MicroRNAs (miRNAs) play a vital role in regulating gene expression. Detecting conserved and novel miRNAs in very large genomic datasets generated using next generation sequencing platforms is a new research area in the field of gene regulation, but finding useful miRNA information from a large wheat genome is a challenging research project. We propose to design a toolchain that will identify conserved miRNAs using various software tools such as Basic Local Alignment Search Tool (BLAST), Bowtie 2, MAFFT and RNAfold. Our toolchain identified 36 wheat conserved miRNA families that matched with 232 experimental sequences. Moreover, we found 87 plant conserved miRNA families that matched between 613 experimental sequences and the miRBase dataset. In addition, we observed significant differential expression for the wheat exposed to the heat stress compared to those exposed to light and UV stresses or no stress (control). / October 2015

The colonial marine bryozoan, Bugula neritina, harbors an uncultured endosymbiont, “Candidatus Endobugula sertula” throughout its life stages. The bacterial symbiont has been proposed to be a source of complex polyketide metabolites, the bryostatins, that chemically defend B. neritina larvae from predation. Within a bryozoan colony, significantly higher amounts of bryostatins are found in ovicell-bearing zooids where the developing larvae are brooded, as compared to ovicell-free zooids. It is hypothesized that signaling between B. neritina and “Ca. Endobugula sertula” may be involved in the regulation of bryostatin production in different zooids, as well as in maintenance of the symbiosis. In this study, suppression subtractive hybridization (SSH) was used to identify differentially expressed host genes during this association. The identified genes suggest that the host plays a role in the distribution and localization of bacterial symbionts in different host zooids, possibly to regulate levels of bryostatin production in the zooids.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Atherosclerosis is a disease of both lipids and inflammatory immune cells. More specifically, elevated plasma levels of low-density lipoproteins (LDL) ultimately lead to migration of circulating monocytes into the artery wall. Lipid-loaded monocytes proliferate and become macrophage foam cells, the hallmark of atherosclerotic lesions. A proposed mechanism for the protective effects of high-density lipoprotein (HDL) is apolipoprotein A-I (apoA-I) acting as a mediator of cholesterol efflux from cells and subsequent foam cell regression. To better understand the biological changes stimulated by apoA-I treatment, differential gene expression analysis of microarray data was performed on spleen cells from mice treated with recombinant HDL (rHDL). LDL receptor null (LDLr-/-) and LDL receptor and apoA-I null (LDLr-/-, apoA-I-/-) mice were fed a Western diet consisting of 0.2% cholesterol and 42% calories as fat (HF) for a total of 12 weeks. After six weeks of diet, a subset of mice for each genotype was subcutaneously injected with 200 micrograms of rHDL (protein weight) three times a week for the remaining six weeks. The control group of mice was subcutaneously injected with 200 micrograms of bovine serum albumin (BSA). Spleen cell RNA was isolated, purified, and analyzed via Illumina BeadArray Microarray Technology. Individual differential gene expression analysis that contrasted treated to non-treated groups for each genotype was performed. LDLr-/-, apoA-I-/- rHDL treated mice showed 281 significantly differentially expressed genes compared to non-treated mice while LDLr-/- mice had 1502 such genes. Of the significant genes, 189 intersected across both genotypes. In LDLr-/-, apoA-I-/-, 73 of these were up-regulated and 116 were down-regulated. LDLr-/- similarly showed 71 of the intersected genes to be up-regulated and 118 to be down-regulated. One-directional gene set pathway analysis was also performed. LDLr-/-, apoA-I-/- treated mice revealed 49 significant pathways while LDLr-/- showed a total of 63. Of these, 21 were up-regulated and 14 were down-regulated in both genotypes. Of the overrepresented, up-regulated pathways, eight of the top ten most significant ones were related to immune cells. Major functions involved receptor, adhesion, and chemokine signaling. Overall, preliminary analysis suggests apoA-I treatment induces similar gene expression changes across different genotypes in mouse spleen cells.

No description available.

