Cis-acting elements and developmental regulators govern toxin gene expression in Bacillus anthracis

Expression of the structural genes for the anthrax toxin proteins is coordinately controlled by host-related signals such as elevated CO2 , and the trans-acting positive regulator, AtxA. No specific binding of AtxA to the toxin gene promoters has been demonstrated and no sequence-based similarities are apparent in the promoter regions of toxin genes. We hypothesized that the toxin genes possess common structural features that are required for positive regulation. To test this hypothesis, I performed an extensive characterization of the toxin gene promoters. I determined the minimal sequences required for atxA-mediated toxin gene expression and compared these sequences for structural similarities. In silico modeling and in vitro experiments indicated significant curvature within these regions. Random mutagenesis revealed that point mutations associated with reduced transcriptional activity, mostly mapped to areas of high curvature. This work enabled the identification of two potential cis-acting elements implicated in AtxA-mediated regulation of the toxin genes. In addition to the growth condition requirements and AtxA, toxin gene expression is under growth phase regulation. The transition state regulator AbrB represses atxA expression to influence toxin synthesis. Here I report that toxin gene expression also requires sigH, a gene encoding the RNA polymerase sigma factor associated with development in B. subtilis. In the well-studied B. subtilis system, σH is part of a feedback control pathway that involves AbrB and the major response regulator of sporulation initiation, Spo0A. My data indicate that in B. anthracis, regulatory relationships exist between these developmental regulators and atxA . Interestingly, during growth in toxin-inducing conditions, sigH and abrB expression deviates from that described for B. subtilis, affecting expression of the atxA gene. These findings, combined with previous observations, suggest that the steady state level of atxA expression is critical for optimal toxin gene transcription. I propose a model whereby, under toxin-inducing conditions, control of toxin gene expression is fine-tuned by the independent effects of the developmental regulators on the expression of atxA . The growth condition-dependent changes in expression of these regulators may be crucial for the correct timing and uninterrupted expression of the toxin genes during infection. ^

Genes to blood pressure: The effects of variation in genes of the renin-angiotensin system on the hormonal and renal control of blood pressure

Objective. Essential hypertension affects 25% of the US adult population and is a leading contributor to morbidity and mortality. Because BP is a multifactorial phenotype that resists simple genetic analysis, intermediate phenotypes within the complex network of BP regulatory systems may be more accessible to genetic dissection. The Renin-Angiotensin System (RAS) is known to influence intermediate and long-term blood pressure regulation through alterations in vascular tone and renal sodium and fluid resorption. This dissertation examines associations between renin (REN), angiotensinogen (AGT), angiotensin-converting enzyme (ACE) and angiotensin II type 1 receptor (AT1) gene variation and interindividual differences in plasma hormone levels, renal hemodynamics, and BP homeostasis.^ Methods. A total of 150 unrelated men and 150 unrelated women, between 20.0 and 49.9 years of age and free of acute or chronic illness except for a history of hypertension (11 men and 7 women, all off medications), were studied after one week on a controlled sodium diet. RAS plasma hormone levels, renal hemodynamics and BP were determined prior to and during angiotensin II (Ang II) infusion. Individuals were genotyped by PCR for a variable number tandem repeat (VNTR) polymorphism in REN, and for the following restriction fragment length polymorphisms (RFLP): AGT M235T, ACE I/D, and AT1 A1166C. Associations between clinical measurements and allelic variation were examined using multiple linear regression statistical models.^ Results. Women homozygous for the AT1 1166C allele demonstrated higher intracellular levels of sodium (p = 0.044). Men homozygous for the AGT T235 allele demonstrated a blunted decrement in renal plasma flow in response to Ang II infusion (p = 0.0002). There were no significant associations between RAS gene variation and interindividual variation in RAS plasma hormone levels or BP.^ Conclusions. Rather than identifying new BP controlling genes or alleles, the study paradigm employed in this thesis (i.e., measured genes, controlled environments and interventions) may provide mechanistic insight into how candidate genes affect BP homeostasis. ^

THE EFFECT OF PERSON-ENVIRONMENT FIT ON THE PSYCHOLOGICAL WELL-BEING OF PSYCHIATRIC AIDES WORKING IN A STATE HOSPITAL (COPING RESOURCES, SOCIAL SUPPORT, OCCUPATIONAL STRESS, LOCUS-OF-CONTROL)

This study addressed two purposes: (1) to determine the effect of person-environment fit on the psychological well-being of psychiatric aides and (2) to determine what role the coping resources of social support and control have on the above relationship. Two hundred and ten psychiatric aides working in a state hospital in Texas responded to a questionnaire pertaining to these issues.^ Person-environment fit, as a measure of occupational stress, was assessed through a modified version of the Work Environment Scale (WES). The WES subscales used in this study were: involvement, autonomy, job pressure, job clarity, and physical comfort. Psychological well-being was measured with the General Well-Being Schedule which was developed by the National Center for Health Statistics. Co-worker and supervisor support were measured through the WES and finally, control was assessed through Rotter's Locus of Control Scale.^ The results of this study were as follows: (1) all person-environment (p-e) dimensions appeared to have linear relationships with psychological well-being; (2) the p-e fit - well-being relationship did not appear to be confounded by demographic factors; (3) all p-e fit dimensions were significantly related to well-being except for autonomy; (4) p-e fit was more strongly related to well-being than the environmental measure alone; (5) supervisor support and non-work related support were found to have additive effects on the relationship between p-e fit and well-being, however no interaction or buffering effects were observed; (6) locus of control was found to have additive effects in the prediction of well-being and showed interactive effects with work pressure, involvement and physical comfort; and (7) the testing of the overall study model which included many of the components mentioned above yielded an R('2) = .27.^ Implications of these findings are discussed, future research suggested and applications proposed. ^

