Characterization of Vrille Function in the Drosophila Circadian Clock

TThe Drosophila circadian clock is comprised of transcriptional feedback loops, which control rhythmic gene expression and are also responsible for daily rhythms in physiology, metabolism and behavior. The core feedback loop, which employs CLOCK-CYCLE (CLK-CYC) activators and PERIOD-TIMELESS (PER-TIM) repressors to drive rhythmic transcription peaking at dusk, is required for circadian timekeeping and overt behavioral rhythms. CLK-CYC also activates an interlocked feedback loop, which uses PAR DOMAIN PROTEIN 1ɛ (PDP1ɛ) activator and VRILLE (VRI) repressor to drive Clk and other rhythmic transcription peaking at dawn. In addition to its role in the clock, vri controls many developmental processes which are essential for early embryonic development, making vri null mutants embryonic lethal. This has hindered the ability to determine the role that vri plays within the circadian clock. The goal of my study is to determine if vri is necessary for circadian time keeping and/or output. To achieve this goal, I first determined which transcripts were important for clock function. I generated an isoform specific vri mutant, which eliminated the expression of mRNAs that were thought to be essential for clock function. It turned out that these mutants are hypomorphic, as residual VRI expression was still observed in the clock cells of these mutants. Further analysis demonstrated that another vri mRNA isoform is expressed rhythmically under the control of the clock. Interestingly, the mutation impacted the abundance of short and long proteins, since the mutated mRNA can produce both VRI protein isoforms using an alternative translation initiation site. Furthermore, the E-boxes that are used by CLK-CYC are important for development, since deletion of these E-boxes leads to embryonic lethality. Then using a conditionally inactivatable transgene to rescue vri developmental lethality, I showed that clock function persists after vri inactivation, but activity rhythms were abolished. The inactivation of vri disrupts multiple output pathways thought to be important for activity rhythms, including Pigment-Dispersing Factor (PDF) accumulation and arborization rhythms in the small ventrolateral neuron (sLNvv) dorsal projection. These results demonstrate that vri acts as a key regulator of clock output, and suggests that the primary function of vri in Drosophila is to drive rhythmic transcription, which is required for overt rhythms

The CD-loop of PAI-2 (SERPINB2) is redundant in the targeting, inhibition and clearance of cell surface uPA activity

<p>Abstract</p> <p>Background</p> <p>Plasminogen activator inhibitor type-2 (PAI-2, SERPINB2) is an irreversible, specific inhibitor of the urokinase plasminogen activator (uPA). Since overexpression of uPA at the surface of cancer cells is linked to malignancy, targeting of uPA by exogenous recombinant PAI-2 has been proposed as the basis of potential cancer therapies. To this end, reproducible yields of high purity protein that maintains this targeting ability is required. Herein we validate the use <it>in vitro </it>of recombinant 6 × His-tagged-PAI-2 lacking the intrahelical loop between C and D alpha-helices (PAI-2 ΔCD-loop) for these purposes.</p> <p>Results</p> <p>We show that PAI-2 ΔCD-loop expressed and purified from the pQE9 vector system presents an easier purification target than the previously used pET15b system. Additionally, PAI-2 ΔCD-loop gave both higher yield and purity than wild-type PAI-2 expressed and purified under identical conditions. Importantly, absence of the CD-loop had no impact on the inhibition of both solution phase and cell surface uPA or on the clearance of receptor bound uPA from the cell surface. Furthermore, uPA:PAI-2 ΔCD-loop complexes had similar binding kinetics (K_D~5 nM) with the endocytosis receptor Very Low Density Lipoprotein Receptor (VLDLR) to that previously published for uPA:PAI-2 complexes.</p> <p>Conclusion</p> <p>We demonstrate that the CD-loop is redundant for the purposes of cellular uPA inhibition and cell surface clearance (endocytosis) and is thus suitable for the development of anti-uPA targeted cancer therapeutics.</p

