Made available in DSpace on 2015-12-10T14:23:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-04-24. Added 1 bitstream(s) on 2015-12-10T14:30:07Z : No. of bitstreams: 1 000852258.pdf: 1391953 bytes, checksum: 0d7e06938cd3251b6670b1cae39a85d1 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Transcrição é o processo catalizado por um complexo enzimático, RNA polimerase (RNAP), responsável pela síntese de RNA mensageiro a partir de uma sequência de DNA. Diferentes estudos experimentais foram realizados para investigar esse processo, como técnicas bioquímicas, de pinça ótica ou magnética, microscopia de força atômica e fluorescência de molécula única. Com os estudos bioquímicos, por exemplo, sabe-se que várias RNAPs podem transcrever uma sequência simultaneamente. O número de diferentes moléculas depende da necessidade da célula, número de RNAP livres, regeneração do promotor e fatores de transcrição. Um dos sistemas mais investigados para o estudo desse processo é a síntese dos genes ribossomais em Escherichia coli. Os genes ribossomais são fundamentais na fisiologia dos organismos, são expressos abundantemente e existem evidências da aceleração da transcrição devido ao comportamento colaborativo entre as RNAPs. Neste trabalho, propomos simular a transcrição múltipla dos genes ribossomais em E. coli com o modelo estocástico e dependente de sequências desenvolvido em nosso laboratório. As reações químicas foram simuladas utilizando o algoritmo de Gillespie. Essa metodologia apresenta uma boa relação entre custo computacional e realismo biológico e inclui alguns parâmetros não utilizados em estudos teóricos prévios. O modelo considera o alongamento em back e forward tracking, identificando os sítios de pausas e colisões entre RNAPs, determinando o tempo de permanência e predizendo a ocorrência de transcrição abortiva ou a aceleração da transcrição devido ao fenômeno colaborativo entre múltiplas RNAPs. A sequência do operon ribossomal rrnB da E. coli foi simulada variando o número de RNAP (R), a força de interação entre RNAPs (F) e a concentração de nucleosídeo trifosfato ([NTP]). Nossos resultados mostraram-se promissores quando utilizamos uma... / The process that produces messenger RNAs from DNA sequences is called transcription, and these reactions are catalyzed by the RNA polimerase enzyme. Many different experimental techniques have been applied to investigate this process including biochemical techniques, optical and magnetic tweezers, atomic force microscopy and single molecule florescence. These biochemical process studies showed that many RNAP molecules operate simultaneously on a single DNA strand. The number of different molecules depends on cellular demands, concentration of free RNAPs, promoter strength and the presence of transcription factors. Escherichia coli ribosomal genes are a popular experimental model to investigate the transcription process. These genes are essential to cell physiology, and they are strongly expressed. There are evidences that some cellular mechanisms collaborate to accelerate their transcription. In this work we investigate the RNAP collaborative transcription in E. coli ribosomal genes using a stochastic and sequence dependent model proposed by our group. The chemical reactions were simulated using a model based on the Gillespie algorithm. This methodology is a good compromise between computational cost and biological realism and includes some ingredients that were missing in previous theoretical studies. The model considers back and forward tracking elongation and it identifies pauses by determining the dwell time on specific sites. The model also predicts abortive transcription and transcription acceleration due to collaborative RNAP interaction. The E. coli rrnB ribosomal operon sequence was simulated by varying (i) the number of RNAP (R) on the DNA strand, (ii) the interaction force between two colliding RNAPs (F) and (iii) the concentration of nucleoside triphosphate ([NTP]). Our results are promising for F =15 pN, R = 50 and [NTP] ... / CNPq: 2013/06683-2

