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Soil salinity is an increasing problem facing agriculture in many parts of the world. Climate change and irrigation practices have led to decreased yields of large areas of farmland due to increased salt levels in the soil. Irrigation introduces salts to the soil that with time accumulate and threaten crop yield. In arid climates like Utah, the practice of irrigation is especially threatening to salt-sensitive crops including alfalfa (Medicago sativa). Plants that have tolerance to salt are needed to feed livestock and the world's population. One approach to address this problem is to introduce genes encoding salt tolerance into the genomes of salt-sensitive plants through genetic engineering, but this approach has limitations. These limitations include the misinformed public perception of genetically modified organisms (GMOs). Even if the GMO salt-tolerant plants could produce palatable foods, in regions of the world with saline soils incongruous with farming, if consumers refuse to purchase the food then the engineering and upfront costs of production are negated. Another fairly new approach involves the isolation and development of salt-tolerant (halophilic) plant-associated bacteria. Several reports are now available demonstrating how the use of halophilic inoculants enhance plant growth in salty soil. This enhanced plant growth is most likely associated with changes in plant gene expression; however, the mechanisms behind this growth stimulation are not yet clear. Halomonas elongata 1H9, a rhizobacteria native to Goshen UT, has been identified as a plant growth-promoting rhizobacteria (PGPR) when used as an inoculant added to alfalfa seedlings grown in salty soils. Plants grown in the presence of this Halomonas sp. and 1% salt demonstrated an average increase of 2.4x the biomass of alfalfa plants grown without inoculum in salty soils. This suggests that this Halomonas sp. positively influences plant salt tolerance, which raises the question as to how the bacteria stimulate plant growth under these conditions. To identify and characterize plant genes induced by Halomonas elongata, transcriptional analysis was performed using RNA-sequencing (RNA-seq). This analysis identified a variety of differentially expressed genes (DEGs) including transcription factors (e.g. MYB14, GATA transcription factor 9, Ethylene-responsive transcription factors ER017 and ER109) and plant enzymes involved in growth and development (e.g. xyloglucan endotransglucosylase and phosphodiesterase). This was followed by gene validation via real-time quantitative PCR (RT-qPCR), the gold standard for RNA-seq validation, however this process was never successfully completed. Suggestions for next steps are included in the discussion section of chapter 3.

Soil microbiomes play pivotal roles to the health of the environment by maintaining metabolic cycles. One question is how will climate change affect soil bacteria over time and what could the repercussions be. To answer these questions, the Harvard Forest Long-Term Warming Experiment was established to mimic predicted climate change by warming plots of land 5℃ above ambient conditions. In 2017, 14 soil core samples were collected from Barre Woods warming experiment to mark 15 years since the establishment of the soil warming in that location. These samples underwent traditional metatranscriptomics to generate an mRNA library as well as a process coined cell-sorted or mini-metagenomics involving the sorting of single bacterial cells from the environment using FACS. This was followed by pooling into groups of 100 cells for more cost efficient genome recovery. 200 high-quality genomes were compiled, 12 of which were taxonomically identified as Acidobacteria. Acidobacteria are an extremely abundant and diverse phylum of bacteria that were found to be very well represented in the soil samples. Due to their abundance in many different soil environments as well as their known importance in many metabolic cycles, they were chosen as the candidate phylum to further investigate. Using a reference-based read mapping approach with the 12 Acidobacteria genomes and metatranscriptomic data, we identified over 3,000 differentially expressed genes within these organisms as a result of soil warming. Due to the diversity within the phylum itself, many of the genomes indicated different patterns of expression making it difficult to identify phylum-wide differential expression trends. However, the sigma70 factor, an important housekeeping gene used as a transcription regulator, was found to be up-regulated in a majority of the genomes. Over 30 different glycoside hydrolase encoding genes and glycosyltransferases were also found to be differentially expressed across the Acidobacteria reference genomes as well as 23 chemotaxis-related genes. Despite identifying four different groups of genes that showed statistically significant differences in expression levels, there may be more changes occurring in these soil bacteria and the soil microbiome as a whole due to climate change than previously measured by read-based analyses of metatranscriptomic data.

