Mikromatriser handlar numera inte bara om att alstra genuttrycksdata i snabb takt, utan det är minst lika viktigt att effektivt ta hand om informationen efteråt. I den här avhandlingen presenteras ett arbetsflöde för att mäta, lagra och analysera genuttrycksdata i asp och poppel (Populus spp.). En Populus} mikromatrisdatabas - UPSC--BASE - tillgänglig för alla intresserade, utvecklades i syfte att samla in och lagra genuttrycksdata. Flertalet analysverktyg gjordes samtidigt tillgängliga, för att möjliggöra ett smidigt arbetsflöde från rådata till biologiska slutsatser. En av de stora utmaningarna i analys av mikromatriser är att kunna särskilja bruset från värdefull biologisk information. Att studera träd som växer utomhus är komplext eftersom de interagerar med omgivningen i mycket större utsträckning än vad som är fallet i växthusets kontrollerade miljö. Det här arbetet visar att det är möjligt, med hjälp av avancerad statistik och god försöksplanering, att följa och jämföra genuttrycket i blad från aspar utomhus under flera år för att dra värdefulla slutsatser om geners reglering. Den lagrade biologiska informationen i UPSC-BASE är avsedd att vara en värdefull tillgång för växtfältet i stort. I databasen finns nästan hundra olika experiment som innefattar alltifrån unga blad till ved, insektsangrepp, kyla och torka samt studier av genmodifierade växter. Informationen kan användas både för jämförande studier inom olika aspförsök, men också för att jämföra med andra växter. För att illustrera möjligheterna, studerades och grupperades gener i blad baserat på hur de uppträder över alla dessa experiment. Dessa grupperingar användes sedan för att definiera gener som är viktiga i bladutvecklingen. Sammanfattningsvis ger arbetet som presenteras i denna avhandling tillgång till verktyg och kunskap för storskaliga studier av genuttryck och den lagrade informationen har bevisats vara en värdefull tillgång för mer ingående studier av geners reglering. / DNA microarray analysis today is not just generation of high-throughput data, much more attention is paid to the subsequent efficient handling of the generated information. In this thesis, a pipeline to generate, store and analyse Populus transcriptional data is presented A public Populus microarray database - UPSC--BASE - was developed to gather and store transcriptomic data. In addition, several tools were provided to facilitate microarray analysis without requirements for expert-level knowledge. The aim has been to streamline the workflow from raw data through to biological interpretation. Differentiating noise from valuable biological information is one of the challenges in DNA microarray analysis. Studying gene regulation in free-growing aspen trees represents a complex analysis scenario as the trees are exposed to, and interacting with, the environment to a much higher extent than under highly controlled conditions in the greenhouse. This work shows that, by using multivariate statistics and experimental planning, it is possible to follow and compare gene expression in leaves from multiple growing seasons, and draw valuable conclusions about gene expression from field-grown samples. The biological information in UPSC-BASE is intended to be a valuable transcriptomic resource also for the wider plant community. The database provides information from almost a hundred different experiments, spanning different developmental stages, tissue types, abiotic and biotic stresses and mutants. The information can potentially be used for both cross-experiment analysis and for comparisons against other plants, such as Arabidopsis or rice. As a demonstration of this, microarray experiments performed on Populus leaves were merged and genes preferentially expressed in leaves were organised in to regulons of co-regulated genes. Those regulons were used to define genes of importance in leaf development in Populus. Taken together, the work presented in this thesis provides tools and knowledge for large-scale transcriptional studies and the stored gene expression information has been proven to be a valuable information resource for in-depth studies about gene regulation.

In this study, I analyzed the Lactococcus lactis subspecies cremoris MG1363 stress response at sub-lethal alcohol levels during exponential growth phase at transcriptomics, proteomics, metabolomics levels. Ethanol, 1-butanol, 1-hexanol were the selected alcohols. Manganese- transporter- and arginine catabolic pathway genes were up-regulated by all alcohols suggesting they evoked oxidative and acidic stress. ATP manganese transporter genes, histidine- and galactose genes were also up-regulated. Purine- and pyrimidine synthesis genes were down-regulated. HPLC analysis displayed decreased biomass yield and glycolytic flux, suggesting increased glycolytic energy production and slowed down overall enzymatic rate. Proteomics analysis displayed differential expressed proteins associated with heat and oxidative stress.