BACKGROUND: Organelle retention is a form of mixotrophy that allows organisms to reap metabolic benefits similar to those of photoautotrophs through capture of algal prey and sequestration of their plastids. Mesodinium rubrum is an abundant and broadly distributed photosynthetic marine ciliate that steals organelles from cryptophyte algae, such as Geminigera cryophila. M. rubrum is unique from most other acquired phototrophs because it also steals a functional nucleus that facilitates genetic control of sequestered plastids and other organelles. We analyzed changes in G. cryophila nuclear gene expression and transcript abundance after its incorporation into the cellular architecture of M. rubrum as an initial step towards understanding this complex system. METHODS: We compared Illumina-generated transcriptomes of the cryptophyte Geminigera cryophila as a free-living cell and as a sequestered nucleus in M. rubrum to identify changes in protein abundance and gene expression. After KEGG annotation, proteins were clustered by functional categories, which were evaluated for over- or under-representation in the sequestered nucleus. Similarly, coding sequences were grouped by KEGG categories/ pathways, which were then evaluated for over- or under-expression via read count strategies. RESULTS: At the time of sampling, the global transcriptome of M. rubrum was dominated (~58-62 %) by transcription from its stolen nucleus. A comparison of transcriptomes from free-living G. cryophila cells to those of the sequestered nucleus revealed a decrease in gene expression and transcript abundance for most functional protein categories within the ciliate. However, genes coding for proteins involved in photosynthesis, oxidative stress reduction, and several other metabolic pathways revealed striking exceptions to this general decline. CONCLUSIONS: Major changes in G. cryophila transcript expression after sequestration by M. rubrum and the ciliate's success as a photoautotroph imply some level of control or gene regulation by the ciliate and at the very least reflect a degree of coordination between host and foreign organelles. Intriguingly, cryptophyte genes involved in protein transport are significantly under-expressed in M. rubrum, implicating a role for the ciliate's endomembrane system in targeting cryptophyte proteins to plastid complexes. Collectively, this initial portrait of an acquired transcriptome within a dynamic and ecologically successful ciliate highlights the remarkable cellular and metabolic chimerism of this system.

Scrapie is a transmissible spongiform encephalopathy (TSE) of sheep and goats, for which there is currently no ante-mortem diagnostic test. A rapid, ante-mortem diagnostic test for scrapie would also potentially be important for other TSEs such as bovine spongiform encephalopathy (BSE) and variant Creutzfeldt Jakob's disease (vCJD). The hypothesis of this study was that there is differential gene expression in the blood and peripheral tissues of scrapie infected animals, and that a panel of differentially expressed genes could be identified and used as surrogate markers of infection. An expression screening approach, using real-time PCR and an EST microarray, was used to identify genes that were differentially expressed between SSBP/1 infected and mock-infected control sheep. The animals used in this study were New Zealand Cheviot sheep of three genotypes, the highly susceptible VRQ/VRQ (incubation time 193 ± 12 days), the intermediately susceptible VRQ/ARR (incubation time 325 ± 36 days) and the disease resistant ARR/ARR (no clinical signs of disease), experimentally infected with scrapie strain SSBP/1 and sacrificed at various time points post infection. No differentially expressed candidates were identified in blood. Other microarray experiments in our group had demonstrated evidence of differential expression in spleen fractions enriched for follicular dendritic cells (FDCs). These data were analysed and candidates were selected for quantitative real-time PCR validation, with a view to assessing the expression of validated candidates in blood as a more targeted approach to identifying markers of infection. The gene Early Growth Response 1 (EGR1) emerged as an interesting candidate as its expression was found to be significantly up-regulated in FDC-enriched spleen samples of VRQ/VRQ and ARR/ARR animals over a number of time points post infection. EGR1 expression was steady among all mock-infected controls. There was, however, no evidence of differential expression of EGR1 in blood. This is the first report of differential expression of EGR1 in preclinical spleen samples in sheep. EGR1 is an attractive candidate for a surrogate marker of preclinical infection, as its levels rise very early after infection and remain elevated for a sustained amount of time in the VRQ/VRQ sheep. Elevated expression is also detectable in VRQ/ARR and in ARR/ARR sheep. Further studies with larger sample numbers would be necessary to more accurately estimate the extent of differential expression and to assess its true worth as a diagnostic marker. Expression studies in samples from other TSEs and non-TSE neuropathological disease would also be necessary to establish whether differential expression of EGR1 is specific to TSE disease.