Sociability is an individual’s tendency to associate with conspecifics in a non-aggressive manner. Sociability can manifest in the formation of social groups that can reduce predation risk and increase feeding success. Studies of social behaviour in insects are typically through the lens of classically know social insects, however many insect species that have been long thought as non-social have been shown to exhibit social behaviour, in particular Drosophila. A previous experiment evolved lineages of high and low sociable fruit flies (Drosophila melanogaster) following 25 generations of artificial selection, after which RNA and DNA was extracted and sequenced. The main goal of this thesis was to integrate analyses of differential gene expression, transcript usage and population genomics to investigate the genetic architecture of sociability in Drosophila. I developed a pipeline to perform differential gene expression analysis by modelling gene expression using a generalized linear mixed-effect model. Here I found a total of 327 genes differentially expressed and 174 genes differentially expressed between the low and high sociable lineages. Next, I developed a pipeline to perform differential transcript usage analysis using a generalized linear mixed-effect model to model transcript usage. I found 619 genes to have transcripts with differential usage and 190 genes to have transcripts with differential usage between the low and high sociable lineages. Lastly, I developed a pipeline for population genomics to identify regions of the genome under selection. I identified genes that are likely under selection and the overlap between these genes and genes/transcripts found to be differentially expressed/used. Overall, I identified potential genes that are involved in the genetic architecture of sociability and can be further candidate tested. / Thesis / Master of Science (MSc)

Malnutrition, including both undernutrition and obesity, affects millions of people globally and is persistently on the rise. Obesity affects ~13% of adults globally and was identified as a risk factor for worse disease outcomes after the H1N1 influenza pandemic of 2009 and has since been shown to aggravate disease outcomes of respiratory viruses like severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and mosquito-borne viruses like West Nile Virus (WNV), chikungunya virus (CHIKV) and Mayaro virus (MAYV) and reduce the vaccine efficacy for influenza and SARS-CoV-2. Obesity is associated with a chronic state of inflammation and dysregulated immune response which has been proposed to be one of the mechanisms driving the severity of coronavirus disease 2019 (COVID-19). These altered signatures or biomarkers might be associated with disease outcome and prognosis. Therefore, animal models reflecting the clinical outcomes and natural immune responses observed in humans are crucial to identifying reliable biomarkers. Using mouse hepatitis virus 1 (MHV-1) as a model for SARS-CoV-2, we established obesity as a risk factor and identified biomarkers and pathways associated with worse disease outcomes. Obesity rates in low and middle-income countries (LMICs) are approaching levels found in high-income countries (HICs). Mosquito-borne viral diseases like dengue, chikungunya, and Zika pose a significant threat to LMICs and cause huge health and economic losses. Obesity was shown to worsen alphavirus pathogenesis, but interestingly, it also reduced their transmission by mosquitoes. Given the global prevalence of obesity and mosquito-borne viruses, it is critical to understand how obesity drives reduced alphavirus transmission. Using a natural transmission cycle between lean and obese mice and mosquitoes, we confirmed that obesity reduced the transmission potential of alphaviruses like CHIKV and MAYV and activated the Toll pathway in mosquito midguts. Various genes and other pathways were also altered in response to obese bloodmeal at various time-points post-bloodmeal; however, one gene, AAEL009965, was downregulated in the mosquito midguts 1-day-post-bloodmeal and its knockdown led to reduced infection rates and titers in mosquitoes. Through this thesis, we aimed to utilize obesity as a tool to identify biomarkers to predict coronavirus disease outcomes and design effective alphavirus transmission control strategies. / Doctor of Philosophy / Obesity, a global epidemic associated with chronic systemic inflammation, has emerged as an independent risk factor following infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The impact of obesity on