The human microbiome has important roles in maintaining health, but dysbiosis of the microbiota can lead to disease. Polymicrobial interactions can result in synergy, producing disease that is worse than the sum of the respective single species infections. Despite this significant impact, synergy is understudied due to the complexity of polymicrobial interactions. Periodontitis is a microbiome-associated disease, and is one of the most common infectious diseases worldwide. Therefore, we have used periodontal disease as a model to study polymicrobial synergy. I have used two complementary approaches to study polymicrobial infections. The opportunistic periodontal pathogen Aggregatibacter actinomycetemcomitans exhibits synergy with streptococci in model murine infections. Because polymicrobial interactions are dependent on organisms’ abilities to sense their environments, I have examined the genetic regulatory mechanisms used by A. actinomycetemcomitans to interact with its environment. Through Northern blot analyses and biochemical approaches, I show that A. actinomycetemcomitans uses non-coding RNAs to regulate amino acid transport. Taking a comparative genomics approach, I demonstrate that A. actinomycetemcomitans DNA uptake systems are evolutionarily linked to genome defense. To describe host-influenced changes in gene expression, I develop a new technique to transcriptionally profile A. actinomycetemcomitans in a murine abscess infection, thereby revealing the importance of specific fermentative and anaerobic respiratory genes for in vivo survival. The long-term goal is to use these studies as a basis to characterize genetic regulatory mechanisms mediating synergy in polymicrobial A. actinomycetemcomitans infections with streptococci and other oral microbes. As a second approach to study polymicrobial infections, I analyze gene expression of healthy and diseased human plaque communities from aggressive periodontitis patients. Profiling ribosome content of healthy and diseased communities, I show that disease communities adopt similar less diverse population structures distinct from healthy populations. In addition to changes in population composition, using community transcriptional profiling I show that a keystone species within diseased communities up-regulates expression of genes involved in making the oral inflammatory molecule butyrate. These studies demonstrate for the first time that microbiome based diseases are marked by gene expression changes in addition to compositional changes. / text

Thesis (Ph. D.)--Miami University, Dept. of Microbiology, 2008. / Title from second page of PDF document. Includes bibliographical references (p. 215-246).

The human bronchial epithelium is composed of multiple, discrete cell types that cooperate to perform mucociliary clearance. While previous studies have shown that cigarette smoke can alter bronchial epithelial gene expression, the underlying effects of this exposure on specific cell types are not well understood. In this thesis, single-cell RNA sequencing was used to profile bronchial epithelial cells from six current smokers and six never smokers. Thirteen cell clusters were identified that were defined by expression of unique combinations of nineteen distinct gene sets. This clustering revealed that smoke exposure induced expression of a toxin metabolism program that specifically associated with ciliated cells. Extensive airway remodeling was also observed, in which smoking was associated with loss of club cells as well as goblet cell expansion and hyperplasia. Additionally, a previously uncharacterized CEACAM5+ KRT8+ epithelial subpopulation was identified in the airways of smokers. While it has been shown that most smoking-associated gene expression alterations can be reversed upon smoking cessation, a subset of these alterations persists in former smokers. The basal layer of the bronchial epithelium is comprised of a multipotent progenitor subpopulation. When abnormalities persist in the bronchial epithelium despite normal tissue turnover, the source of these abnormalities may be traced to this progenitor population and its program of differentiation. Therefore, basal cells were procured from three current smokers and three never smokers, differentiated in vitro, and profiled by RNA sequencing at eight time points spanning the differentiation procedure. Twenty-seven unique sets of co-expressed genes associated with differentiation were identified and functionally characterized, a subset of which were abnormally expressed in smoker cells. Robust expression of genes involved with the unfolded protein response was specifically detected in smoker basal cells. Additionally, a smoking-associated delay in the onset of expression of genes involved with ciliogenesis was observed. These data therefore indicate that smoking has long-term consequences on the differentiated state of the airway epithelium. Collectively, the observations outlined in this thesis demonstrate that smoking drives a complex landscape of alterations that affects the function and composition of the human bronchial epithelium. / 2020-10-24T00:00:00Z