Made available in DSpace on 2016-04-01T17:55:12Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-11-06. Added 1 bitstream(s) on 2016-04-01T18:01:06Z : No. of bitstreams: 1 000860189.pdf: 924771 bytes, checksum: 86cbef733632ef64b946cc2d2006b9b0 (MD5) / Estudos recentes em indivíduos da espécie humana e camundongos relataram que o exercício físico pode alterar a microbiota intestinal. Evidências indicaram que essa comunidade microbiana está envolvida na homeostase do metabolismo do hospedeiro. Ademais, estudos com substâncias ergogênicas são escassos na literatura sobre a microbiota intestinal. Objetivou-se avaliar o impacto do exercício físico agudo e do condicionamento aeróbio associado a suplementação com cromo ou L-carnitina na microbiota intestinal de potras. Nosso modelo experimental utilizou potras da raça Mangalarga Machador em estágio de condicionamento físico incipiente, distribuídas em 4 grupos: Controle (sem exercício), Exercício, L-carnitina e Cromo. As potras foram submetidas a dois testes de esforço incremental e condicionadas em esteira por 42 dias na intensidade de 70-80% do limiar de lactato. Amostras fecais foram obtidas antes e 48 horas após do exercício agudo e do programa de condicionamento. As populações bacterianas foram caracterizadas pelo sequenciamento da região V4 do gene 16S rRNA por meio da plataforma Illumina MiSeq e um total de 5.224.389 de sequências foi obtido de 48 amostras. Para o total de sequências, os três filos mais abundantes foram Firmicutes (50,22%), Verrucomicrobia (15,13%), seguido por 13,80% de bactérias que não foram classificadas no nível filo. Os gêneros com maior abundância relativa foram: Clostridiales não classificados (17,06%), 5 genus incertae sedis (12,98%) e bactérias não classificadas (12,95%). Nossos resultados indicaram que a atividade física intensa e o condicionamento aeróbio podem alterar a composição e a estrutura da população bacteriana intestinal de potras. O cromo ou a L-carnitina na comparação intra grupos provocaram alterações moderadas na diversidade da microbiota fecal de potras. Entretanto, os resultados dos grupos suplementados não apresentaram alteração em... / Recent studies performed in humans and rats reported that exercise could alter the intestinal microbiota. Evidence indicated that this microbial community is involved in the metabolic homeostasis of the host. Moreover, studies about ergogenic substances are scarce in the literature on intestinal microbiota. This study aimed to assess the impact of acute exercise and aerobic conditioning, associated either with chromium or L-carnitine supplementation, on the intestinal microbiota of fillies. Twelve Mangalarga Marchador fillies in incipient fitness stage were distribuided into four groups: control (no exercise), exercise, L-carnitine and chromium. The fillies underwent two incremental exercise tests, before and after training on a treadmill for 42 days at 70-80% of the lactate threshold intensity. Fecal samples were obtained before and 48 hours after the acute exercise (incremental exercise test). Bacterial populations were characterized by sequencing the V4 region of the 16S rRNA gene using the MiSeq Illumina platform, and 5,224,389 sequences were obtained from 48 samples. The results showed that the three most abundant phyla were Firmicutes (50.22%), Verrucomicrobia (15.13%), followed by bacteria that were not classified at the phylum level (13.80%). The genera with the highest relative abundance were unclassified Clostridiales (17.06%), 5 genus incertae sedis (12.98%) and unclassified bacteria (12.95%). Our results indicated that intense physical activity and aerobic conditioning might change the composition and structure of the intestinal bacterial population in fillies. The intra-group comparison showed that chromium or L-carnitine caused moderate changes in the fecal microbiota of fillies. However, the results of the supplemented groups were not different from the exerciseonly group. Thus, further studies using a larger sampling are needed to further investigate these changes

Orientador: Fausto Foresti / Coorientador: José Carlos Pansonato Alves / Banca: Diogo Teruo Hashimoto / Banca: Vanessa Paes da Cruz / Resumo: No presente estudo, foram analisadas citogeneticamente cinco espécies de peixes pertencentes ao gênero Trichomycterus: T. diabolus, T. iheringi, T. zonatus, Trichomycterus cf. mimonha e Trichomycterus sp., provenientes de diferentes bacias hidrográficas brasileiras. Foram utilizadas as técnicas de citogenética clássica (coloração com Giemsa, localização das RONs pela marcação com nitrato de Prata e bandamento C) e cito-moleculares (Hibridação Fluorescente in situ - FISH com sondas de DNAr 5S e 18S e o snDNA U2). Todas as espécies apresentaram número diploide de 2n=54 cromossomos com variações em sua fórmula