COVID-19 severity, largely based on epidemiological data or research with non-natural hosts of SARS-CoV-2, necessitates the use of natural models of obesity to study the host's response to coronaviruses. Here we used mouse models of diet-induced obesity and infected them with a mouse coronavirus to account for the host's natural immune response against the virus. We showed that obesity exacerbates disease outcomes and identified various genes associated with disease severity in obese mice that correspond to the gene expression patterns in severe COVID-19 patients. These findings underscore the critical role of obesity in disease outcomes and pave the way for further research in this area. Obesity also worsens the disease outcome following infection with mosquito-borne alphaviruses like chikungunya (CHIKV) and Mayaro (MAYV), however, its impact on the vector's transmission potential is poorly understood. Using a natural transmission cycle between lean and obese mice and mosquitoes, we showed that obesity reduced alphavirus transmission by mosquitoes. We found altered expression of specific genes and pathways, in mosquito midguts exposed to alphavirus-infected obese bloodmeal compared to the lean ones and their knockdown led to altered infection rates in mosquitoes suggestive of their role in viral infection of mosquitoes. We also established insulin as a potential antiviral factor in obese bloodmeal. Therefore, these studies provide the basic framework for understanding the impact of obesity on viral transmission and pathogenesis by using relevant animal models. Furthermore, these studies utilize obesity as a tool to identify predictors of severe disease outcomes and identify genes that could be used as potential transmission control strategies for various mosquito-borne viruses.

Mecanismos que regem o desenvolvimento do olho composto e lóbulo óptico tem sido amplamente estudados em Drosophila melanogaster onde a retina é formada a partir de um disco imaginal anexado com o cérebro e os lóbulos opticos a partir do primórdio óptico externo. Através de histologia comparativa e análise de expressão gênica no desenvolvimento do sistema visual em Apis mellifera nós procuramos elucidar questões sobre plasticidade do desenvolvimento subjacente a fortes diferenças sexo- e casta-específico no olho assim como contribuir com aspectos evo-devo. O desenvolvimento dos lóbulos ópticos ocorre por dobramento neuroepitelial a partir de um centro de diferenciação no cérebro larval. Deste centro, a medula, lamina e lóbula surgem ao mesmo tempo em operárias e zangões. Dois passos marcam a diferenciação da lâmina (i) sua origem a partir da diferenciação de neuroblastos da camada mais externa da medula, isso coincidindo com o primeiro pico de expressão de roughest, e (ii) 24 horas mais tarde o aparecimento dos omatideos hexagonais coincidindo com o segundo pico de expressão de roughest. Com a inclusão de genes candidatos relacionados com o desenvolvimento do olho e lóbulos ópticos em insetos [small optic lobe (sol), eyes absent (eya), minibrain (mnb), sine oculis (so), embryonic lethal, abnormal vision (elav) e epidermal growth factor receptor (egfr)] nós encontramos distintos picos de expressão para sol, eya, mnb e so em níveis de transcritos e tempo de aparição do pico diferindo entre operárias e zangões. Enquanto estes quatro genes mostraram relativa sincronia durante o desenvolvimento em zangões, o mesmo não ocorreu em operárias. Além disso, em operárias sol é muito mais expresso na pré-pupa do que em zangões. Ambos os sexo mostraram padrões muito similares de expressão de elav, exceto por um atraso em zangões. Em contraste, a expressão de egfr ocorre antes em zangões. Durante a phase chave no desenvolvimento do sistema visual, uma análise global do transcriptoma, por meio de micro-arranjos mostrou vários genes relacionados com ciclo celular entre os diferencialmente expressos. Em conclusão, a relação entre tempo e eventos morfológicos com os padrões de expressão gênica revelou diferenças possivelmente relacionadas com mecanismos subjacentes ao desenvolvimento do sistema visual altamente dimorfico de Apis mellifera. / Developmental mechanisms governing compound eye development in insects have been broadly studied in Drosophila melanogaster, where the retina is formed from an imaginal disc attached to the larval brain. However little is known about eye development in other insects, most of which do not have such imaginal eye discs. Through a comparative histological and gene expression analysis of eye development in the honey bee, Apis