Batatas (Solanum tuberosum) juntamente com arroz, trigo e milho, são as culturas mais consumida no mundo. A inclusão de novas variedades de S. tuberosum para consumo humano e sob a forma de ração necessita avaliação quanto sua segurança. Normas internacionais citam glicoalcalóides (GA) como tóxicos-chave em avaliação de novas variedades de batatas. Portanto, o objetivo do trabalho foi propor um modelo de avaliação da segurança de novas variedades de batata, incluindo geneticamente modificados, baseado no transcriptoma e análise comparativa de variedades com histórico de segurança. Adicionalmente, pretendeu-se determinar genes referência para análise transcricional de tubérculos. E ainda, caracterizar os genes glycoalkaloid metabolism (GAMEs), que fazem parte da biossíntese de GA e relacionar sua expressão com o total de glicoalcalóides (TGA) em diferentes genótipos de tubérculos de batatas. Para tanto, construiu-se um banco de dados com RNAseq de 90 tubérculos de batatas com grande variabilidade genética e natural. O banco de dados também foi utilizado para ranquear candidatos a genes normalizadores, baseado no intervalo interquartil (IQR), que, posteriormente, tiveram sua estabilidade calculada pelo: geNorm, NormFinder e BestKeeper. O conteúdo de TGA foi medido em cromatografia líquida de alta eficiência com espectrômetro de massa (CLAE-EM) e a expressão dos GAMEs foi determinada por reação de transcriptase reversa quantitativa seguida da reação em cadeia da polimerase (RT-qPCR). Para análise da região promotora utilizou-se: Plant Pan – Plant Promoter Analysis Navigator. Através dos resultados obtidos e analisados, podemos afirmar que o modelo, quando devidamente validado e finalizado, pode ser considerado bastante promissor. Os genes C2, SEC3 e CUL3A foram considerados como excelentes normalizadores para tubérculos de batata. A expressão dos GAMEs foi maior em tubérculos com maior conteúdo de TGA, e com exceção do GAME7, os genes GAME4, GAME6, GAME8ab, GAME11 e GAME12 demonstraram ser estatisticamente (α = 0,05) mais expressos em amostras com maior teor de TGA e menos em amostras com menor TGA, confirmando a relação destes genes com a produção de glicoalcalóides. Na análise de fatores de transcrição dos GAMEs muitos elementos cis relacionados a estresse biótico e abiótico, e regulação por luz foram encontrados, além de muitas cópias desses elementos cis nas regiões promotoras de todos os GAMEs. Os resultados obtidos no presente trabalho são importantes para um melhor entendimento da formação e regulação dos GA em tubérculos de batatas, auxiliando na predição e prevenção de formação deste composto. / Potato (Solanum tuberosum) is the third most important food crop in the world after rice and wheat in terms of human consumption. The introduction of new varieties of S. tuberosum for food or feed requires the checking of not only substances of pro-nutritional functioning on the human body, but should examine the content of toxic compounds, such as glycoalkaloids (GA), as an international norm recommendation for new potatoes varieties evaluation, including genetic modified. For this reason, the goal of the present work was build a food safety assessment model based on transcriptomics, and comparative with history of safe human consumption. Besides, we intended to determined reference genes for transcriptional analysis of potato tuber, and characterized glycoalkaloid metabolism (GAME) genes that partake of GA biosynthesis pathway. The correlation between the expression of GAME genes and total glycoalkaloids (TGA) content in different genotypes of potato tubers was evaluated. To do so, a database was build with RNASeq data from 90 potato tubers with large genotype and natural variability. These database were also used to ranking the candidates to be reference genes based on Interquartile Range (IQR). The stability of candidates for reference genes were evaluated by geNorm, NormFinder and BestKeeper. The content of TGA were measured on high performance liquid chromatography acoplated on mass spectrometry (LC-MS). Later, Reverse Transcriptase quantitative Polymerase Chain Reactions (RT-qPCR) determined the expression of GAME genes. For the promoter region analysis of GAMEs were used “Plant Pan – Plant Promoter Analysis Navigator”. It is possible affirmed that the food safety assessment model after a properly validation will be promising. The best reference genes for transcriptional analysis studies of potato tubers were C2, SEC3 and CUL3A. The expression of GAME4, GAME6, GAME7, GAME8ab, GAME11, and GAME12 were higher in tubers with high TGA content, and, exception for GAME7, the other GAME genes demonstrate statically (α = 0,05) higher expression on samples with high TGA and lower on samples with low TGA content, confirming that there is a relation between GAME genes and GA biosynthesis. On transcription factors analysis of GAME genes several cis-elements related to abiotic and biotic stress, and light regulated were found, also many copies of these factors in all GAME promoter region. All these results are very helpful to better understand GA formation and regulation on potato tubers, helping to predict and prevent the formation of these toxic.