cariotípica. Trichomycterus diabolus, T. iheringi, T. zonatus e Trichomycterus cf. mimonha apresentaram seu cariótipo com 34m + 12sm + 8st e Trichomycterus sp. apresentou 36m + 10sm + 8st, além da ocorrência de cromossomos supranumerários. As espécies também revelaram diferenças em relação ao tamanho dos dois primeiros pares cromossômicos do cariótipo, onde Trichomycterus sp. e T. diabolus apresentaram o primeiro e segundo metacêntrico de tamanho semelhante e maiores em relação aos demais cromossomos, enquanto em T. zonatus, Trichomycterus cf. mimonha e T. iheringi apenas o primeiro metacêntrico foi considerado o maior par. O bandamento C evidenciou blocos heterocromáticos instersticiais nos pares 2, 3, 7, 8, 19 e 23 de T. iheringi e no par 18 de T. diabolus, sendo que as demais espécies apresentaram blocos centroméricos de diferentes tamanhos espalhados por quase todo o cariótipo. As RONs foram identificadas em apenas um par cromossômico e foram coincidentes com a hibridação fluorescente in situ realizada com a sonda para DNAr 18S, com exceção de T. zonatus e Trichomycterus sp. que apresentaram marcações centroméricas no primeiro par e em um pequeno metacêntrico, respectivamente. A hibridação com a sonda para DNAr 5S revelou resultados mais diversos, sendo detectado um par para... / Abstract: In the present study five species of fish from the genus Trichomycterus were analyzed cytogenetically, including T. diabolus, T. iheringi, T. zonatus, Trichomycterus cf. mimonha and Trichomycterus sp., collected at different Brazilian basins. The analyses involved classical (Giemsa conventional staining, localization of NORs for silver nitrate marking and C-banding) and molecular cytogenetic techniques (fluorescent in situ hybridization with ribosomal 5S and 18S, and U2 snDNA probes). All species showed diploid number of 2n = 54 chromosomes with variations in karyotype formula. Trichomycterus diabolus, T. iheringi, T. zonatus and Trichomycterus cf. mimonha presented their karyotype with 34m + 12sm + 8st and Trichomycterus sp. presented 36m + 10sm + 8st, besides the occurrence of supernumerary chromosomes. The species also reveals differences in relation to the size of the first two chromosome pairs of the karyotype, where Trichomycterus sp. and T. diabolus presented the first and second metacentric with similar size and larger than the other chromosomes, while in T. zonatus, Trichomycterus cf. mimonha and T. iheringi only the first metacentric was considered the largest pair. The constitutive heterochromatin showed interstitial blocks in pairs 2, 3, 7, 8, 19 and 23 in T. iheringi and the par 18 in T. diabolus and the other species presented centromeric blocks with different sizes spread throughout the greater part of the karyotype. NORs were identified in only one chromosome pair and were coincident with the fluorescent in situ hybridization using probes of 18S rDNA, except for T. zonatus and Trichomycterus sp. that showed additional centromeric signals on the first pair and other small metacentric, respectively. FISH using the probes of 5S rDNA was differentially distributed in the different species, with one chromosome pairs detected in T. diabolus, two pairs in T. iheringi, T. zonatus and three ... / Mestre

Orientador: Maria Inês Tiraboschi Ferro / Coorientador: Flávia Maria de Souza Carvalho / Coorientador: Poliana Fernanda Giachetto / Banca: Priscila Lupino Gratão / Banca: Paula Macedo Nobile / Resumo: A cana-de-açúcar é uma cultura fortemente influenciada pela seca, o que é um fator limitante para a produção sucroenergética. No Brasil, esta cultura está se expandindo para regiões com deficiência hídrica bastante acentuada, e uma das formas de contornar este problema é utilizar cultivares que sejam mais adaptadas a este tipo de estresse. Por isso, o objetivo deste trabalho foi analisar o comportamento fisiológico e molecular de plantas de cana-de-açúcar submetidas a distintos períodos de deficiência hídrica, a fim de contribuir com novos conhecimentos na área e auxiliar no desenvolvimento e cultivo de plantas mais bem adaptadas a essa condição. Neste trabalho, foram utilizadas duas cultivares de cana-de-açúcar contrastantes à seca (SP81-3250 e RB855453), tolerante e sensível, respectivamente. As plantas foram cultivadas em casa de vegetação e submetidas a três controlados potenciais hídricos do solo (controle, déficit hídrico moderado e déficit hídrico severo) a partir de 60 dias após o plantio. Essas