Orchestration of gene expression across the seasons : Hypothalamic gene expression in natural photoperiod throughout the year in the Siberian hamster

Funding for work in the laboratory of PB was supported by Scottish Government Rural and Environment Science and Analytical Services Division, BBSRC (grant BB/M001504/1), British Society for Neuroendocrinology (research visit grant to IP). Work in the laboratory of SS was supported by a grant from the DFG (Ste 331/8-1). We thank Siegried Hilken, Marianne Brüning, Dr. Esther Lipokatic-Takacs and Dr. Frank Scherbarth at UVMH for technical assistance. We thank Graham Horgan of Bioinformatics, Statistics Scotland for assistance with some of statistical tests.

Learning Models of Gene Expression from Synthetic DNA Sequences

Thesis (Ph.D.)--University of Washington, 2016-06

Effector ExoU from the type III secretion system is an important modulator of gene expression in lung epithelial cells in response to Pseudomonas aeruginosa infection

Pseudomonas aeruginosa is an important pathogen in immunocompromised patients and secretes a diverse set of virulence factors that aid colonization and influence host cell defenses. An important early step in the establishment of infection is the production of type III-secreted effectors translocated into host cells by the bacteria. We used cDNA microarrays to compare the transcriptomic response of lung epithelial cells to P. aeruginosa mutants defective in type IV pili, the type III secretion apparatus, or in the production of specific type III-secreted effectors. Of the 18,000 cDNA clones analyzed, 55 were induced or repressed after 4 It of infection and could be classified into four different expression patterns. These include (i) host genes that are induced or repressed in a type III secretion-independent manner (32 clones), (ii) host genes induced specifically by ExoU (20 clones), and (iii) host genes induced in an ExoU-independent but type III secretion dependent manner (3 clones). In particular, ExoU was essential for the expression of immediate-early response genes, including the transcription factor c-Fos. ExoU-dependent gene expression was mediated in part by early and transient activation of the AN transcription factor complex. In conclusion, the present study provides a detailed insight into the response of epithelial cells to infection and indicates the significant role played by the type III virulence mechanism in the initial host response.

Antagonistic interaction between abscisic acid and jasmonate-ethylene signaling pathways modulates defense gene expression and disease resistance in Arabidopsis

The plant hormones abscisic acid (ABA), jasmonic acid (JA), and ethylene are involved in diverse plant processes, including the regulation of gene expression during adaptive responses to abiotic and biotic stresses. Previously, ABA has been implicated in enhancing disease susceptibility in various plant species, but currently very little is known about the molecular mechanisms underlying this phenomenon. In this study, we obtained evidence that a complex interplay between ABA and JA-ethylene signaling pathways regulate plant defense gene expression and disease resistance. First, we showed that exogenous ABA suppressed both basal and JA-ethylene-activated transcription from defense genes. By contrast, ABA deficiency as conditioned by the mutations in the ABA1 and ABA2 genes, which encode enzymes involved in ABA biosynthesis, resulted in upregulation of basal and induced transcription from JA-ethylene responsive defense genes. Second, we found that disruption of AtMYC2 (allelic to JASMONATE INSENSITIVE1 [JIN1]), encoding a basic helix-loop-helix Leu zipper transcription factor, which is a positive regulator of ABA signaling, results in elevated levels of basal and activated transcription from JA-ethylene responsive defense genes. Furthermore, the jin1/myc2 and aba2-1 mutants showed increased resistance to the necrotrophic fungal pathogen Fusarium oxysporum. Finally, using ethylene and ABA signaling mutants, we showed that interaction between ABA and ethylene signaling is mutually antagonistic in vegetative tissues. Collectively, our results indicate that the antagonistic interactions between multiple components of ABA and the JA-ethylene signaling pathways modulate defense and stress responsive gene expression in response to biotic and abiotic stresses.

Dynamics of gene expression in an artificial genome

Complex systems techniques provide a powerful tool to study the emergent properties of networks of interacting genes. In this study we extract models of genetic regulatory networks from an artificial genome, represented by a sequence of nucleotides, and analyse how variations in the connectivity and degree of inhibition of the extracted networks affects the resulting classes of behaviours. For low connectivity systems were found to be very stable. Only with higher connectivity was a significant occurrence of chaos found. Most interestingly, the peak in occurrence of chaos occurs perched on the edge of a phase transition in the occurrence of attractors.

Regulation of the expression of secretory component by human airway epithelial cells in vitro

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Quantification of functionalised gold nanoparticle-targeted knockdown of gene expression in HeLa cells

Introduction: Gene therapy continues to grow as an important area of research, primarily because of its potential in the treatment of disease. One significant area where there is a need for better understanding is in improving the efficiency of oligonucleotide delivery to the cell and indeed, following delivery, the characterization of the effects on the cell. Methods: In this report, we compare different transfection reagents as delivery vehicles for gold nanoparticles functionalized with DNA oligonucleotides, and quantify their relative transfection efficiencies. The inhibitory properties of small interfering RNA (siRNA), single-stranded RNA (ssRNA) and single-stranded DNA (ssDNA) sequences targeted to human metallothionein hMT-IIa are also quantified in HeLa cells. Techniques used in this study include fluorescence and confocal microscopy, qPCR and Western analysis. Findings: We show that the use of transfection reagents does significantly increase nanoparticle transfection efficiencies. Furthermore, siRNA, ssRNA and ssDNA sequences all have comparable inhibitory properties to ssDNA sequences immobilized onto gold nanoparticles. We also show that functionalized gold nanoparticles can co-localize with autophagosomes and illustrate other factors that can affect data collection and interpretation when performing studies with functionalized nanoparticles. Conclusions: The desired outcome for biological knockdown studies is the efficient reduction of a specific target; which we demonstrate by using ssDNA inhibitory sequences targeted to human metallothionein IIa gene transcripts that result in the knockdown of both the mRNA transcript and the target protein. © 2014 Jiwaji et al.