The relationship between a pathogen and its host is a complex series of events that occurs at the molecular level and is controlled by transcriptional and protein interactions. To facilitate the understanding of these mechanisms in Aspergillus flavus and Zea mays, three approaches were taken: 1) the development of a predicted interactome for Z. mays (PiZeaM), 2) the development of co-expression networks for Z. mays and A. flavus from RNA-seq data, and 3) the development of causal inference networks depicting interactions between the host and the pathogen. PiZeaM is the genome-wide roadmap of protein-protein interactions that occur within Z. mays. PiZeaM helps create a novel map of the interactions in Z. mays in response to biotic and abiotic stresses. To further support the predicted interactions, an analysis of microarray-based gene expression was used to produce a gene co-expression network. PiZeaM was able to capture conserved resistance pathways involved involved in the response to pathogens, abiotic stress and development. Gene Co-expression networks were developed by the simultaneous use of correlations to develop networks for differentially expressed genes, resistance marker genes, pathogenicity genes, and genes involved is secondary metabolism in Z. mays and A. flavus. From these networks, correlation and anti-correlation of host and pathogen gene expression was detected, revealing genes that potentially interact at different stages of pathogenesis. Finally, causal gene regulatory relationships were inferred using partial correlation analysis of Z. mays infected with A. flavus over a 3 day period. The gene regulatory network (GRN) sheds light on the specifics of the mechanisms of pathogenesis and resistance that govern the Z. mays-A. flavus interaction. The direct product of this research is the understanding of key transcription factors and signaling genes involved in resistance. This body of research highlights how PPIs and GRNs can be utilized to identify biomarkers and gene functions in both Z. mays and A. flavus.

Pneumonia is the greatest cause of childhood mortality outside the neonatal period, yet the pathogen-specific aetiology of childhood pneumonia remains poorly defined. Vaccine probe studies estimate that approximately one third of children <5 years of age with radiographic endpoint consolidation have pneumococcal pneumonia in settings prior to the introduction of pneumococcal conjugate vaccination (PCV), such as much of South Asia. 10-valent PCV was introduced to the Nepali infant immunisation schedule in August 2015. I investigated childhood pneumococcal pneumonia in Nepal. I aimed to describe the prevalence of pneumococcal infection in children with suspected invasive bacterial disease admitted to Patan Hospital, Kathmandu, Nepal; to assess the impact of 10- valent PCV on pneumococcal pneumonia; and to assess two potential diagnostic tests for pneumococcal pneumonia based on the childhood response to pneumococci - assay of antibodies from lymphocyte supernatant (ALS) and analysis of differential gene expression (transcriptomics) - in children with pneumonia at Patan Hospital. Pneumococci were the second-most most prevalent pathogen isolated from the blood of children between 2005 and 2016. Interrupted time series analyses of data from children admitted with pneumonia from March 2014 - December 2016, showed a small increase (approximately 4%) in the odds of admission with pneumonia in comparison to non-pneumonia admis- sions temporally associated with 10-valent PCV introduction. However, it was not possible to adjust these time series analyses for extreme events including