Cardiac hypertrophy defines an adaptive process brought about in response to sustained increases in haemodynamic work. Cardiomyocytes undergo an initial compensatory phase in which enlargement and contractility alterations normalise wall stress and maintain adequate perfusion of organs. In pathological hypertrophy, this deteriorates to a decompensated state characterised by ventricular dysfunction and predisposition to heart failure. In contrast, physiological hypertrophy and associated enhanced cardiac functioning arising from chronic exercise training does not progress to heart failure. Determination of the molecular pathways underlying myocardial hypertrophy remains a challenge for cardiovascular research. The objective of the work presented in this thesis was to identify genes differentially expressed during pathological and physiological hypertrophy in order to enhance our knowledge of the mechanistic processes involved. A reverse Northern hybridisation method was applied to profile the expression of specifically selected genes in the hypertrophic models examined. Functional categories represented in the gene panel assembled included cardiac contractile and cytoskeletal markers, matrix metalloproteinases, vasoactive pathway factors, calcium handling genes, ion channels, cardiac regulatory factors, signalling pathway intermediates, apoptotic factors and histone deacetylases. In order to investigate pathological hypertrophy, a deoxycorticosterone acetate-salt (DOCA-salt) rat model was utilised. DOCA-salt treated rats used in this study demonstrated a 1.4-fold increase in heart weight to body weight ratio compared to controls. Impaired cardiac function indicative of a decompensated pathological phenotype in the DOCA-salt treated group was demonstrated by way of decreased chamber size, impaired myocardial compliance and significantly reduced cardiac output. Reverse Northern hybridisation analysis of 95 selected genes identified a number of candidates with differential expression in hearts of DOCA-salt treated rats. Increased gene expression was demonstrated for the collagenase MMP1 and stress-activated signal transduction factor Sin1. In contrast, the sarcoplasmic reticulum calcium ATPase SERCA-2 and anti-apoptotic factor BCL2l-10 genes exhibited decreased expression. To investigate changes in gene expression associated with physiological hypertrophy, use was made of an endurance run-trained rat model. The run-trained rats used in this study demonstrated a 24.1% increase in heart weight to body weight ratio and improvements in performance consistent with physiological cardiac adaptation. These performance indicators included improvements in systolic volume, cardiac output, myocardial compliance and bio-energetic function. Reverse Northern hybridisation expression analysis of 56 genes identified a number of differentially expressed mRNA transcripts in run-trained hypertrophied hearts. Four genes shown to demonstrate reduced expression in the run-trained rat model were interleukin-1 receptor associated kinase (IRAK1) and the developmentally expressed transcription factors Nkx-2.3, dHAND, and IRX-2. Based upon the reverse Northern hybridisation results, four genes were selected for Western blotting analysis of rat cardiac tissue. Of these, MMP1 and a putative isoform of Sin1 exhibited increased levels in DOCA-salt treated hypertrophic left ventricular tissue, results that correlate with the findings of increased mRNA expression for these two genes. Therefore, this study identified MMP1 and Sin1 as candidates involved in pathological but not physiological hypertrophy. This finding is in accord with other recent investigations demonstrating that pathological hypertrophy and physiological hypertrophy are associated with distinct molecular phenotypes. An aside to the major objective of identifying genes differentially regulated in left ventricular hypertrophy involved the application of the P19CL6 cell in vitro model of cardiomyogenesis to compare protein expression during hypertrophy and development. The Sin1 isoform, found to be up-regulated during DOCA-salt induced hypertrophy, was also shown to be more abundant in differentiating, than non-differentiating, P19CL6 cells. This result is consistent with the developing paradigm that implicates 'fetal' genes in the hypertrophic remodelling process.

