Muscle signaling in exercise intolerance: Insights from the McArdle mouse model

We recently generated a knock-in mouse model (PYGM p.R50X/p.R50X) of McArdle disease (myophosphorylase deficiency). One mechanistic approach to unveil the molecular alterations caused by myophosphorylase deficiency, which is arguably the paradigm of 'exercise intolerance', is to compare the skeletal-muscle tissue of McArdle, heterozygous, and healthy (wild type (wt)) mice. We analyzed in quadriceps muscle of p.R50X/p.R50X (n=4), p.R50X/wt (n=6) and wt/wt mice (n=5) (all male, 8 wk-old) molecular markers of energy-sensing pathways, oxidative phosphorylation (OXPHOS) and autophagy/proteasome systems, oxidative damage and sarcoplamic reticulum (SR) Ca handling. We found a significant group effect for total AMPK (tAMPK) and ratio of phosphorylated (pAMPK)/tAMPK (P=0.012 and 0.033), with higher mean values in p.R50X/p.R50X mice vs. the other two groups. The absence of massive accumulation of ubiquitinated proteins, autophagosomes or lysosomes in p.R50X/p.R50X mice suggested no major alterations in autophagy/proteasome systems. Citrate synthase activity was lower in p.R50X/p.R50X mice vs. the other two groups (P=0.036) but no statistical effect existed for respiratory chain complexes. We found higher levels of 4-hydroxy-2-nonenal-modified proteins in p.R50X/p.R50X and p.R50X/wt mice compared with the wt/wt group (P=0.011). Sarco(endo)plasmic reticulum ATPase 1 (SERCA1) levels detected at 110kDa tended to be higher in p.R50X/p.R50X and p.R50X/wt mice compared with wt/wt animals (P=0.076), but their enzyme activity was normal. We also found an accumulation of phosphorylated SERCA1 in p.R50X/p.R50X animals. Myophosphorylase deficiency causes alterations in sensory energetic pathways together with some evidence of oxidative damage and alterations in Ca handling but with no major alterations in OXPHOS capacity or autophagy/ubiquitination pathways, which suggests that the muscle tissue of patients is likely to adapt overall favorably to exercise training interventions.

Molecular analysis of the anaerobic rumen fungus Orpinomyces - insights into an AT-rich genome

Matthew J. Nicholson, Michael K. Theodorou and Jayne L. Brookman. (2005). Molecular analysis of the anaerobic rumen fungus Orpinomyces - insights into an AT-rich genome. Microbiology, 151 (1), 121-133. Sponsorship: BBSRC RAE2008

Novel insights into the role of the GABAB receptor in depression and anxiety

The GABAB receptor is a functional heterodimer comprising the GABAB1 and GABAB2 subunits, with the GABAB1 subunit displaying two major isoforms, GABAB(1a) and GABAB(1b). Preclinical findings have strongly implicated the GABAB receptor in stress-related psychiatric disorders, however, the precise contribution of the GABAB receptor in depression and anxiety disorders remains unknown. Emerging data suggest that the interaction between adverse environmental conditions, such as early life stress, and a specific genetic composition can increase the risk to develop psychiatric disorders in adulthood. This thesis investigated the role of the GABAB receptor alone or in combination with early-life stress (maternal separation), in modulating antidepressant like and anxiety-related behaviours. Pharmacological blockade of the GABAB receptor with CGP52432 had antidepressant-like behavioural effects. Moreover, mice lacking the GABAB(1b) receptor subunit isoform exhibited antidepressant-like behaviours in adulthood but anxiety-like behaviour in early-life. In response to maternal separation, GABAB(1a)-/- mice exhibited early-life stress-induced anhedonia, a core symptom of depression, while GABAB(1b)-/- mice exhibited a more resilient phenotype. Moreover, when compared with wildtype or GABAB(1a)-/- mice, GABAB(1b)-/- mice that underwent maternal separation exhibited enhanced stressinduced neuronal activation in the hippocampus and in the nucleus accumbens (NAcc), a critical area for anhedonia thus suggesting that enhanced stress-induced neuronal activation in the hippocampus and NAcc in GABAB(1b)-/- mice may be important for their antidepressant-like phenotype and their resilience to stress-induced anhedonia. Pharmacological blockade of GABAB receptor and GABAB(1b) receptor subunit isoform loss of function increased adult hippocampal cell proliferation, thus suggesting that increased hippocampal neurogenesis could be a potential mechanism for the antidepressant-like effects of GABAB receptor antagonists and GABAB(1b) receptor subunit isoform disruption. Finally, this thesis investigated whether the expression of several genes involved in hippocampal neurogenesis or the antidepressant response were altered in the mouse hippocampus following chronic treatment with a GABAB receptor antagonist.