plantas foram avaliadas molecular e fisiologicamente em três épocas distintas: 30, 60 e 90 dias após a aplicação dos tratamentos, sendo este um dos poucos trabalhos disponíveis até o momento sobre a resposta de plantas de cana-de-açúcar sob déficit hídrico prolongado. A tecnologia de RNA-Seq foi utilizada para obtenção dos transcriptomas de folhas dos dois genótipos de cana. A montagem de novo desses transcriptomas foi realizada, o que permitiu identificar os genes exclusivos ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The sugarcane culture is strongly influenced by drought, which is a limiting factor for sugar and energy production. In Brazil, this culture is expanding to regions with very severe water stress and one way to workaround this issue is to use cultivars that are more adapted to this form of stress. Therefore, the objective of this study has been to analyze the physiological and molecular behavior of sugarcane plants submitted to different periods of water stress in order to contribute to new knowledge in the area and assist in the development and cultivation of better-adapted plants to this condition. In this study, we have employed two cultivars of sugarcane that are contrasting to drought (SP81-3250 and RB855453), tolerant and sensitive, respectively. Plants have been cultivated in a greenhouse and submitted to three controlled potential soil hydric (control, moderate drought and severe water deficit) from 60 days after planting.These plants have been evaluated molecular and physiologically in three different periods: 30, 60 and 90 days after treatment application, which is one of the few studies available to date on the response of sugarcane plants under prolonged drought. RNA-Seq technology has been used to obtain the transcriptomes leaves of the two genotypes of sugarcane.The de novo assembly has been performed which has allowed identifying exclusive genes of both cultivars and analyzing the exclusive tolerant cultivar involved in defense response to prolonged drought. The physiological parameters evaluated have shown significant changes in response to water stress.The de novo assembly resulted in 161,295 isoforms, with 86,087 different genes. To record, the transcripts have been were aligned against sequences of Sorghum bicolor, Arabidopsis thaliana, and sugarcane sequences available in public databases. Through the analysis of differentially ... (Complete abstract click electronic access below) / Mestre

O Papaya ringspot virus (PRSV) é uma espécie do gênero Potyvirus, sendo que a maioria dos isolados pertence a duas estirpes distintas: "papaya" (P) e "watermelon" (W). O Papaya ringspot virus - estirpe W (PRSV-W) tem sido considerado um dos vírus mais importantes no cultivo de cucurbitáceas pela predominância e pelos prejuízos significativos que causa no Brasil. O controle do mosaico da abobrinha-de-moita, causado pelo PRSV-W, tem sido obtido de forma satisfatória através da premunização com as três estirpes fracas, designadas PRSV-W–1, PRSV-W–2 e PRSV-W-3. O principal objetivo desse estudo foi comparar essas estirpes fracas com estirpes severas PRSV-W-C, PRSV-W-PR e PRSV-W-PE, com base na seqüência de nucleotídeos do gene que codifica a proteína da capa protéica e na mobilidade dessa proteína em SDS-PAGE. A seqüência de nucleotídeos da capa protéica das estirpes fracas PRSV-W-1 e PRSV-W-2 apresentou 100 % de homologia. Quando comparadas com a estirpe fraca PRSV-W-3, a homologia foi de 98 %. As estirpes fracas PRSV-W–1 e PRSV-W–2 apresentaram 95 % de homologia com as estirpes severas PRSV-W-C e PRSV-W-PE. Essas duas estirpes severas, por sua vez, apresentaram respectivamente, 93 e 95 % de homologia com a estirpe fraca PRSV-W-3. O alinhamento das seqüências de nucleotídeos entre as estirpes fracas e as severas evidenciou a inserção de 6 nucleotídeos na região conservada desse gene nas estirpes fracas. Essa inserção refletiu na adição de dois amino ácidos (Asn e Asp) na seqüência de amino ácidos deduzidos dessa proteína. A mobilidade da capa protéica em SDS-PAGE foi semelhante para todas as estirpes estudadas. / Papaya ringspot virus (PRSV), a species of the enus Potyvirus, is classified into two strains: "papaya" (P) and "watermelon" (W). Papaya ringspot virus - type W (PRSV-W) has been considered one of the most important viruses infecting cucurbits in Brazil due to its predominance and significative damage caused on the crops. The control of the disease caused