Preimplantation genetic diagnosis: Analysis of gene expression, nuclear DNA and cytoplasmic DNA

Preimplantation genetic diagnosis (PGD) following in vitro fertilization (IVF) offers couples at risk for transmitting genetic disorders the opportunity to identify affected embryos prior to replacement. In particular, embryo gender determination permits screening for X-linked diseases of unknown etiology. Analysis of embryos can be performed by polymerase chain reaction (PCR) amplification of material obtained by micromanipulation. This approach provides an alternative to the termination of an established pregnancy following chorionic villi sampling or amniocentesis. ^ Lately, the focus of preimplantation diagnosis and intervention has been shifting toward an attempt to correct cytoplasmic deficiencies. Accordingly, it is the aim of this investigation to develop methods to permit the examination of single cells or components thereof for clinical evaluation. In an attempt to lay the groundwork for precise therapeutic intervention for age related aneuploidy, transcripts encoding proteins believed to be involved in the proper segregation of chromosomes during human oocyte maturation were examined and quantified. Following fluorescent rapid cycle RT-PCR analysis it was determined that the concentration of cell cycle checkpoint gene transcripts decreases significantly as maternal age increases. Given the well established link between increasing maternal age and the incidence of aneuploidy, these results suggest that the degradation of these messages in aging oocytes may be involved with inappropriate chromosome separation during meiosis. ^ In order to investigate the cause of embryonic rescue observed following clinical cytoplasmic transfer procedures and with the objective of developing a diagnostic tool, mtDNA concentrations in polar bodies and subcellular components were evaluated. First, the typical concentration of mtDNA in human and mouse oocytes was determined by fluorescent rapid cycle PCR. Some disparity was noted between the copy numbers of individual cytoplasmic samples which may limit the use of the current methodology for the clinical assessment of the corresponding oocyte. ^

Development of Gene Expression Markers of Acute Heat-Light Stress in Reef-Building Corals of the Genus Porites

Coral reefs are declining worldwide due to increased incidence of climate-induced coral bleaching, which will have widespread biodiversity and economic impacts. A simple method to measure the sub-bleaching level of heat-light stress experienced by corals would greatly inform reef management practices by making it possible to assess the distribution of bleaching risks among individual reef sites. Gene expression analysis based on quantitative PCR (qPCR) can be used as a diagnostic tool to determine coral condition in situ. We evaluated the expression of 13 candidate genes during heat-light stress in a common Caribbean coral Porites astreoides, and observed strong and consistent changes in gene expression in two independent experiments. Furthermore, we found that the apparent return to baseline expression levels during a recovery phase was rapid, despite visible signs of colony bleaching. We show that the response to acute heat-light stress in P. astreoides can be monitored by measuring the difference in expression of only two genes: Hsp16 and actin. We demonstrate that this assay discriminates between corals sampled from two field sites experiencing different temperatures. We also show that the assay is applicable to an Indo-Pacific congener, P. lobata, and therefore could potentially be used to diagnose acute heat-light stress on coral reefs worldwide.

Development of gene expression-based biomarkers of exposure to metals and pesticides in the freshwater amphipod Hyalella azteca

Ecological risk assessment (ERA) is a framework for monitoring risks of exposure and adverse effects of environmental stressors to populations or communities of interest. One tool of ERA is the biomarker, which is a characteristic of an organism that reliably indicates exposure to or effects of a stressor like chemical pollution. Traditional biomarkers which rely on characteristics at the tissue level and higher often detect only acute exposures to stressors. Sensitive molecular biomarkers may detect lower stressor levels than traditional biomarkers, which helps inform risk mitigation and restoration efforts before populations and communities are irreversibly affected. In this study I developed gene expression-based molecular biomarkers of exposure to metals and insecticides in the model toxicological freshwater amphipod Hyalella azteca. My goals were to not only create sensitive molecular biomarkers for these chemicals, but also to show the utility and versatility of H. azteca in molecular studies for toxicology and risk assessment. I sequenced and assembled the H. azteca transcriptome to identify reference and stress-response gene transcripts suitable for expression monitoring. I exposed H. azteca to sub-lethal concentrations of metals (cadmium and copper) and insecticides (DDT, permethrin, and imidacloprid). Reference genes used to create normalization factors were determined for each exposure using the programs BestKeeper, GeNorm, and NormFinder. Both metals increased expression of a nuclear transcription factor (Cnc), an ABC transporter (Mrp4), and a heat shock protein (Hsp90), giving evidence of general metal exposure signature. Cadmium uniquely increased expression of a DNA repair protein (Rad51) and increased Mrp4 expression more than copper (7-fold increase compared to 2-fold increase). Together these may be unique biomarkers distinguishing cadmium and copper exposures. DDT increased expression of Hsp90, Mrp4, and the immune response gene Lgbp. Permethrin increased expression of a cytochrome P450 (Cyp2j2) and decreased expression of the immune response gene Lectin-1. Imidacloprid did not affect gene expression. Unique biomarkers were seen for DDT and permethrin, but the genes studied were not sensitive enough to detect imidacloprid at the levels used here. I demonstrated that gene expression in H. azteca detects specific chemical exposures at sub-lethal concentrations, making expression monitoring using this amphipod a useful and sensitive biomarker for risk assessment of chemical exposure.