earthquakes (April 2015) and clean fuel shortages/increased air pollution (winter 2015/2016). In contrast, the indirect cohort method (a case-control approach in vaccinated vs unvaccinated children) showed vaccine effectiveness of 84% on the odds of nasopharyngeal carriage of vaccine-type pneumococci, but no effectiveness on pneumonia in these early data. Assay of IgG ALS pneumococcal capsular polysaccharides was complicated by what appear to be non-specific binding to capsular polysaccharides (of both S. pneumoniae, particularly serotype 3, and H. influenzae). Assay of IgG ALS to the best-performing of five pneumococcal proteins assessed had a sensitivity of 88% and specificity of 71% for the discrimination of pneumococcal pneumonia from other bacterial pneumonia. A transcriptomic signature discriminated between pneumococcal pneumonia and other bacterial pneumonia with a sensitivity of 91% and specificity of 100%. This thesis therefore contributes to knowledge of the clinical epidemiology of pneumococcal disease in South Asia. These data may also contribute to public health policy-making in the region. In addition, the development of two diagnostic tests for the aetiology of childhood pneumonia may be useful for future studies of childhood pneumonia aetiology.

Doctor of Philosophy / Biology / Michael A. Herman / Nematodes play an important role in various habitats where numerous factors serve to shape their communities. One such factor is the potentially pathogenic nematode-prey interaction. This project is focused on the elucidation of the genes that the bacterivorous nematode Caenorhabditis elegans employs to respond to the emerging nosocomial bacterial pathogen Stenotrophomonas maltophilia. A virulent S. maltophilia strain JCMS requires the action of several C. elegans conserved innate immune pathways that serve to protect the nematode from other pathogenic bacteria. However, insulin-like DAF-2/16 signaling pathway mutants that are typically pathogen resistant are susceptible to JCMS, and several DAF-2/16 regulated genes are not significantly differentially expressed between JCMS and avirulent E. coli OP50. We have determined the complete set of mRNA transcripts under different bacterial treatments to identify genes that might explain this JCMS specific DAF-2/16 pathway evasion. The identified set included 438 differentially expressed transcripts among pairwise comparisons of wild-type nematodes fed OP50, JCMS or avirulent S. maltophilia K279a. Candidate genes were nominated from this list of differentially expressed genes using a probabilistic functional connection model. Six of seven genes that were highly connected within a gene network generated from this model showed a significant effect on nematode survival by mutation. Of these genes, C48B4.1, mpk-2, cpr-4, clec-67 and lys-6 are needed for combating JCMS, while dod-22 was solely involved in K279a response. Only dod-22 had a documented role in innate immunity, which merits our approach in the identification of gene candidates. To a lesser extent, we have also focused on the identification of virulence factors and the mode of action employed by S. maltophilia. JCMS virulence requires rpfF, xps and involves living bacteria that accumulate in the intestinal lumen. Additionally, the bacterial secretion encoding genes cs, p773, p1176, pi1y1 and xdi are involved in JCMS evasion of daf-2. In summary, we have discovered a novel host-pathogen interaction between C. elegans and S. maltophilia JCMS, revealed genes that are involved in each partner of the interaction, and established a new animal model for the study of S. maltophilia mode of action.