mellifera, we intended to elucidate questions about developmental plasticity underlying the marked sex and castespecific differences in eye size, as well as to contribute to evo-devo aspects. Optic lobe development occurs by neuroepithelial folding initiating from a differentiation center in the larval brain. From this center, the medula, lamina and lobula arise at the same time in drones and workers. Two steps mark the differentiation of the lamina (i) its origin from neuroblasts differentiating in the outer layer of the medula, this coinciding with the first peak of roughest expression during the feeding stage of the fifth larval instar, and (ii) 24 hours later, the appearance of hexagonal ommatidia, coinciding with a second peak in roughest expression. Upon including further candidate genes related to insect eye development [small optic lobe (sol), eyes absent (eya), minibrain (mnb), sine oculis (so), embryonic lethal, abnormal vision (elav) and epidermal growth factor receptor (egfr)] we found distinct expression peaks for sol, eya, mnb and so, with timing and relative transcript levels differing between drones and workers. Whereas these four genes showed a relatively synchronous pattern of expression in drones in the fifth larval instar, this was not so in workers. Furthermore, in prepupae sol was higher expressed in workers than the other three genes, and also in comparison to drones. Both sexes showed a strikingly similar expression pattern for elav, except for some delay in drones. In contrast, egfr expression was found to occur earlier in drones. Through a global transcriptom analysis, done at a key step of larval development, several genes were reveled as diffetentially expressed, many of these regulating cell cycle steps. In conclusion, the relationship in the timing of morphological events with gene expression patterns revealed differences possibly related to mechanisms underlying development of the highly dimorphic compound eye in the honey bee.

Neuronal differentiation encompasses an elaborate developmental program which until recently was difficult to study in vitro. The advent of several cell lines able to differentiate in culture proved to be the turning point for gaining an understanding of molecular neuroscience. In particular the olfactory epithelium provides an attractive tool with which to investigate fundamental questions relating to neuronal differentiation, as it displays a unique capacity to regenerate and to retain a neurogenetic potential from its genesis and throughout adult life. The coordinated regulation of gene expression is fundamental to the control of neuronal differentiation. In order to reveal active processes at the molecular level and to dissect key components of molecular pathways, differential gene expression studies provide a foundation for the elucidation of dynamic molecular mechanisms. This thesis identified genes involved in neuronal differentiation by utilising a clonal olfactory receptor neuronal cell line (OLF442). Gene expression levels were identified using differential display and oligonucleotide array technology before and after serum deprivation. Differential display revealed two kinases whose expression levels were elevated during the differentiation of OLF442, identified as focal adhesion kinase (FAK) related non-kinase (FRNK) and mammalian ste20 like (MST)2 kinase. Furthermore, analysis of the oligonucleotide array data confirmed the expression of genes involved in altering presentation of extracellular matrix molecules, in mediating cytoskeletal rearrangements, and in ceasing the cell cycle, supporting the use of OLF442 as a model for studying differentiation. The differentiation of OLF442 results from the synchronisation of multiple transduction cascades and cellular responses as evidenced by the microarray data. A protein that can synchronise such signalling is the non-receptor protein tyrosine kinase, FAK. Thus the finding of the endogenous FAK inhibitor FRNK by differential display was intriguing as there was no difference in the expression level of FAK induced by differentiation, contrasting that of FRNK. This induced FRNK expression was derived autonomously as it was not responsive to the caspase-3 inhibitor, DEVD-CHO. This is particularly pertinent since the primary role of FRNK is to act as an inhibitor of FAK by competing with its substrates and reducing the phosphorylation of both FAK and its associated proteins. Differential display also revealed the upregulation of another kinase, which had 90% homology with rat MST2 kinase within the 