Characterization of Vrille Function in the Drosophila Circadian Clock

TThe Drosophila circadian clock is comprised of transcriptional feedback loops, which control rhythmic gene expression and are also responsible for daily rhythms in physiology, metabolism and behavior. The core feedback loop, which employs CLOCK-CYCLE (CLK-CYC) activators and PERIOD-TIMELESS (PER-TIM) repressors to drive rhythmic transcription peaking at dusk, is required for circadian timekeeping and overt behavioral rhythms. CLK-CYC also activates an interlocked feedback loop, which uses PAR DOMAIN PROTEIN 1ɛ (PDP1ɛ) activator and VRILLE (VRI) repressor to drive Clk and other rhythmic transcription peaking at dawn. In addition to its role in the clock, vri controls many developmental processes which are essential for early embryonic development, making vri null mutants embryonic lethal. This has hindered the ability to determine the role that vri plays within the circadian clock. The goal of my study is to determine if vri is necessary for circadian time keeping and/or output. To achieve this goal, I first determined which transcripts were important for clock function. I generated an isoform specific vri mutant, which eliminated the expression of mRNAs that were thought to be essential for clock function. It turned out that these mutants are hypomorphic, as residual VRI expression was still observed in the clock cells of these mutants. Further analysis demonstrated that another vri mRNA isoform is expressed rhythmically under the control of the clock. Interestingly, the mutation impacted the abundance of short and long proteins, since the mutated mRNA can produce both VRI protein isoforms using an alternative translation initiation site. Furthermore, the E-boxes that are used by CLK-CYC are important for development, since deletion of these E-boxes leads to embryonic lethality. Then using a conditionally inactivatable transgene to rescue vri developmental lethality, I showed that clock function persists after vri inactivation, but activity rhythms were abolished. The inactivation of vri disrupts multiple output pathways thought to be important for activity rhythms, including Pigment-Dispersing Factor (PDF) accumulation and arborization rhythms in the small ventrolateral neuron (sLNvv) dorsal projection. These results demonstrate that vri acts as a key regulator of clock output, and suggests that the primary function of vri in Drosophila is to drive rhythmic transcription, which is required for overt rhythms

Epigenetic regulation of airway inflammation in asthma

Research Doctorate - Doctor of Philosophy (PhD)Asthma is an inflammatory disease that manifests in the airways. There are an estimated 300 million people worldwide currently suffer from asthma. Common asthma symptoms include dyspnea and wheezing. These are consequences of the reversible airflow obstruction associated with airway inflammation. The symptoms can be mild or can be as severe as life threatening depending on nature of underlying inflammation. Although heredity plays a role in the disease pathogenesis, the high and rising prevalence of asthma, particularly in recent decades highlights a strong influence of the environment. To this end, epigenetic phenomena including alteration of DNA methylation and chromatin structure are likely contributors to the pathogenesis of asthma as well as a plausible source of phenotype heterogeneity. Especially subtle alteration of DNA methylation patterns which occur early in life may impact on disease development. However, the exact role of epigenetic mechanisms in the pathogenesis of asthma and inflammatory phenotypes of asthma are not well understood. This thesis investigates; 1) Alterations in infant peripheral blood DNA methylation profiles associated with pre-natal exposure to maternal asthma, 2) The role of chromatin structure by analysing histone acetyl-transferases (HAT) and histone de-acetylases (HDAC) activity of peripheral blood monocytes in inflammatory phenotypes of adult asthma, 3) Alterations in the DNA methylation profile of peripheral blood monocytes associated with inflammatory phenotype of adult asthma. The primary findings of this thesis are: 1) Maternal asthma during pregnancy is associated with alterations in peripheral blood DNA methylation in infants’. 2) Inflammatory phenotypes of asthma are associated with differential DNA methylation in peripheral blood monocytes. Gene network analyses of these differentially methylated genes revealed distinct molecular pathways, suggesting possible implications in the disease pathogenesis. 3) Neutrophilic asthma is associated with lower HDAC activity and higher HAT activity of peripheral blood monocytes compared to both eosinophilic and paucigranulocytic asthma. Collectively, the findings of this thesis emphasised the significance of epigenetic factors playing a role in the development of asthma and inflammatory phenotypes of asthma. An association of peripheral blood methylation profiles of infants with maternal asthma suggests a potential inheritance of the disease susceptibility. The characteristic alterations of DNA methylation in blood monocytes suggest an underlying epigenetic basis for the inflammatory phenotypes while the differences in HAT/HDAC activity in monocytes further emphasise a role for the epigenome in the development of inflammatory phenotypes. The findings of this thesis warrant further investigation and may help us get one step closer to understanding the role of epigenetics in airway inflammation in asthma