Batatas (Solanum tuberosum) juntamente com arroz, trigo e milho, são as culturas mais consumida no mundo. A inclusão de novas variedades de S. tuberosum para consumo humano e sob a forma de ração necessita avaliação quanto sua segurança. Normas internacionais citam glicoalcalóides (GA) como tóxicos-chave em avaliação de novas variedades de batatas. Portanto, o objetivo do trabalho foi propor um modelo de avaliação da segurança de novas variedades de batata, incluindo geneticamente modificados, baseado no transcriptoma e análise comparativa de variedades com histórico de segurança. Adicionalmente, pretendeu-se determinar genes referência para análise transcricional de tubérculos. E ainda, caracterizar os genes glycoalkaloid metabolism (GAMEs), que fazem parte da biossíntese de GA e relacionar sua expressão com o total de glicoalcalóides (TGA) em diferentes genótipos de tubérculos de batatas. Para tanto, construiu-se um banco de dados com RNAseq de 90 tubérculos de batatas com grande variabilidade genética e natural. O banco de dados também foi utilizado para ranquear candidatos a genes normalizadores, baseado no intervalo interquartil (IQR), que, posteriormente, tiveram sua estabilidade calculada pelo: geNorm, NormFinder e BestKeeper. O conteúdo de TGA foi medido em cromatografia líquida de alta eficiência com espectrômetro de massa (CLAE-EM) e a expressão dos GAMEs foi determinada por reação de transcriptase reversa quantitativa seguida da reação em cadeia da polimerase (RT-qPCR). Para análise da região promotora utilizou-se: Plant Pan – Plant Promoter Analysis Navigator. Através dos resultados obtidos e analisados, podemos afirmar que o modelo, quando devidamente validado e finalizado, pode ser considerado bastante promissor. Os genes C2, SEC3 e CUL3A foram considerados como excelentes normalizadores para tubérculos de batata. A expressão dos GAMEs foi maior em tubérculos com maior conteúdo de TGA, e com exceção do GAME7, os genes GAME4, GAME6, GAME8ab, GAME11 e GAME12 demonstraram ser estatisticamente (α = 0,05) mais expressos em amostras com maior teor de TGA e menos em amostras com menor TGA, confirmando a relação destes genes com a produção de glicoalcalóides. Na análise de fatores de transcrição dos GAMEs muitos elementos cis relacionados a estresse biótico e abiótico, e regulação por luz foram encontrados, além de muitas cópias desses elementos cis nas regiões promotoras de todos os GAMEs. Os resultados obtidos no presente trabalho são importantes para um melhor entendimento da formação e regulação dos GA em tubérculos de batatas, auxiliando na predição e prevenção de formação deste composto. / Potato (Solanum tuberosum) is the third most important food crop in the world after rice and wheat in terms of human consumption. The introduction of new varieties of S. tuberosum for food or feed requires the checking of not only substances of pro-nutritional functioning on the human body, but should examine the content of toxic compounds, such as glycoalkaloids (GA), as an international norm recommendation for new potatoes varieties evaluation, including genetic modified. For this reason, the goal of the present work was build a food safety assessment model based on transcriptomics, and comparative with history of safe human consumption. Besides, we intended to determined reference genes for transcriptional analysis of potato tuber, and characterized glycoalkaloid metabolism (GAME) genes that partake of GA biosynthesis pathway. The correlation between the expression of GAME genes and total glycoalkaloids (TGA) content in different genotypes of potato tubers was evaluated. To do so, a database was build with RNASeq data from 90 potato tubers with large genotype and natural variability. These database were also used to ranking the candidates to be reference genes based on Interquartile Range (IQR). The stability of candidates for reference genes were evaluated by geNorm, NormFinder and BestKeeper. The content of TGA were measured on high performance liquid chromatography acoplated on mass spectrometry (LC-MS). Later, Reverse Transcriptase quantitative Polymerase Chain Reactions (RT-qPCR) determined the expression of GAME genes. For the promoter region analysis of GAMEs were used “Plant Pan – Plant Promoter Analysis Navigator”. It is possible affirmed that the food safety assessment model after a properly validation will be promising. The best reference genes for transcriptional analysis studies of potato tubers were C2, SEC3 and CUL3A. The expression of GAME4, GAME6, GAME7, GAME8ab, GAME11, and GAME12 were higher in tubers with high TGA content, and, exception for GAME7, the other GAME genes demonstrate statically (α = 0,05) higher expression on samples with high TGA and lower on samples with low TGA content, confirming that there is a relation between GAME genes and GA biosynthesis. On transcription factors analysis of GAME genes several cis-elements related to abiotic and biotic stress, and light regulated were found, also many copies of these factors in all GAME promoter region. All these results are very helpful to better understand GA formation and regulation on potato tubers, helping to predict and prevent the formation of these toxic.