3' UTR. Both mouse MST2 kinase (sequence submitted to GenBank, accession number AY058922) and the closely related family member MST1 kinase were sequenced and cloned. Moreover, evidence to support an autonomously expressed carboxyl-terminal domain of MST2 kinase is presented in Chapter 3 and provides a unique way in which MST2 may regulate its own activity. To further understand the role of MST in neuronal differentiation, a series of stable OLF442 transfections (with mutant and wild-type MST constructs) were carried out. MST was localised with cytoplasmic structures that may represent actin stress fibres, indicating a potential cytoskeletal role during neuronal differentiation. This indicated that MST1 may play a role in the morphological processes involved in neuronal differentiation. The identification of two kinases by differential display provided the motivation to understand the cellular context of OLF442 and to determine the phosphorylation status of the mitogen-activated protein kinase (MAPK) signalling cascades. Differentiation of OLF442 induced high-level phosphorylation of a putative B-Raf isoform, MEK2 and ERK1/2. Interestingly, there was a switch between preferential phosphorylation of MEK1 in undifferentiated OLF442 to preferential phosphorylation of MEK2 following differentiation. SAPK/JNK was also phosphorylated, as was the transcription factor c-Jun, which is a common substrate of both the ERK and SAPK/JNK signalling modules. The mapping of the cellular context of differentiating OLF442 has identified a promising model of a novel MAPK module. This consists of FAK signalling through Rap1 to ERK providing sustained activation, which is buffered or terminated by the expression of the endogenous FAK inhibitor FRNK. Furthermore, MST kinase could potentially play a role in regulating the cytoskeletal re-arrangements that are necessary for neuronal differentiation. MST kinase may signal transiently via the SAPK pathway to provide concomitant activation of c-Jun that is required for neuronal differentiation. An understanding of the gene expression pattern of the normal neuronal differentiation program allows a greater understanding of potential developmental aberrations. This could provide an opportunity for therapies to be conceived, while understanding the complexity of neuronal determination could also provide opportunities for stem cell transplantation.

Mecanismos que regem o desenvolvimento do olho composto e lóbulo óptico tem sido amplamente estudados em Drosophila melanogaster onde a retina é formada a partir de um disco imaginal anexado com o cérebro e os lóbulos opticos a partir do primórdio óptico externo. Através de histologia comparativa e análise de expressão gênica no desenvolvimento do sistema visual em Apis mellifera nós procuramos elucidar questões sobre plasticidade do desenvolvimento subjacente a fortes diferenças sexo- e casta-específico no olho assim como contribuir com aspectos evo-devo. O desenvolvimento dos lóbulos ópticos ocorre por dobramento neuroepitelial a partir de um centro de diferenciação no cérebro larval. Deste centro, a medula, lamina e lóbula surgem ao mesmo tempo em operárias e zangões. Dois passos marcam a diferenciação da lâmina (i) sua origem a partir da diferenciação de neuroblastos da camada mais externa da medula, isso coincidindo com o primeiro pico de expressão de roughest, e (ii) 24 horas mais tarde o aparecimento dos omatideos hexagonais coincidindo com o segundo pico de expressão de roughest. Com a inclusão de genes candidatos relacionados com o desenvolvimento do olho e lóbulos ópticos em insetos [small optic lobe (sol), eyes absent (eya), minibrain (mnb), sine oculis (so), embryonic lethal, abnormal vision (elav) e epidermal growth factor receptor (egfr)] nós encontramos distintos picos de expressão para sol, eya, mnb e so em níveis de transcritos e tempo de aparição do pico diferindo entre operárias e zangões. Enquanto estes quatro genes mostraram relativa sincronia durante o desenvolvimento em zangões, o mesmo não ocorreu em operárias. Além disso, em operárias sol é muito mais expresso na pré-pupa do que em zangões. Ambos os sexo mostraram padrões muito similares de expressão de elav, exceto por um atraso em zangões. Em contraste, a expressão de egfr ocorre antes em zangões. Durante a phase chave no desenvolvimento do sistema visual, uma análise global do transcriptoma, por meio de micro-arranjos mostrou vários genes relacionados com ciclo celular entre os diferencialmente expressos. Em conclusão, a relação entre tempo e eventos morfológicos com os