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.

Chemistry and synthesis of 3(2H)-furanones and 4,5-dihydro analogues: mechanistic investigations and stereochemical control

The subject matter of this thesis relates to the chemistry of the five-membered oxygen heterocycles – 3(2H)-furanones and their 4, 5-dihydro analogues. Chapter one of the thesis is a review of the relevant chemistry of the compounds: their synthesis and key transformations. In chapter two, new research on 3(2H)-furanones is outlined in two parts. The first describes an investigation into the cyclisation of α'-trialkylsilyloxyenone adducts with arenesulfenyl and selenenyl chlorides into the corresponding sulfur and selenium substituted 3(2H)-furanones without the involvement of a Lewis acid catalyst. The study, largely involving in situ NMR techniques, identified key features associated with the formation and reaction of the chlorosulfide and chloroselenide intermediates, including operation of the Thorpe-Ingold effect. The knowledge gained in this study was applied (the second part) to the synthesis of vinyl substituted furanone systems from α'-trialkylsilyloxydienones where choice of the reaction conditions and electrophilic reagent was a key feature. An important difference in the behaviour of arenesulfenyl and selenenyl halides towards conjugated dienes emerged from this work. This phase of the research concluded with a new synthesis of geiparvarin, a natural product possessing anti-tumour properties.

Mechanistic study on spraying of blended biodiesel using phase Doppler anemometry

Droplet size and dynamics of blended palm oil-based fatty acid methyl ester (FAME) and diesel oil spray were mechanistically investigated using a phase Doppler anemometry. A two-fluid atomizer was applied for dispersing viscous blends of blended biodiesel oil with designated flow rates. It was experimentally found that the atomizer could generate a spray with large droplets with Sauter mean diameters of ca. 30 mm at low air injection pressure. Such large droplets traveled with a low velocity along their trajectory after emerging from the nozzle tip. The viscosity of blended biodiesel could significantly affect the atomizing process, resulting in the controlled droplet size distribution. Blended biodiesel with a certain fraction of palm oil-based FAME would be consistently atomized owing to its low viscosity. However, the viscosity could exert only a small effect on the droplet velocity profile with the air injection pressure higher than 0.2 MPa.

Osmolyte-induced folding of an intrinsically disordered protein: folding mechanism in the absence of ligand.

Understanding the interconversion between thermodynamically distinguishable states present in a protein folding pathway provides not only the kinetics and energetics of protein folding but also insights into the functional roles of these states in biological systems. The protein component of the bacterial RNase P holoenzyme from Bacillus subtilis (P protein) was previously shown to be unfolded in the absence of its cognate RNA or other anionic ligands. P protein was used in this study as a model system to explore general features of intrinsically disordered protein (IDP) folding mechanisms. The use of trimethylamine N-oxide (TMAO), an osmolyte that stabilizes the unliganded folded form of the protein, enabled us to study the folding process of P protein in the absence of ligand. Transient stopped-flow kinetic traces at various final TMAO concentrations exhibited multiphasic kinetics. Equilibrium "cotitration" experiments were performed using both TMAO and urea during the titration to produce a urea-TMAO titration surface of P protein. Both kinetic and equilibrium studies show evidence of a previously undetected intermediate state in the P protein folding process. The intermediate state is significantly populated, and the folding rate constants are relatively slow compared to those of intrinsically folded proteins similar in size and topology. The experiments and analysis described serve as a useful example for mechanistic folding studies of other IDPs.

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.

Thermodynamic and dynamic contributions to future changes in regional precipitation variance: focus on the Southeastern United States

© 2014, Springer-Verlag Berlin Heidelberg.The frequency and severity of extreme events are tightly associated with the variance of precipitation. As climate warms, the acceleration in hydrological cycle is likely to enhance the variance of precipitation across the globe. However, due to the lack of an effective analysis method, the mechanisms responsible for the changes of precipitation variance are poorly understood, especially on regional scales. Our study fills this gap by formulating a variance partition algorithm, which explicitly quantifies the contributions of atmospheric thermodynamics (specific humidity) and dynamics (wind) to the changes in regional-scale precipitation variance. Taking Southeastern (SE) United States (US) summer precipitation as an example, the algorithm is applied to the simulations of current and future climate by phase 5 of Coupled Model Intercomparison Project (CMIP5) models. The analysis suggests that compared to observations, most CMIP5 models (~60 %) tend to underestimate the summer precipitation variance over the SE US during the 1950–1999, primarily due to the errors in the modeled dynamic processes (i.e. large-scale circulation). Among the 18 CMIP5 models analyzed in this study, six of them reasonably simulate SE US summer precipitation variance in the twentieth century and the underlying physical processes; these models are thus applied for mechanistic study of future changes in SE US summer precipitation variance. In the future, the six models collectively project an intensification of SE US summer precipitation variance, resulting from the combined effects of atmospheric thermodynamics and dynamics. Between them, the latter plays a more important role. Specifically, thermodynamics results in more frequent and intensified wet summers, but does not contribute to the projected increase in the frequency and intensity of dry summers. In contrast, atmospheric dynamics explains the projected enhancement in both wet and dry summers, indicating its importance in understanding future climate change over the SE US. The results suggest that the intensified SE US summer precipitation variance is not a purely thermodynamic response to greenhouse gases forcing, and cannot be explained without the contribution of atmospheric dynamics. Our analysis provides important insights to understand the mechanisms of SE US summer precipitation variance change. The algorithm formulated in this study can be easily applied to other regions and seasons to systematically explore the mechanisms responsible for the changes in precipitation extremes in a warming climate.

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.

Clinical Insights Into the Biology and Treatment of Pancreatic Cancer.

Pancreatic cancer is a devastating disease with a universally poor prognosis. In 2015, it is estimated that there will be 48,960 new cases of pancreatic cancer and that 40,560 people will die of the disease. The 5-year survival rate is 7.2% for all patients with pancreatic cancer; however, survival depends greatly on the stage at diagnosis. Unfortunately, 53% of patients already have metastatic disease at diagnosis, which corresponds to a 5-year survival rate of 2.4%. Even for the 9% of patients with localized disease confined to the pancreas, the 5-year survival is still modest at only 27.1%. These grim statistics highlight the need for ways to identify cohorts of individuals at highest risk, methods to screen those at highest risk to identify preinvasive pathologic precursors, and development of effective systemic therapies. Recent clinical and translational progress has emphasized the relationship with diabetes, the role of the stroma, and the interplay of each of these with inflammation in the pathobiology of pancreatic cancer. In this article, we will discuss these relationships and how they might translate into novel management strategies for the treatment of this disease.