Novel insights into the inflammatory basis of gastrointestinal disease

It has become clear that inflammation is beneficial to man, there are situations though that the inflammatory response causes damage to the host that is harmful to health. When the inflammatory response fails or is too strong, the health of the host is damaged and disease can occur. The implication of intestinal disease caused by an ineffective immune response is of great social and economic burden to society. The overarching purpose of this thesis is to assess inflammatory signalling targets associated with immune mediated disorders such as IBD, IBS and inflammatory liver disease. By assessing these targets and modifying their function I hope to contribute and expand further the pre-existing information on these disorders and improve the therapeutic interventions available in these debilitating conditions. I will assess the role of inflammation in disorders of the GI tract and liver IBD, IBS, hepatic inflammatory injury and furthermore, I will use pharmaceutical agents to activate and suppress components of the immune system. I will examine the inflammatory response in experimental models of disease for IBD and liver injury, I will attempt to alter these pathways using pharmaceutical intervention to delineate the disease causing mechanism that may lead to clinically relevant therapeutic interventions. In regards to IBS, I will attempt to improve the existing knowledge that exists in relation to the pathogenesis of this functional bowel disorder. I will attempt to define a mechanism by which the low grade mucosal inflammation that has been demonstrated by others arises and what this inflammation is induced by. The overall aim of this thesis is to attempt to further understand the mechanisms behind GI and liver disease. Looking at the inflammatory response in these specific conditions and how they can be altered may lead to exciting new therapies for inflammatory conditions in the gastrointestinal tract.

A policy analysis of key employability interventions for the educationally disadvantaged in further education: Comparative insights for Irish education

The aim of this study is to garner comparative insights so as to aid the development of the discourse on further education (FE) conceptualisation and the relationship of FE with educational disadvantage and employability. This aim is particularly relevant in Irish education parlance amidst the historical ambiguity surrounding the functioning of FE. The study sets out to critically engage with the education/employability/economy link (eee link). This involves a critique of issues relevant to participation (which extends beyond student activity alone to social relations generally and the dialogic participation of the disadvantaged), accountability (which extends beyond performance measures alone to encompass equality of condition towards a socially just end) and human capital (which extends to both collective and individual aspects within an educational culture). As a comparative study, there is a strong focus on providing a way of conceptualising and comparatively analysing FE policy internationally. The study strikes a balance between conceptual and practical concerns. A critical comparative policy analysis is the methodology that structures the study which is informed and progressed by a genealogical method to establish the context of each of the jurisdictions of England, the United States and the European Union. Genealogy allows the use of history to diagnose the present rather than explaining how the past has caused the present. The discussion accentuates the power struggles within education policy practice using what Fairclough calls a strategic critique as well as an ideological critique. The comparative nature of the study means that there is a need to be cognizant of the diverse cultural influences on policy deliberation. The study uses the theoretical concept of paradigmatic change to critically analyse the jurisdictions. To aid with the critical analysis, a conceptual framework for legislative functions is developed so as to provide a metalanguage for educational legislation. The specific contribution of the study, while providing a manner for understanding and progressing FE policy development in a globalized Ireland, is to clear the ground for a more well-defined and critically reflexive FE sector to operate and suggests a number of issues for further deliberation.