Motivational interviewing and feedback for college drinkers: Handling missing values with complete case analysis and multiple imputation

Objective: In this secondary data analysis, three statistical methodologies were implemented to handle cases with missing data in a motivational interviewing and feedback study. The aim was to evaluate the impact that these methodologies have on the data analysis. ^ Methods: We first evaluated whether the assumption of missing completely at random held for this study. We then proceeded to conduct a secondary data analysis using a mixed linear model to handle missing data with three methodologies (a) complete case analysis, (b) multiple imputation with explicit model containing outcome variables, time, and the interaction of time and treatment, and (c) multiple imputation with explicit model containing outcome variables, time, the interaction of time and treatment, and additional covariates (e.g., age, gender, smoke, years in school, marital status, housing, race/ethnicity, and if participants play on athletic team). Several comparisons were conducted including the following ones: 1) the motivation interviewing with feedback group (MIF) vs. the assessment only group (AO), the motivation interviewing group (MIO) vs. AO, and the intervention of the feedback only group (FBO) vs. AO, 2) MIF vs. FBO, and 3) MIF vs. MIO.^ Results: We first evaluated the patterns of missingness in this study, which indicated that about 13% of participants showed monotone missing patterns, and about 3.5% showed non-monotone missing patterns. Then we evaluated the assumption of missing completely at random by Little's missing completely at random (MCAR) test, in which the Chi-Square test statistic was 167.8 with 125 degrees of freedom, and its associated p-value was p=0.006, which indicated that the data could not be assumed to be missing completely at random. After that, we compared if the three different strategies reached the same results. For the comparison between MIF and AO as well as the comparison between MIF and FBO, only the multiple imputation with additional covariates by uncongenial and congenial models reached different results. For the comparison between MIF and MIO, all the methodologies for handling missing values obtained different results. ^ Discussions: The study indicated that, first, missingness was crucial in this study. Second, to understand the assumptions of the model was important since we could not identify if the data were missing at random or missing not at random. Therefore, future researches should focus on exploring more sensitivity analyses under missing not at random assumption.^

DETERMINING THE GENOTYPE-PHENOTYPE CONNECTION IN SYNTHETIC INDUCIBLE GENE EXPRESSION SYSTEMS

Introduction Gene expression is an important process whereby the genotype controls an individual cell’s phenotype. However, even genetically identical cells display a variety of phenotypes, which may be attributed to differences in their environment. Yet, even after controlling for these two factors, individual phenotypes still diverge due to noisy gene expression. Synthetic gene expression systems allow investigators to isolate, control, and measure the effects of noise on cell phenotypes. I used mathematical and computational methods to design, study, and predict the behavior of synthetic gene expression systems in S. cerevisiae, which were affected by noise. Methods I created probabilistic biochemical reaction models from known behaviors of the tetR and rtTA genes, gene products, and their gene architectures. I then simplified these models to account for essential behaviors of gene expression systems. Finally, I used these models to predict behaviors of modified gene expression systems, which were experimentally verified. Results Cell growth, which is often ignored when formulating chemical kinetics models, was essential for understanding gene expression behavior. Models incorporating growth effects were used to explain unexpected reductions in gene expression noise, design a set of gene expression systems with “linear” dose-responses, and quantify the speed with which cells explored their fitness landscapes due to noisy gene expression. Conclusions Models incorporating noisy gene expression and cell division were necessary to design, understand, and predict the behaviors of synthetic gene expression systems. The methods and models developed here will allow investigators to more efficiently design new gene expression systems, and infer gene expression properties of TetR based systems.

Rabies control in the developing world: Results of a cross-sectional study in Bohol, Philippines

Rabies remains a significant problem in much of the developed world, where canine rabies is not well controlled, and the bite of an infected dog is the most common means of transmission. The Philippines continues to report several hundred cases of human rabies every year, and many more cases go undetected. In recent years, the province of Bohol has been targeted by the Philippine government and the World Health Organization for a rabies eradication program. ^ The primary objective of this dissertation research was to describe factors associated with dog vaccination coverage and knowledge, attitudes, and practices regarding rabies among households in Bohol, Philippines. Utilizing a cross-sectional cluster survey design, we sampled 460 households and 541 dogs residing within dog-owning households. ^ Multivariate linear regression was used to examine potential associations between knowledge, attitudes, and practices (KAPs) and variables of interest. Forty-six percent of households knew that rabies was spread through the bite of an infected dog. The mean knowledge score was 8.36 (SD: ± 3.4; range: 1–24). We found that having known someone with rabies was significantly associated with an almost one point increase in the knowledge score (β = 0.88; p = 0.02). The mean attitudes score was 5.65 (SD: ± 0.63; range: 2–6), and the mean practices score was 7.07 (SD: ± 1.7; range: 2–9). Both the attitudes score and the practices score were positively and significantly associated with only the knowledge score and no other covariates. ^ Multivariate logistic regression was used to examine associations between dog vaccination coverage and variables of interest. Approximately 71% of owned dogs in Bohol were reported as vaccinated at some time during their lives. We found that a dog's age was significantly associated with vaccination, and the odds of vaccination increased in a linear fashion with age. We also found that dogs had approximately twice the odds of being vaccinated if they were confined both day and night to the household premises or if the owner was employed; however, these results were only marginally significant (p = 0.07) in the multivariate model. ^ Finally, a systematic review was conducted on canine rabies vaccination and dog population demographics in the developing world. We found few studies on this topic, especially in countries where the burden of rabies is greatest. Overall, dog ownership is high. Dogs are quite young and do not live very long due to disease and accidents. The biggest deterrent to vaccination is the rapid dog population turnover. ^ It is our hope that this work will be used to improve dog rabies vaccination programs around the world and save lives, both human and canine.^