<p>Lichen-forming fungi employ a successful mode of nutrition as symbiotic partners with green algae and/or cyanobacteria (the photobiont). Nearly one fifth of all known fungi are obligate lichen formers, yet we know little of how they find compatible partners and establish long-lived symbiotic relationships. The combined growth of these symbionts forms a body (thallus) with emergent properties unlike either of the symbionts individually grown. Based on other well-studied eukaryotic systems, the development of a lichen thallus must rely upon the successful identification and collaboration of these two very different organisms. Identifying the molecular basis of microbe recognition and interactions remains one of the greatest challenges in studying symbiotic systems. </p><p>In this thesis, I determine the stage in which to begin looking for lichen symbiosis specific genes, and then examine mycobiont and photobiont genes that, when compared to the aposymbiotic state, are upregulated in the symbiotic state. Using the symbiosis between the mycobiont <italic>Cladonia grayi</italic> and the photobiont <italic>Asterochloris</italic> sp., as well as scanning electron microscopy observations of the earliest stages of contact between <italic>C. grayi</italic> and <italic>Asterochloris</italic> sp., I determined that the mycobiont undergoes a specific change in phenotypic growth in response to <italic>Asterochloris</italic> sp. This change is particular to the lichen symbiosis, and is not observed with algal shaped inanimate objects or algae other than <italic>Asterochloris</italic>. I then used this phenotypically defined stage that is exclusive to lichen symbiosis to begin studying the the genetic and molecular mechanisms underlying the development of a stratified lichen thallus. Using suppression subtractive hybridization to determine differential gene expression, fungal and algal libraries were made of upregulated genes in the first 2 stages of lichen symbiosis. The symbiotic expression levels of select genes were then verified using quantitative PCR. Lastly, a candidate gene for involvement in lichen symbiosis was transformed into <italic>Saccharomyces cerevisiae</italic> to test for protein function.</p><p>Further results of this study show that the fungal protein products of genes upregulated in lichen symbiosis show significant matches to proteins putatively involved in fungal self and non-self recognition, lipid metabolism, negative regulation of glucose repressible genes, an oxidoreductase, a dioxygenase, and a conserved hypothetical protein. Algal genes that are upregulated in lichen symbiosis include a chitinase-like protein, an amino acid metabolism protein, a dynein related protein, and a protein arginine methyltransferase. Furthermore, genes that are expressed in the early stages of lichen symbiosis are common varying metabolic pathways. Furthermore stages 1 and 2 of development are marked not by a drastic change in transcriptional products, but instead by an overall change in genes that are already expressed. Finally, the <italic>Cladonia~grayi Lip3</italic>was cloned in its entirety from genomic DNA and cDNA, was predicted to be secreted using signal peptide prediction software, and shown to be a functioning secreted extracellular lipase in yeast.</p><p>I conclude that many genes are involved in the interactions of symbionts and the development of a stratified lichen thallus, and that many more genes remain to be discovered. Furthermore, the possibility that genes exist in either symbiont that are specific to lichen symbiosis remains, and that their discovery awaits the creation of better genomic tools for \textit{Cladonia~grayi} and <italic>Asterochloris</italic>.</p> / Dissertation

Yellow rust disease is one of the most important problems in wheat production. It causes substantial yield losses throughout the world. There are resistant and susceptible wheat varieties to various yellow rust pathotypes. In this thesis genes that are induced in wheat, in virulence and avirulence conditions upon yellow rust inoculations were investigated. Consequently, it was aimed to identify genes that may be playing critical roles in the disease resistance mechanism. The strategy was to construct subtracted cDNA libraries from resistant and susceptible plants and analyse the sequences obtained from these libraries. The subtraction approach in this study differs from the common subtraction designs implicated in plant-pathogen interactions / instead of comparing a compatible or an incompatible interaction with a control, one of the subtractions in this study is done taking a compatible interaction as the tester and an incompatible one as the driver, and the second subtraction, vice versa. Therefore, it was intended to compare the transcriptomes from compatible and incompatible plant-pathogen interactions directly. Suppression Subtractive Hybridization method was used to construct subtracted cDNA libraries. Two subtractions were performed / SSH1 (D-R), taking a compatible interaction as the tester sample and an incompatible one as the driver sample, and SSH2 (R-D), taking an incompatible interaction as the tester sample and a compatible one as the driver. In the end, two subtracted cDNA libraries, SSH1 (D-R) library (1536 clones) and SSH2 (R-D) library (1152 clones) were obtained and the libraries were sequenced. Sequence results were subjected to BlastN and BlastX analysis. We looked for a group of genes that were frequently emphasized in plant disease related studies when we searched within the Blast N homology results of the two libraries. We found out that 19 such genes are present in our libraries. We discussed supposed induction of these genes in the interactions investigated in our study. The fact that these genes were found to be present in our libraries enhances the reliability of our results suggesting that the gene sequences we found indeed belong to genes differentially expressed in the respective comparisons investigated in our study. As such, it also implies that other sequences that were found similar to genes of known functions may represent candidate genes as subjects of further studies investigating wheat-yellow rust interactions.