Novel insights into the expression and function of p38δ MAPK in oesophageal squamous cell carcinoma

Oesophageal cancer is an aggressive malignancy which is resistant to conventional therapy and has a poor prognosis. A greater understanding of the underlying molecular biology of oesophageal cancer and the identification of novel targets is necessary for the future treatment of this disease. This thesis focuses specifically on the ill-defined and understudied p38δ mitogen-activated protein kinase (MAPK) and its function(s) in oesophageal squamous cell carcinoma (OESCC). In contrast to the three other p38 isoforms (p38α, -β and –γ which have to-date been relatively well-studied), p38δ MAPK signalling is poorly understood. Thus, this research elucidates some of the role(s) played by p38δ MAPK in cancer progression. This work outlines how loss of p38δ MAPK expression confers greater tumourigenicity in oesophageal cancer. Restoration of p38δ MAPK expression, however, has anti-proliferative and anti-migratory effects and decreases OESCC capacity for anchorageindependent growth. Using a novel application of an enzyme-substrate fusion approach, the effect of phosphorylated p38δ (p-p38δ) MAPK expression is also considered. The work goes onto describe the effect(s) of p38δ MAPK status on the chemosensitivity of OESCC to conventional cisplatin and 5-fluorouracil (CF) versus the effectiveness of doxorubicin, cisplatin and 5-fluorouracil (ACF). ACF treatment of p38δ MAPK-negative OESCC results in decreased proliferation, migration and recovery, and increased apoptosis when compared with CF treatment. This thesis examines the potential mechanisms by which p38δ MAPK expression is lost in OESCC and identifies epigenetic regulation as the probable cause of differential p38δ MAPK expression. Also analysed is the role p38δ MAPK and p-p38δ MAPK play in the cell cycle. In summary, this research identifies p38δ MAPK as a possible molecular target and a potential predictor of response to chemotherapy in OESCC patients.

The humoral response to HIV-1: new insights, renewed focus.

During the past 2 decades, significant advances in our understanding of the humoral immune response to human immunodeficiency virus type 1 (HIV-1) infection have been made, yet a tremendous amount of work lies ahead. Despite these advances, strategies to reliably induce antibodies that can control HIV-1 infection are still critically needed. However, recent advances in our understanding of the kinetics, specificity, and function of early humoral responses offer alternative new approaches to attain this goal. These results, along with the new broadly neutralizing antibody specificities, the role for other antibody functions, the increased understanding of HIV-1-induced changes to B cell biology, and results from the RV144 "Thai" trial showing potential modest sterilizing protection by nonneutralizing antibody responses, have renewed focus on the humoral system. In this review, recent advances in our understanding of the earliest humoral responses are discussed, highlighting presentations from the meeting on the Biology of Acute HIV Infection.

Two Antarctic penguin genomes reveal insights into their evolutionary history and molecular changes related to the Antarctic environment.

BACKGROUND: Penguins are flightless aquatic birds widely distributed in the Southern Hemisphere. The distinctive morphological and physiological features of penguins allow them to live an aquatic life, and some of them have successfully adapted to the hostile environments in Antarctica. To study the phylogenetic and population history of penguins and the molecular basis of their adaptations to Antarctica, we sequenced the genomes of the two Antarctic dwelling penguin species, the Adélie penguin [Pygoscelis adeliae] and emperor penguin [Aptenodytes forsteri]. RESULTS: Phylogenetic dating suggests that early penguins arose ~60 million years ago, coinciding with a period of global warming. Analysis of effective population sizes reveals that the two penguin species experienced population expansions from ~1 million years ago to ~100 thousand years ago, but responded differently to the climatic cooling of the last glacial period. Comparative genomic analyses with other available avian genomes identified molecular changes in genes related to epidermal structure, phototransduction, lipid metabolism, and forelimb morphology. CONCLUSIONS: Our sequencing and initial analyses of the first two penguin genomes provide insights into the timing of penguin origin, fluctuations in effective population sizes of the two penguin species over the past 10 million years, and the potential associations between these biological patterns and global climate change. The molecular changes compared with other avian genomes reflect both shared and diverse adaptations of the two penguin species to the Antarctic environment.

Early clopidogrel versus prasugrel use among contemporary STEMI and NSTEMI patients in the US: insights from the National Cardiovascular Data Registry.

