Simulating the Mechanics of Protein-Induced DNA Looping and Protein-Constrained DNA Buckling.

The bending and twisting mechanics of DNA are known to play a crucial role in many biological processes, yet fundamental details, even in relatively simple systems, remain unclear. The overall objective of this dissertation is to advance this knowledge in the context of two important systems including 1) the role of protein flexibility in an elementary gene regulatory protein that loops DNA, and 2) the structure and biological function(s) of DNA buckling during packaging and ejection in bacterial viruses. To address this objective, we contribute new modeling techniques by extending an elastic rod model for DNA. For protein-induced DNA looping, we use the model to reinterpret two seminal studies on Lac repressor looped DNA including the stability and topology of loops. For viral packaging, we contribute a model for mechanical contact between DNA and a cavity of arbitrary geometry, and also the first multi-scale model for DNA under extreme compression, to examine a buckled DNA toroid recently discovered in a protein cavity below the bacteriophage phi29 viral capsid. This example further motivates an extensive study of the mechanics of constrained DNA buckles inside the portal cavities of a family of viruses. The theoretical results for looped DNA successfully predict experimental observations and reveal that an extended protein conformation was active, yet overlooked, in classic experiments by the Muller-Hill laboratory. A detailed analysis of phi29 reveals that a DNA toroid can form under biologically-relevant force levels supplied by a packing motor. Computed DNA density maps compare favorably with the experimental data. Upon simulating the dynamic ejection of the toroid from the cavity, we reveal that large reaction forces/torques develop at the portal that could be used to signal genome release. Using Greenhill's equation, we show that DNA buckling is also feasible in a number of other bacteriophages including T7 and P22 that contain large portal cavities. Simulating DNA buckles in these cavities shows that large reaction forces develop on the portal walls that could signal a motor to terminate packing. Despite differences in size and shape, the cavities possess the same energy density.PHDMechanical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/99845/1/adhirsh_1.pd

Ecto 5′-Nucleotidase and Nonspecific Alkaline Phosphatase: TWO AMP-HYDROLYZING ECTOENZYMES WITH DISTINCT ROLES IN HUMAN AIRWAYS

In human airways, extracellular adenosine regulates epithelial functions supporting mucociliary clearance, an important airway defense mechanism against bacterial infection. Thus, defining the mechanisms of adenosine generation is critical for elucidating the role of this nucleoside in airway homeostasis. In this study, we identified the source of adenosine on the mucosal surface of human airway epithelia. Polarized primary cultures of human nasal or bronchial epithelial cells were assayed for transepithelial transport, cytosolic and cell surface adenosine production. Ussing chamber experiments indicated that serosal 1 microM [(3)H]adenosine was not transported to the mucosal compartment. Messenger RNA for the cytosolic AMP-specific 5'-nucleotidase (CN-I) was not detected in human bronchial epithelial cells, suggesting that mucosal adenosine did not originate from intracellular pools. In contrast, extracellular 0.1 mm ATP was rapidly dephosphorylated into adenosine on the mucosal epithelial surface. We identified two ectonucleotidases that mediated the conversion of AMP to adenosine: ecto 5'-nucleotidase (ecto 5'-NT, CD73) and alkaline phosphatase (AP). Both mucosal and serosal epithelial surfaces displayed ecto 5'-NT activity (K(m) = 14 microM, V(max) = 0.5 nmol x min(-1) x cm(-2)), whereas AP activity was restricted to the mucosal surface (K(m,)(high) = 36 microM, V(max) = 1.2 nmol x min(-1) x cm(-2); K(m,)(low) = 717 microM, V(max) = 2.8 nmol x min(-1) x cm(-2)). In bronchial cultures and tissues, ecto 5'-NT accounted for >80% of total activity toward 0.01 mm AMP, compared with <15% for 5 mm AMP. The proximal airway AP isoform was identified as nonspecific AP (NS AP) by levamisole sensitivity and mRNA expression. The two ectoenzymes presented opposite airway distributions, ecto 5'-NT and NS AP mRNA dominating in higher and lower airways, respectively. Collectively, these experiments support a major role for extracellular nucleotide catalysis and for ecto 5'-NT and NS AP in the regulation of adenosine concentrations on airway surfaces

