the Y. pseudotuberculosis and Y. enterocolitica species. The HRMA assay developed in this study can be used as an alternative for the genotyping and the differentiation of Y. pseudotuberculosis and Y. enterocolitica. This method can also complement sequence-based methods and facilitate epidemiological studies of these two Yersinia species.

A genética forense vem se desenvolvendo cada vez mais, com novas tecnologias e implementação de novos conjuntos de marcadores de DNA com maiores níveis de informatividade. Os marcadores genéticos são amplamente usados na identificação humana, pois permitem distinguir indivíduos com alta acurácia. Duas classes de marcadores muito utilizadas atualmente são os STRs (Short Tandem Repeats) e os SNPs (Single Nucleotide Polymorphisms). Os STRs são altamente informativos e, portanto, úteis para a prática forense. Kits mais novos como GlobalFiler (Thermo Fisher Scientific) e PowerPlex Fusion System (Promega) apresentam a análise de mais de 20 loci STRs de uma só vez. Já os SNPs, por possuírem sua informatividade mais reduzida (necessita de mais loci analisados), são menos utilizados, porém apresentam vantagem em amostras degradadas de DNA; assim, conjuntos de identificação como o 52-plex desenvolvido pelo consórcio SNPforID e o conjunto IISNPs, vêm sendo estudados em várias populações do mundo. Com o desenvolvimento de técnicas de sequenciamento de nova geração (NGS Next Generation Sequencing) para análise de DNA, a obtenção de perfis de DNA se tornou mais acurada. Algumas plataformas permitem gerar perfis de até 96 indivíduos simultaneamente. Este estudo tem por objetivo principal analisar 171 marcadores genéticos (Amelogenina, Y-INDEL, 30 STRSs e 139 SNPs) em 340 indivíduos miscigenados da região da cidade de Ribeirão Preto (SP) utilizando a plataforma de sequenciamento de nova geração MiSeq Personal Sequencer (Illumina Inc.), bem como calcular as frequências alélicas e genotípicas, verificar a aderência ao equilíbrio de HardyWeinberg e estimar parâmetros forenses para os diferentes conjuntos de marcadores. Análises de ancestralidade foram realizadas para os conjuntos de SNPs. Para o preparo das bibliotecas de amostras a serem sequenciadas, foi utilizado o kit HaloPlex (Agilent Technologies, Inc), onde foram incluídos os marcadores dos kits GlobalFiler e PowerPlex Fusion System, e os SNPs existentes no conjunto do consórcio SNPforID (52-plex) e IISNPs (92 SNPs). De todos os marcadores incluídos no ensaio, apenas um SNP (rs763869) presente no conjunto SNPforID não pôde ser analisado devido a questões técnicas. Dos 139 SNPs analisados apenas seis apresentaram desvios significativos em relação ao equilíbrio de Hardy-Weinberg,número este esperado devido ao acaso. Os conjuntos de SNPs apresentam elevada informatividade com Probabilidade de Match de 6,48 x 10-21 (52-plex) a 4,91 x 10-38 (IISNP), e Poder de Exclusão de 0,9997 (52-plex) e 0,99999997 (IISNP). De modo geral, as inferências de ancestralidade obtida utilizando estes conjuntos, indicaram elevada contribuição europeia (superior a 70%) e baixa contribuição ameríndia (inferior a 10%) na população, enquanto que as análises de mistura individual se mostraram consistentes, com a maioria dos indivíduos apresentando elevada ancestralidade europeia. Os resultados dos marcadores relativos ao sexo (Amelogenina, Y-INDEL e DYS391) foram consistentes com o sexo dos doadores das amostras. As frequências alélicas e parâmetros forenses foram calculados para os STRs, revelando uma alta informatividade. A Probabilidade de Match combinada e o Poder de Exclusão combinado foram de 1,19 x 10-36 e 0,999999999997 respectivamente. Dos 29 STRs autossômicos presentes, seis apresentaram desvios ao equilíbrio de Hardy-Weinberg, refletindo possíveis falhas no sequenciamento e genotipagem destes marcadores / The field of forensic genetics has developed increasingly with the implementation of new sets of DNA markers with higher levels of informativeness. The genetic markers are widely used in human identification as they allow distinguishing individuals with high accuracy. Two of the most commonly used markers are the Short Tandem Repeats (STRs) and the Single Nucleotide Polymorphisms (SNPs). Newer kits such as GlobalFiler (Thermo Fisher Scientific) and PowerPlex Fusion System (Promega) can analyze more than 20 STRs loci at once. When comparing with STRs, the SNPs are less informative and many more loci are needed to reach the same informativeness of STR kits. However, they