BACKGROUND: P2Y12 antagonist therapy improves outcomes in acute myocardial infarction (MI) patients. Novel agents in this class are now available in the US. We studied the introduction of prasugrel into contemporary MI practice to understand the appropriateness of its use and assess for changes in antiplatelet management practices. METHODS AND RESULTS: Using ACTION Registry-GWTG (Get-with-the-Guidelines), we evaluated patterns of P2Y12 antagonist use within 24 hours of admission in 100 228 ST elevation myocardial infarction (STEMI) and 158 492 Non-ST elevation myocardial infarction (NSTEMI) patients at 548 hospitals between October 2009 and September 2012. Rates of early P2Y12 antagonist use were approximately 90% among STEMI and 57% among NSTEMI patients. From 2009 to 2012, prasugrel use increased significantly from 3% to 18% (5% to 30% in STEMI; 2% to 10% in NSTEMI; P for trend <0.001 for all). During the same period, we observed a decrease in use of early but not discharge P2Y12 antagonist among NSTEMI patients. Although contraindicated, 3.0% of patients with prior stroke received prasugrel. Prasugrel was used in 1.9% of patients ≥75 years and 4.5% of patients with weight <60 kg. In both STEMI and NSTEMI, prasugrel was most frequently used in patients at the lowest predicted risk for bleeding and mortality. Despite lack of supporting evidence, prasugrel was initiated before cardiac catheterization in 18% of NSTEMI patients. CONCLUSIONS: With prasugrel as an antiplatelet treatment option, contemporary practice shows low uptake of prasugrel and delays in P2Y12 antagonist initiation among NSTEMI patients. We also note concerning evidence of inappropriate use of prasugrel, and inadequate targeting of this more potent therapy to maximize the benefit/risk ratio.

Using Behavioral Insights For a Better Understanding of Economic Decisions (Regarding Savings, Inequality and Dishonesty)

This dissertation consists of three essays on behavioral economics, with a general aim of enriching our understanding of economic decisions using behavioral insights and experimental methodology. Each essay takes on one particular topic with this general aim.

The first chapter studies savings behavior of the poor. In this project, partnering with a savings product provider in Kenya, we tested the extent to which behavioral interventions and financial incentives can increase the saving rate of individuals with low and irregular income. Our experiment lasted for six months and included a total of twelve conditions. The control condition received weekly reminders and balance reporting via text messages. The treatment conditions received in addition one of the following interventions: (1) reminder text messages framed as if they came from the participant’s kid (2) a golden colored coin with numbers for each week of the trial, on which participants were asked to keep track of their weekly deposits (3) a match of weekly savings: The match was either 10% or 20% up to a certain amount per week. The match was either deposited at the end of each week or the highest possible match was deposited at the start of each week and was adjusted at the end. Among these interventions, by far the most effective was the coin: Those in the coin condition saved on average the highest amount and more than twice as those in the control condition. We hypothesize that being a tangible track-keeping object; the coin made subjects remember to save more often. Our results support the line of literature suggesting that saving decisions involve psychological aspects and that policy makers and product designers should take these influences into account.

The second chapter is related to views towards inequality. In this project, we investigate how the perceived fairness of income distributions depends on the beliefs about the process that generated the inequality. Specifically, we examine how two crucial features of this process affect fairness views: (1) Procedural justice - equal treatment of all, (2) Agency - one's ability to determine his/her income. We do this in a lab experiment by varying the equality of opportunity (procedural justice), and one's ability to make choices, which consequently influence subjects’ ability to influence their income (agency). We then elicit ex-post redistribution decisions of the earnings as a function of these two elements. Our results suggest both agency and procedural justice matter for fairness. Our main findings can be summarized as follows: (1) Highlighting the importance of agency, we find that inequality resulting from risk is considered to be fair only when risk is chosen freely; (2) Highlighting the importance of procedural justice, we find that introducing inequality of opportunity significantly increases redistribution, however the share of subjects redistributing none remain close to the share of subjects redistributing fully revealing an underlying heterogeneity in the population about how fairness views should account for inequality of opportunity.

The third chapter is on morality. In this project, we study whether religious rituals act as an internal reminder for basic moral principles and thus affect moral judgments. To this end, we conducted two survey experiments in Turkey and Israel to specifically test the effect of Ramadan and Yom Kippur. The results from the Turkish sample how that Ramadan has a significant effect on moral judgments to some extent for those who report to believe in God. Those who believe in God judged the moral acceptability of ten out of sixty one actions significantly differently in Ramadan, whereas those who reported not to believe in God significantly changed their judgments only for one action in Ramadan. Our results extends the hypothesis established by lab experiments that religious reminders have a significant effect on morality, by testing it in the field in the natural environment of religious rituals.