by this virus has been efficiently achieved by means of cross protection with three mild strains, namely PRSV-W-1, PRSV-W-2, and PRSV-W-3. The main purpose of the present study was to compare these mild strains with the severe strains PRSV-W-C, PRSV-W-PR, and PRSV-W-PE, based on the nucleotide sequence of the coat protein gene and on the mobility of the capsid protein in SDS-PAGE. The nucleotide sequence of the coat protein gene of the mild strains PRSV-W-1 and PRSV-W-2 showed 100 % of homology. When compared with the coat protein gene of the mild strain PRSV-W-3, the homology was 98 %. The mild strains PRSV-W-1 and PRSV-W-2 showed 95 % of homology with the severe strains PRSV-W-C and PRSV-W-PE. These two severe strains, otherwise, showed respectively, 93 and 95 % of homology with the mild strain PRSV-W-3. The alignment of the nucleotide sequences of the mild and the severe strains indicated an insertion of 6 nucleotides in the conserved region of the coat protein gene of the mild strains. This insertion reflected on the addition of two amino acids (Asn e Asp) in the amino acid deduced sequence of this protein. The mobility of the coat protein in SDS-PAGE was similar for all the strains studied.

Numerous troponin T (TnT) isoforms are generated by alternative RNA splicing primarily in its NH2-terminal hypervariable region, but the functions of these isoforms are not completely understood. In this dissertation work, calcium and terbium binding behavior of several forms of TnT were investigated by spectroscopic and radioactive techniques. Chicken breast muscle TnT binds calcium and terbium through its NH2-terminal Tx motif (HEEAH)n with high affinity (10-6 mM) and fast on-rate (106 - 107 M-1 s-1). Chicken leg muscle TnT and a human cardiac TnT NH2-terminal fragment, which both lack the Tx motif on their NH2-terminal regions, do not have affinities for calcium in the physiological range. Computational predictions on TnT N47 suggest that the TnT NH2-terminal region might fold into an elongated structure with at least one high affinity metal ion binding pocket comprised primarily of the Tx motif sequence and several lower affinity binding sites. In addition, calcium binding to TnT N47 might alter its conformation and flexibility. Luminescence resonance energy transfer measurements and other experimental observations are consistent with the computational predictions suggesting the computational simulated atomic model is reasonable. TnT mutations are responsible for 15% of familiar hypertrophic cardiomyopathy (FHC) cases with a phenotype of relatively mild hypertrophy, but a high incidence of sudden death. Detection of those genetic mutations would facilitate the clinical diagnosis and initiation of treatment at an early stage. This dissertation also investigated a novel hybridization proximity assay (HYPA) combining molecular beacon and luminescence resonance energy transfer (LRET) technologies. Experimental results suggest that a shared stem probe design produces a more consistent response upon hybridization, whereas the internally labeled probe was less consistent, but can yield the highest responses. Using the optimally designed molecular probes, the HYPA provides a detection of alterations in nucleic acid structure of as little as a single nucleotide. This novel HYPA is expected to expand its applications in the analysis and screening of genetic diseases.

High-throughput technologies have continuously driven the generation of different biomolecular data, including the genomics, epigenomics, transcriptomics, and other omics data in the last two decades. The developments and advances have revolutionized medical research. In this dissertation, a collection of computational analyses and tools, based on different types of biomolecular data with particular applications on human diseases are presented including 1) a cascade ensemble model based on the Dirichlet process mixture model for reconstructing tumor subclonality from tumor DNA sequencing data; 2) a meta-analysis of gene expression and DNA methylation data from prefrontal cortex samples of patients with neuropsychiatric disorders indicating a stress-related epigenetic mechanism; 3) 2DImpute, an imputation algorithm that is designed to alleviate the sparsity problem in single-cell RNA-sequencing data; and 4) a pan-cancer transformation from adipose-derived stromal cells to metastasis-associated fibroblasts revealed by single cell analysis.