DETC2008-49886 PIEZOELECTRIC T-BEAM MICROACTUATORS

ABSTRACT This paper introduces a novel T-beam actuator fabricate

Implementing Diversity Training Targeting Faculty Microaggressions and Inclusion: Practical Insights and Initial Findings

Despite the importance of faculty diversity training for advancing an inclusive society, little research examines whether participation improves inclusion perceptions and belongingness. Integrating training and diversity education literature concepts, this study examines the effectiveness of training targeting microaggressions in six STEM (Science, Technology, Engineering, Mathematics) oriented departments at a research-intensive university. Reactions data collected at the end of face-to-face training suggested that participation generally increased inclusion understanding. Self-assessments on inclusion concepts collected from 45% of participants before and three weeks after training suggest participation increases perceptions of the importance of inclusion, microaggression allyship awareness, inclusive behaviors, and organizational identification. Compared to white men, women and minorities reported a greater increase in satisfaction with their department affiliation. While self-assessment results are exploratory and have limitations, analysis suggests that diversity training may enhance knowledge of microaggressions, allyship, inclusive behaviors, and belongingness perceptions. We provide insights for evaluating and implementing diversity training interventions

Regulation of the Epithelial Sodium Channel by Serine Proteases in Human Airways

The epithelial sodium channel (ENaC) constitutes the rate-limiting step for sodium absorption across airway epithelia, which in turn regulates airway surface liquid (ASL) volume and the efficiency of mucociliary clearance. This role in ASL volume regulation suggests that ENaC activity is influenced by local factors rather than systemic signals indicative of total body volume homeostasis. Based on reports that ENaC may be regulated by extracellular serine protease activity in Xenopus and mouse renal epithelia, we sought to identify proteases that serve similar functions in human airway epithelia. Homology screening of a human airway epithelial cDNA library identified two trypsin-like serine proteases (prostasin and TMPRSS2) that, as revealed by in situ hybridization, are expressed in airway epithelia. Functional studies in the Xenopus oocyte expression system demonstrated that prostasin increased ENaC currents 60--80%, whereas TMPRSS2 markedly decreased ENaC currents and protein levels. Studies of primary nasal epithelial cultures in Ussing chambers revealed that inhibition of endogenous serine protease activity with aprotinin markedly decreased ENaC-mediated currents and sensitized the epithelia to subsequent channel activation by exogenous trypsin. These data, therefore, suggest that protease-mediated regulation of sodium absorption is a function of human airway epithelia, and prostasin is a likely candidate for this activity

Airway drug pharmacokinetics via analysis of exhaled breath condensate

Although the airway surface is the anatomic target for many lung disease therapies, measuring drug concentrations and activities on these surfaces poses considerable challenges. We tested whether mass spectrometric analysis of exhaled breath condensate (EBC) could be utilized to non-invasively measure airway drug pharmacokinetics and predicted pharmacological activities

Identifying Barriers to Statin Therapy in Diabetic Patients in a Large Ambulatory Practice

Aim 1: To understand the root cause(s) for gaps in statin therapy within a large ambulatory care practice. Aim 2: To improve provider awareness of indications for statin therapy, as recommended by the ACC/AHA guidelines. Aim 3: To increase the proportion of patients with diabetes at our practice that are on statin therapy

Benefits and Effects of Interactive Gaming Exercise as Compared to Traditional Exercise Techniques: An Investigation of the Relationship Between Physical Activity Levels and Mode of Activity in College Students

Gemstone Team F.I.T.N.E.S.S. (Fun Interactive Techniques for New Exercise and Sport Styles)The objective of this research project was to determine the relationship between mode of activity and motivation to exercise. Specifically, the research aimed to discover if interactive video games are effective in motivating sedentary college students to exercise. The introduction discusses the problems of physical inactivity and obesity, the increased use of video games, and a proposed solution using interactive technology. For a ten-week period, sedentary University of Maryland – College Park students joined either the traditional exercise or interactive gaming group, with heart rates and activity levels documented throughout. Together, quantitative heart rate and activity data and qualitative participant feedback, illustrated that it was too difficult to collect significant data supporting a conclusion that one activity was a significantly better means to raise heart rates and motivate students to exercise. However, the data did support the importance of continuing research on potential benefits of interactive gaming for a sedentary population