This thesis is part of a broader collaborative research agenda with both colleagues and advisors. The programming, analyses, and writing, as well as any errors in this work, are my own.

New insights into Hoogsteen base pairs in DNA duplexes from a structure-based survey.

Hoogsteen (HG) base pairs (bps) provide an alternative pairing geometry to Watson-Crick (WC) bps and can play unique functional roles in duplex DNA. Here, we use structural features unique to HG bps (syn purine base, HG hydrogen bonds and constricted C1'-C1' distance across the bp) to search for HG bps in X-ray structures of DNA duplexes in the Protein Data Bank. The survey identifies 106 A•T and 34 G•C HG bps in DNA duplexes, many of which are undocumented in the literature. It also uncovers HG-like bps with syn purines lacking HG hydrogen bonds or constricted C1'-C1' distances that are analogous to conformations that have been proposed to populate the WC-to-HG transition pathway. The survey reveals HG preferences similar to those observed for transient HG bps in solution by nuclear magnetic resonance, including stronger preferences for A•T versus G•C bps, TA versus GG steps, and also suggests enrichment at terminal ends with a preference for 5'-purine. HG bps induce small local perturbations in neighboring bps and, surprisingly, a small but significant degree of DNA bending (∼14°) directed toward the major groove. The survey provides insights into the preferences and structural consequences of HG bps in duplex DNA.

Ventricular conduction and long-term heart failure outcomes and mortality in African Americans: insights from the Jackson Heart Study.

BACKGROUND: QRS prolongation is associated with adverse outcomes in mostly white populations, but its clinical significance is not well established for other groups. We investigated the association between QRS duration and mortality in African Americans. METHODS AND RESULTS: We analyzed data from 5146 African Americans in the Jackson Heart Study stratified by QRS duration on baseline 12-lead ECG. We defined QRS prolongation as QRS≥100 ms. We assessed the association between QRS duration and all-cause mortality using Cox proportional hazards models and reported the cumulative incidence of heart failure hospitalization. We identified factors associated with the development of QRS prolongation in patients with normal baseline QRS. At baseline, 30% (n=1528) of participants had QRS prolongation. The cumulative incidences of mortality and heart failure hospitalization were greater with versus without baseline QRS prolongation: 12.6% (95% confidence interval [CI], 11.0-14.4) versus 7.1% (95% CI, 6.3-8.0) and 8.2% (95% CI, 6.9-9.7) versus 4.4% (95% CI, 3.7-5.1), respectively. After risk adjustment, QRS prolongation was associated with increased mortality (hazard ratio, 1.27; 95% CI, 1.03-1.56; P=0.02). There was a linear relationship between QRS duration and mortality (hazard ratio per 10 ms increase, 1.06; 95% CI, 1.01-1.12). Older age, male sex, prior myocardial infarction, lower ejection fraction, left ventricular hypertrophy, and left ventricular dilatation were associated with the development of QRS prolongation. CONCLUSIONS: QRS prolongation in African Americans was associated with increased mortality and heart failure hospitalization. Factors associated with developing QRS prolongation included age, male sex, prior myocardial infarction, and left ventricular structural abnormalities.

Structure of the polyisoprenyl-phosphate glycosyltransferase GtrB and insights into the mechanism of catalysis.

The attachment of a sugar to a hydrophobic polyisoprenyl carrier is the first step for all extracellular glycosylation processes. The enzymes that perform these reactions, polyisoprenyl-glycosyltransferases (PI-GTs) include dolichol phosphate mannose synthase (DPMS), which generates the mannose donor for glycosylation in the endoplasmic reticulum. Here we report the 3.0 Å resolution crystal structure of GtrB, a glucose-specific PI-GT from Synechocystis, showing a tetramer in which each protomer contributes two helices to a membrane-spanning bundle. The active site is 15 Å from the membrane, raising the question of how water-soluble and membrane-embedded substrates are brought into apposition for catalysis. A conserved juxtamembrane domain harbours disease mutations, which compromised activity in GtrB in vitro and in human DPM1 tested in zebrafish. We hypothesize a role of this domain in shielding the polyisoprenyl-phosphate for transport to the active site. Our results reveal the basis of PI-GT function, and provide a potential molecular explanation for DPM1-related disease.