Background: The physiological and pathophysiological roles of secreted membrane-enclosed vesicles known as extracellular vesicles (EVs) have become increasingly recognized, making the EV field a quickly evolving area of research. EVs and their encapsulated molecular material including microRNAs are key mediators of intercellular communication, making EVs analogous to a message in a bottle. This discovery has fundamentally changed the study of gene regulation, and understanding the central role of EVs and their cargo in health and disease will generate new opportunities for basic biology, diagnostics, and disease treatment. EV release and the packaging of molecular material into EVs can be altered by stressors such as air pollution exposure. Exposure to air pollution is associated with significant morbidity among individuals with asthma, especially children who participate in more frequent outdoor activities and are more susceptible to exposure due to their narrower airways and higher breathing rate. Thus, sensitive biomarkers of air pollution exposure are needed to identify children at risk of worsened symptoms and asthma exacerbations. Given their role in cell-to-cell communication, EVs also represent a plausible molecular mechanism in the etiology of disorders such as aging-related cognitive decline. Individuals with mild cognitive impairment and experiencing increased rates of cognitive decline are more likely to develop Alzheimer’s disease and other dementias, signifying the importance of identifying and treating cognitive impairment early. More precise identification of the neurobiological processes of cognitive decline in aging populations may provide critical insights into the precursors of late-life dementias and identify health interventions that can delay cognitive impairment or therapeutic targets for treatment. This dissertation evaluates the utility of EV-encapsulated microRNAs (EV-miRNAs) as biomarkers of environmental exposure (i.e., air pollution) and assesses their role in disease risk (i.e., cognitive decline) in two separate studies. First, in Chapters 2-3, using a cohort of children with asthma in the greater Boston area, we describe saliva EVs isolated from these children using a high-throughput method and explore the potential of salivary EV-miRNAs as easy-to-measure biomarkers of exposure to fine particulate matter (PM2.5), nitrogen dioxide (NO2), and ozone (O₃). Then, in Chapter 4, we evaluate the association between EV-miRNAs and cognitive function and rates of cognitive decline in a cohort of elderly men and discuss the utility of circulating EV-miRNAs as biomarkers of risk. Furthermore, we discuss the pathways that these EV-miRNAs target if they play a causal role in cognitive decline which could have implications for development of therapeutics. Methods: Drawing from the School Inner-City Asthma Study (SICAS), we isolated salivary EVs and EV-miRNA from children with asthma for analysis in relation to ambient exposure to PM₂.₅, NO₂, and O₃. In accordance with the recommended minimal experimental requirements for the definition of EVs, in Chapter 2 we employ multiple orthogonal methods to describe the EVs that were isolated from cell-free saliva using a high-throughput polymer-based reagent (ExoQuick-TC). In Chapter 3, we utilize EV-miRNA data generated via RNA sequencing and ambient air pollution data estimated using a validated spatiotemporal high-resolution model. We perform differential expression analyses to examine the effect of high exposure to PM₂.₅, NO₂, and O₃ on saliva EV-miRNA abundance. In Chapter 4, we leverage data from the Normative Aging Study, a longitudinal cohort of elderly men, to investigate whether circulating EV-miRNAs are associated with cognitive function and rates of cognitive decline. We used linear models to assess the relationship between plasma EV-miRNAs and cognitive function and linear mixed models to evaluate the relationship between plasma EV-miRNAs and rates of cognitive decline. We performed gene ontology functional enrichment and pathway enrichment analyses to identify the biological pathways that these EV-miRNAs would target if they play causal roles in cognitive decline. Results:In Chapter 2, we demonstrate that EVs can be isolated from human saliva using ExoQuick-TC. The saliva EVs isolated from ExoQuick (N=180) ranged in size but were mostly ~55 nm in diameter and expressed tetraspanins CD9 and CD63, canonical markers for EVs, but did not highly express the tetraspanin CD81. In Chapter 3, in a subset of the SICAS cohort (N=69), we show that relatively high (>19.37 parts per billion) short-term ambient NO2 exposure and relatively high (>38.38 parts per million) prior-day O3 exposure are associated with down-regulation of several saliva EV-miRNAs. We did not detect differential expression of any EV-miRNAs in relation to PM₂.₅ exposure over multiple time windows of exposure. Finally, in Chapter 4, multivariable linear and linear mixed models demonstrated a relationship between several plasma EV-miRNAs and global cognitive function and rates of global cognitive decline, measured by the Mini-Mental State Examination. Functional enrichment and pathway enrichment analyses revealed that the biological pathways targeted by these miRNAs are relevant in neurodegeneration, including pathways regulating synaptic function and plasticity and neuronal death. We found no association between EV-miRNAs and cognitive function or cognitive decline as assessed by cognitive tests measuring specific domains of cognitive function. Conclusion: This work demonstrates the opportunities that EV-miRNAs can create for advancing environmental health research. EV-miRNAs may serve as sensitive biomarkers of environmental exposures as well as biomarkers of risk and may play mechanistic roles in disease. We also make recommendations for integrating EV research into the field of environmental health. Future studies should continue to evaluate the potential of EV-miRNAs and seek to identify EV-miRNAs that can serve as mechanistic biomarkers between exposures and effect across all stages of life to (1) increase our understanding of the consequences of circulating miRNA changes and the contribution of the environment to heterogeneous disorders, (2) advance development of non-invasive diagnostics to allow for longitudinal monitoring, and (3) pave the way for new opportunities for disease prevention and treatment.