Elevated expression of the NLRP3 inflammasome in neutrophilic asthma

Asthma is a heterogeneous inflammatory airways disorder where interleukin (IL)-1β is thought to be a key mediator, especially in the neutrophilic subtype of asthma. The generation of active IL-1β requires proteolytic cleavage typically mediated through the formation of a caspase-1-containing inflammasome. This study hypothesised that an IL-1β endotype associated with the nucleotide-binding domain, leucine-rich repeat-containing family protein (NLRP)3/apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC)/caspase-1 inflammasome is characteristic of patients with the neutrophilic subtype of asthma. Participants with asthma (n=85) and healthy controls (n=27) underwent clinical assessment, spirometry and sputum induction. Sputum was processed for differential cell count, gene expression and protein mediators. NLRP3 and caspase-1 expression was also determined by immunocytochemistry. Sputum macrophages were isolated (n=8) and gene expression of NLRP3 and IL-1β determined. There was significantly elevated gene expression of NLRP3, caspase-1, caspase-4, caspase-5 and IL-1β in participants with neutrophilic asthma. Protein levels of IL-1β were significantly higher in those with neutrophilic asthma and correlated with sputum IL-8 levels. Sputum macrophages, as well as sputum neutrophils in neutrophilic asthma, expressed NLRP3 and caspase-1 protein. NLRP3 inflammasome is upregulated in neutrophilic asthma and may regulate the inflammation process observed in this asthma phenotype through production of IL-1β

Novel immune genes associated with excessive inflammatory and antiviral responses to rhinovirus in COPD

Abstract Background Rhinovirus (RV) is a major cause of chronic obstructive pulmonary disease (COPD) exacerbations, and primarily infects bronchial epithelial cells. Immune responses from BECs to RV infection are critical in limiting viral replication, and remain unclear in COPD. The objective of this study is to investigate innate immune responses to RV infection in COPD primary BECs (pBECs) in comparison to healthy controls. Methods Primary bronchial epithelial cells (pBECs) from subjects with COPD and healthy controls were infected with RV-1B. Cells and cell supernatant were collected and analysed using gene expression microarray, qPCR, ELISA, flow cytometry and titration assay for viral replication. Results COPD pBECs responded to RV-1B infection with an increased expression of antiviral and pro-inflammatory genes compared to healthy pBECs, including cytokines, chemokines, RNA helicases, and interferons (IFNs). Similar levels of viral replication were observed in both disease groups; however COPD pBECs were highly susceptible to apoptosis. COPD pBECs differed at baseline in the expression of 9 genes, including calgranulins S100A8/A9, and 22 genes after RV-1B infection including the signalling proteins pellino-1 and interleukin-1 receptor associated kinase 2. In COPD, IFN-β/λ1 pre-treatment did not change MDA-5/RIG-I and IFN-β expression, but resulted in higher levels IFN-λ1, CXCL-10 and CCL-5. This led to reduced viral replication, but did not increase pro-inflammatory cytokines. Conclusions COPD pBECs elicit an exaggerated pro-inflammatory and antiviral response to RV-1B infection, without changing viral replication. IFN pre-treatment reduced viral replication. This study identified novel genes and pathways involved in potentiating the inflammatory response to RV in COPD.</p

Characteristic DNA methylation profiles in peripheral blood monocytes are associated with inflammatory phenotypes of asthma.

Epigenetic changes including DNA methylation caused by environmental exposures may contribute to the heterogeneous inflammatory response in asthma. Here we investigate alterations in DNA methylation of purified blood monocytes that are associated with inflammatory phenotypes of asthma. Peripheral blood was collected from adults with eosinophilic asthma (EA; n = 21), paucigranulocytic asthma (PGA; n = 22), neutrophilic asthma (NA; n = 9), and healthy controls (n = 10). Blood monocytes were isolated using ficoll density gradient and immuno-magnetic cell separation. Bisulfite converted genomic DNA was hybridized to Illumina Infinium Methylation27 arrays and analyzed for differential methylation using R/Bioconductor packages; networks of gene interactions were identified using the STRING database. Compared with healthy controls, differentially methylated CpG loci were identified in EA (n = 413), PGA (n = 495), and NA (n = 89). We found that 223, 237, and 72 loci were significantly hypermethylated in EA, PGA, and NA, respectively. Nine genes were common to all three phenotypes and showed increased methylation in asthma. Three pathway networks were identified in EA, involved in purine metabolism, calcium signaling, and ECM-receptor interaction. In PGA, two networks were identified, involved in neuroactive ligand-receptor interaction and ubiquitin mediated proteolysis. In NA, one network was identified involving sFRP1 as a key node, over representing the Wnt signaling pathway. We have identified characteristic alterations in DNA methylation that are associated with inflammatory phenotypes of asthma and may contribute to the disease mechanisms. This network-based characterization may help in the development of epigenetic biomarkers and therapeutic targets for asthma