padrões de expressão gênica revelou diferenças possivelmente relacionadas com mecanismos subjacentes ao desenvolvimento do sistema visual altamente dimorfico de Apis mellifera. / Developmental mechanisms governing compound eye development in insects have been broadly studied in Drosophila melanogaster, where the retina is formed from an imaginal disc attached to the larval brain. However little is known about eye development in other insects, most of which do not have such imaginal eye discs. Through a comparative histological and gene expression analysis of eye development in the honey bee, Apis mellifera, we intended to elucidate questions about developmental plasticity underlying the marked sex and castespecific differences in eye size, as well as to contribute to evo-devo aspects. Optic lobe development occurs by neuroepithelial folding initiating from a differentiation center in the larval brain. From this center, the medula, lamina and lobula arise at the same time in drones and workers. Two steps mark the differentiation of the lamina (i) its origin from neuroblasts differentiating in the outer layer of the medula, this coinciding with the first peak of roughest expression during the feeding stage of the fifth larval instar, and (ii) 24 hours later, the appearance of hexagonal ommatidia, coinciding with a second peak in roughest expression. Upon including further candidate genes related to insect eye development [small optic lobe (sol), eyes absent (eya), minibrain (mnb), sine oculis (so), embryonic lethal, abnormal vision (elav) and epidermal growth factor receptor (egfr)] we found distinct expression peaks for sol, eya, mnb and so, with timing and relative transcript levels differing between drones and workers. Whereas these four genes showed a relatively synchronous pattern of expression in drones in the fifth larval instar, this was not so in workers. Furthermore, in prepupae sol was higher expressed in workers than the other three genes, and also in comparison to drones. Both sexes showed a strikingly similar expression pattern for elav, except for some delay in drones. In contrast, egfr expression was found to occur earlier in drones. Through a global transcriptom analysis, done at a key step of larval development, several genes were reveled as diffetentially expressed, many of these regulating cell cycle steps. In conclusion, the relationship in the timing of morphological events with gene expression patterns revealed differences possibly related to mechanisms underlying development of the highly dimorphic compound eye in the honey bee.

Philosophiae Doctor - PhD / The growth and development of apple (Malus x domestica Borkh.) fruit occurs over a period of about 150 days after anthesis to full ripeness. During this period morphological and physiological changes occur defining fruit quality. These changes are a result of spatial and temporal patterns of gene expression during fruit development as regulated by environmental, genetic and environmental-by-genetic factors. A number of previous studies partially characterised the transcriptomes of apple leaf, fruit pulp, whole fruit, and peel plus pulp tissues, using cDNA micro arrays and other PCR based technologies. These studies, however, remain limited in throughput and specificity for transcripts of low abundance. Hence, the aim of this project was to apply a high throughput technique to characterise the full mRNA transcriptome of the ‘Golden Delicious’ fruit peels and pulp tissues in order to understand the molecular mechanisms underlying the morphophysiological changes that occur during fruit development.

Hlavním cílem této diplomové práce je analýza diferenciální exprese genů na základě negativního binomického modelu. Úvodní část je věnována teoretickému základu, pojednává o sekvenování RNA, sekvenování nové generace, výhodách a možném využití, formátu fastQ aj. Následující část už se zabývá samotnou praktickou částí, zde byl vybrán vhodný set genů, které budou později analyzovány a příslušná data byla stažena. Tato data byla zarovnána k lidskému genomu verze 37 Burrowsovou-Wheelerovou transformací s využitím bowtie mapovače, byly tak vytvořeny soubory ve formátu SAM. Toto soubory dat byly později setříděny pomocí nástroje SAMtools. Následně byly v programovém prostředí Matlab (verze R2013b) vytvořeny anotované objekty genů s využitím služby Ensembl´s BioMart. Dále byla určena genová exprese a byly odhadnuty faktory velikosti knihovny. Na závěr byly odhadnuty parametry negativního binomického rozložení a byla vyhodnocena diferenciální exprese genů.