are advantageous when using degraded DNA samples. The identification sets such as the 52-plex developed by the SNPforID Consortium and the IISNPs have been analyzed in many worldwide populations. With the development of next generation sequencing techniques (NGS Next Generation Sequencing), obtaining DNA profiles has become more accurate and some platforms allow generating profiles of up to 96 individuals simultaneously. The main goal of this study is to analyze 171 markers (Amelogenin, Y-INDEL, 30 STRs and 139 SNPs) in 340 admixed individuals from Ribeirão Preto, SP, using the NGS platform MiSeq Personal Sequencer (Illumina Inc.). This will allow the calculation of allele and genotype frequencies, the verification of adherence to Hardy-Weinbergs equilibrium and the estimation of forensic parameters for each set of marker. Ancestry analysis was performed for the sets of SNPs. The HaloPlex kit (Agilent Technologies, Inc) was used for library preparation including the STRs from the kits GlobalFiler and PowerPlex Fusion System and the SNPs from the SNPforID consortium (52-plex) and IISNPs (92 SNPs) identification sets. A single SNP (rs763869) from the SNPforID set was not analyzed due to technical issues. Only six of the 139 analyzed SNPs presented significant deviation from the Hardy-Weinberg equilibrium expectations, which is expected by chance alone. The SNPs sets exhibited high informativeness, with matchprobability ranging from 6.48 x 10-21 (52-plex) to 4.91 x 10-38 (IISNPs) and exclusion power of 0.9997 (52-plex) and 0.99999997 (IISNPs). In general, ancestry estimates obtained using these sets indicated a high European contribution (higher than 70%) and low Amerindian contribution (less than 10%) in the population sample, while the individual admixture analyses exhibited were highly consistent, with the majority of individuals presenting high European ancestry. The results of the sex markers (Amelogenin, Y-INDEL and DYS391) were in agreement with the reported sexes from sample donors. The allele frequencies and forensic parameters calculated for the STRs revealed high informativeness. The combined match probability and the combined exclusion power were 1.19 x 10-36 and 0.999999999997 respectively. Six of the 29 autosomal STRs presented significant deviations from the HardyWeinberg equilibrium expectations, reflecting possible failures in sequencing and genotyping of these markers

A genética forense vem se desenvolvendo cada vez mais, com novas tecnologias e implementação de novos conjuntos de marcadores de DNA com maiores níveis de informatividade. Os marcadores genéticos são amplamente usados na identificação humana, pois permitem distinguir indivíduos com alta acurácia. Duas classes de marcadores muito utilizadas atualmente são os STRs (Short Tandem Repeats) e os SNPs (Single Nucleotide Polymorphisms). Os STRs são altamente informativos e, portanto, úteis para a prática forense. Kits mais novos como GlobalFiler (Thermo Fisher Scientific) e PowerPlex Fusion System (Promega) apresentam a análise de mais de 20 loci STRs de uma só vez. Já os SNPs, por possuírem sua informatividade mais reduzida (necessita de mais loci analisados), são menos utilizados, porém apresentam vantagem em amostras degradadas de DNA; assim, conjuntos de identificação como o 52-plex desenvolvido pelo consórcio SNPforID e o conjunto IISNPs, vêm sendo estudados em várias populações do mundo. Com o desenvolvimento de técnicas de sequenciamento de nova geração (NGS Next Generation Sequencing) para análise de DNA, a obtenção de perfis de DNA se tornou mais acurada. Algumas plataformas permitem gerar perfis de até 96 indivíduos simultaneamente. Este estudo tem por objetivo principal analisar 171 marcadores genéticos (Amelogenina, Y-INDEL, 30 STRSs e 139 SNPs) em 340 indivíduos miscigenados da região da cidade de Ribeirão Preto (SP) utilizando a plataforma de sequenciamento de nova geração MiSeq Personal Sequencer (Illumina Inc.), bem como calcular as frequências alélicas e genotípicas, verificar a aderência ao equilíbrio de HardyWeinberg e estimar parâmetros forenses para os diferentes conjuntos de marcadores. Análises de ancestralidade foram realizadas para os conjuntos de SNPs. Para o preparo das bibliotecas de amostras a serem sequenciadas, foi utilizado o kit HaloPlex (Agilent Technologies, Inc), onde foram incluídos os marcadores dos kits GlobalFiler e PowerPlex Fusion System, e os SNPs existentes no conjunto do consórcio SNPforID (52-plex) e IISNPs (92 SNPs). De todos os marcadores incluídos no ensaio, apenas um SNP (rs763869) presente no conjunto SNPforID não pôde ser analisado devido a questões técnicas. Dos 139 SNPs analisados apenas seis apresentaram desvios significativos em relação ao equilíbrio de Hardy-Weinberg,número este esperado devido ao acaso. Os conjuntos de SNPs apresentam elevada informatividade com Probabilidade de Match de 6,48 x 10-21 (52-plex) a 4,91 x 10-38 (IISNP), e Poder de Exclusão de 0,9997 (52-plex) e 0,99999997 (IISNP). De modo geral, as inferências de ancestralidade obtida utilizando estes conjuntos, indicaram elevada contribuição europeia (superior a 70%) e baixa contribuição ameríndia (inferior a 10%) na população, enquanto que as análises de mistura individual se mostraram consistentes, com a maioria dos indivíduos apresentando elevada ancestralidade europeia. Os resultados dos marcadores relativos ao sexo (Amelogenina, Y-INDEL e DYS391) foram consistentes com o sexo dos doadores das amostras. As frequências alélicas e parâmetros forenses foram calculados para os STRs, revelando uma alta informatividade. A Probabilidade de Match combinada e o Poder de Exclusão combinado foram de 1,19 x 10-36 e 0,999999999997 respectivamente. Dos 29 STRs autossômicos presentes, seis apresentaram desvios ao equilíbrio de Hardy-Weinberg, refletindo possíveis falhas no sequenciamento e genotipagem destes marcadores / The field of forensic genetics has developed increasingly with the implementation of new sets of DNA markers with higher levels of informativeness. The genetic markers are widely used in human identification as they allow distinguishing individuals with high accuracy. Two of the most commonly used markers are the Short Tandem Repeats (STRs) and the Single Nucleotide Polymorphisms (SNPs). Newer kits such as GlobalFiler (Thermo Fisher Scientific) and PowerPlex Fusion System (Promega) can analyze more than 20 STRs loci at once. When comparing with STRs, the SNPs are less informative and many more loci are needed to reach the same informativeness of STR kits. However, they are advantageous when using degraded DNA samples. The identification sets such as the 52-plex developed by the SNPforID Consortium and the IISNPs have been analyzed in many worldwide populations. With the development of next generation sequencing techniques (NGS Next Generation Sequencing), obtaining DNA profiles has become more accurate and some platforms allow generating profiles of up to 96 individuals simultaneously. The main goal of this study is to analyze 171 markers (Amelogenin, Y-INDEL, 30 STRs and 139 SNPs) in 340 admixed individuals from Ribeirão Preto, SP, using the NGS platform MiSeq Personal Sequencer (Illumina Inc.). This will allow the calculation of allele and genotype frequencies, the verification of adherence to Hardy-Weinbergs equilibrium and the estimation of forensic parameters for each set of marker. Ancestry analysis was performed for the sets of SNPs. The HaloPlex kit (Agilent Technologies, Inc) was used for library preparation including the STRs from the kits GlobalFiler and PowerPlex Fusion System and the SNPs from the SNPforID consortium (52-plex) and IISNPs (92 SNPs) identification sets. A single SNP (rs763869) from the SNPforID set was not analyzed due to technical issues. Only six of the 139 analyzed SNPs presented significant deviation from the Hardy-Weinberg equilibrium expectations, which is expected by chance alone. The SNPs sets exhibited high informativeness, with matchprobability ranging from 6.48 x 10-21 (52-plex) to 4.91 x 10-38 (IISNPs) and exclusion power of 0.9997 (52-plex) and 0.99999997 (IISNPs). In general, ancestry estimates obtained using these sets indicated a high European contribution (higher than 70%) and low Amerindian contribution (less than 10%) in the population sample, while the individual admixture analyses exhibited were highly consistent, with the majority of individuals presenting high European ancestry. The results of the sex markers (Amelogenin, Y-INDEL and DYS391) were in agreement with the reported sexes from sample donors. The allele frequencies and forensic parameters calculated for the STRs revealed high informativeness. The combined match probability and the combined exclusion power were 1.19 x 10-36 and 0.999999999997 respectively. Six of the 29 autosomal STRs presented significant deviations from the HardyWeinberg equilibrium expectations, reflecting possible failures in sequencing and genotyping of these markers