Differential DNA methylation profiles of infants exposed to maternal asthma during pregnancy

Background: Asthma is a complex disease that involves both genetic factors and environmental exposures. Aberrant epigenetic modifications, such as DNA methylation, may be important in asthma development. Fetal exposure to maternal asthma during critical periods of in utero development may lead to epigenetic alterations that predispose infants to a greater risk of developing asthma themselves. We investigated alterations in the DNA methylation profile of peripheral blood from infants exposed to maternal asthma during pregnancy. Methods: Peripheral blood was collected from 12-month-old infants born to women with (n = 25) and without (n = 15) doctor diagnosed asthma during pregnancy. Genomic DNA was extracted, bisulfite converted, and hybridized to Infinium Methylation 27 arrays (Illumina), containing over27,000 CpGs from 14,495 genes. CpG loci in only autosomal genes were classified as differentially methylated at the 99% level (P < 0.01, |DiffScore| > 22 and delta beta >0.06). Results: There were 70 CpG loci, corresponding to 67 genes that were significantly differentially methylated. Twelve CpG loci (11 genes) showed greater than 10% comparative difference in DNA methylation, including hyper-methylated loci of FAM181A, MRI1, PIWIL1, CHFR, DEFA1, MRPL28, AURKA, and hypo-methylated loci of NALP1L5, MAP8KIP3, ACAT2, and PM20D1 in maternal asthma. Methylation of MAPK8IP3 was significantly negatively correlated with maternal blood eosinophils (r = −0.38; P = 0.022), maternal eNO (r = −0.44; P = 0.005), and maternal serum total IgE (r = −0.39, P = 0.015). Methylation of AURKA negatively correlated with maternal hemoglobin (r = −0.43; P = 0.008), infants height (r = −0.51; P < 0.001) and weight (r = −0.36; P = 0.021). Methylation of PM20D1 was lower in infants born to mothers with asthma on inhaled corticosteroid treatment. Methylation of PM20D1 was lower and MRI1 was higher in infants born to atopic mothers without asthma. Conclusions: In an Australian study population, exposure to maternal asthma during pregnancy is associated with differential methylation profiles of infants' peripheral blood DNA, which may act as risk factors for future asthma development

Novel immune genes associated with excessive inflammatory and antiviral responses to rhinovirus in COPD

Background: Rhinovirus (RV) is a major cause of chronic obstructive pulmonary disease (COPD) exacerbations, and primarily infects bronchial epithelial cells. Immune responses from BECs to RV infection are critical in limiting viral replication, and remain unclear in COPD. The objective of this study is to investigate innate immune responses to RV infection in COPD primary BECs (pBECs) in comparison to healthy controls. Methods: Primary bronchial epithelial cells (pBECs) from subjects with COPD and healthy controls were infected with RV-1B. Cells and cell supernatant were collected and analysed using gene expression microarray, qPCR, ELISA, flow cytometry and titration assay for viral replication. Results: COPD pBECs responded to RV-1B infection with an increased expression of antiviral and pro-inflammatory genes compared to healthy pBECs, including cytokines, chemokines, RNA helicases, and interferons (IFNs). Similar levels of viral replication were observed in both disease groups; however COPD pBECs were highly susceptible to apoptosis. COPD pBECs differed at baseline in the expression of 9 genes, including calgranulins S100A8/A9, and 22 genes after RV-1B infection including the signalling proteins pellino-1 and interleukin-1 receptor associated kinase 2. In COPD, IFN-β/λ1 pre-treatment did not change MDA-5/RIG-I and IFN-β expression, but resulted in higher levels IFN-λ1, CXCL-10 and CCL-5. This led to reduced viral replication, but did not increase pro-inflammatory cytokines. Conclusions: COPD pBECs elicit an exaggerated pro-inflammatory and antiviral response to RV-1B infection, without changing viral replication. IFN pre-treatment reduced viral replication. This study identified novel genes and pathways involved in potentiating the inflammatory response to RV in COPD