Carbonic anhydrase is a ubiquitous zinc-metalloenzyme that catalyzes the interconversion of carbon dioxide and carbonate and has been found to play a wide range of roles in animals, plants and bacteria. Cotton genomic and cDNA libraries were screened for the plastidial isoform of carbonic anhydrase. The nucleotide sequences of two 1.2 Kb partial cDNA clones were determined. These clones exhibit high homology to carbonic anhydrases from other dicot plants and possess all the expected peptide motifs. For example, serine and threonine rich chloroplastic targeting peptide and conserved zinc binding residues are both present. These clones were utilized to isolate two carbonic anhydrase genes that were shown to encode different isoforms by PCR and RFLP analysis.

The study of protein synthesis, and the study of gene expression in general, has accelerated in recent years. Following the advent of next-generation RNA sequencing, powerful library preparation paradigms were developed to capture regulatory activity on a genome-wide scale. In particular, ribosome profiling has emerged as a widely-used measurement of translation. In this method, the state of ribosome association across the transcriptome is obtained by isolation and sequencing of the regions of RNA bound by ribosomes, revealing a snapshot of ribosome positions from which gene-specific densities can be calculated. In combination with RNA sequencing for a measurement of baseline transcription in the same samples, ribosome profiling offers a metric of “translation efficiency”, or TE, corresponding to the average ribosome load per given transcript. Ribosome profiling has advanced the study of translation considerably. However, low throughput in the generation of ribosome profiling and RNA sequencing libraries limits the scale of the experiments that can be performed, while issues in the interpretation of aligned ribosome-protected footprints complicate their analysis, especially in systems of complex regulation. The analysis of such regulatory systems would be greatly aided by a high-throughput sequencing method that can capture translational regulation, but current methods of measuring genome-wide translation are inherently limited in scale. This thesis addresses the key issues presented above in separate chapters. Chapter 2 discusses the analysis of elongation and initiation from ribosome profiling and RNA sequencing data in a mouse model of Fragile X Syndrome. In this chapter, several methods of measuring and modeling variability in the distribution of ribosomes along a coding sequence are used alongside analyses of differential RPF and RNA abundances and their ratio, RFApm, which we distinguish from TE to emphasize its dependence on factors other than initiation rate. The chapter summarizes current information regarding the observed effects of FMRP, and proposes a model congruent with these observations and more-recently published studies. Chapters 3 and 4 present approaches to modeling or inferring translational regulatory networks, either by a novel library preparation paradigm or computational inference from publicly-available data. Chapter 3 presents riboPLATE-seq, a high-throughput RNA-seq library construction method based on the existing PLATE-seq method. The method recapitulates significant findings from ribosome profiling and RNA sequencing at a fraction of the per-sample cost, with further advantages in scalability, and could be implemented in a large-scale screen of translational regulators to create a network of their specific targets. Chapter 4 presents an approach to inferring translational regulation from integrative analysis of public ribosome profiling and RNA sequencing data, tailoring the powerful inference engine ARACNe to measure translational interactions. This yields a comprehensive network of translational regulation, assigning target genes to the set of RNA-binding proteins.