Reactions of dioxygen and nitric oxide with iron(II) compounds : models for chemistry occuring in the active sites of non-heme iron enzymes

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 1995.Vita.Includes bibliographical references.by Andrew L. Feig.Ph.D

An Upstream Hfq Binding Site in the fhlA mRNA Leader Region Facilitates the OxyS-fhlA Interaction

To survive, bacteria must be able to adapt to environmental stresses. Small regulatory RNAs have been implicated as intermediates in a variety of stress-response pathways allowing dynamic gene regulation. The RNA binding protein Hfq facilitates this process in many cases, helping sRNAs base pair with their target mRNAs and initiate gene regulation. Although Hfq has been identified as a critical component in many RNPs, the manner by which Hfq controls these interactions is not known.To test the requirement of Hfq in these mRNA-sRNA complexes, the OxyS-fhlA system was used as a model. OxyS is induced in response to oxidative stress and down regulates the translation of fhlA, a gene encoding a transcriptional activator for formate metabolism. Biophysical characterization of this system previously used a minimal construct of the fhlA mRNA which inadvertently removed a critical element within the leader sequence of this mRNA that effected thermodynamics and kinetics for the interaction with Hfq.Herein, we report thermodynamic, kinetic and structural mapping studies during binary and ternary complex formation between Hfq, OxyS and fhlA mRNA. Hfq binds fhlA mRNA using both the proximal and distal surfaces and stimulates association kinetics between the sRNA and mRNA but remains bound to fhlA forming a ternary complex. The upstream Hfq binding element within fhlA is similar to (ARN)(x) elements recently identified in other mRNAs regulated by Hfq. This work leads to a kinetic model for the dynamics of these complexes and the regulation of gene expression by bacterial sRNAs

Disruption of Intrinsic Motions as a Mechanism for Enzyme Inhibition

AbstractClostridium difficile (C. diff) is one of the most common and most severe hospital-acquired infections; its consequences range from lengthened hospital stay to outright lethality. C. diff causes cellular damage through the action of two large toxins TcdA and TcdB. Recently, there has been increased effort toward developing antitoxin therapies, rather than antibacterial treatments, in hopes of mitigating the acquisition of drug resistance. To date, no analysis of the recognition mechanism of TcdA or TcdB has been attempted. Here, we use small molecule flexible docking followed by unbiased molecular dynamics to obtain a more detailed perspective on how inhibitory peptides, exemplified by two species HQSPWHH and EGWHAHT function. Using principal component analysis and generalized masked Delaunay analysis, an examination of the conformational space of TcdB in its apo form as well as forms bound to the peptides and UDP-Glucose was performed. Although both species inhibit by binding in the active site, they do so in two very different ways. The simulations show that the conformational space occupied by TcdB bound to the two peptides are quite different and provide valuable insight for the future design of toxin inhibitors and other enzymes that interact with their substrates through conformational capture mechanisms and thus work by the disruption of the protein’s intrinsic motions

Adaptation of Clostridium difficile toxin A for use as a protein translocation system

a b s t r a c t A cellular delivery system is a useful biotechnology tool, with many possible applications. Two derivatives of Clostridium difficile toxin A (TcdA) have been constructed (GFP-TcdA and Luc-TcdA), by fusing reporter genes to functional domains of TcdA, and evaluated for their ability to translocate their cargo into mammalian cells. The cysteine protease and receptor binding domains of TcdA have been examined and found to be functional when expressed in the chimeric construct. Whereas GFP failed to internalize in the context of the TcdA fusion, significant cellular luciferase activity was detected in vero cell lysates after treatment with Luc-TcdA. Treatment with bafilomycin A1, which inhibits endosomal acidification, traps the luciferase activity within endosomes. To further understand these results, clarified lysates were subjected to molecular weight sieving, demonstrating that active luciferase was released from Luc-TcdA after translocation and internal processing

Catalyzing collaborations: Prescribed interactions at conferences determine team formation

Collaboration plays a key role in knowledge production. Here, we show that patterns of interaction during conferences can be used to predict who will subsequently form a new collaboration, even when interaction is prescribed rather than freely chosen. We introduce a novel longitudinal dataset tracking patterns of interaction among hundreds of scientists during multi-day conferences encompassing different scientific fields over the span of 5 years. We find that participants who formed new collaborations interacted 63% more on average than those who chose not to form new teams, and that those assigned to a higher interaction scenario had more than an eightfold increase in their odds of collaborating. We propose a simple mathematical framework for the process of team formation that incorporates this observation as well as the effect of memory beyond interaction time. The model accurately reproduces the collaborations formed across all conferences and outperforms seven other candidate models. This work not only suggests that encounters between individuals at conferences play an important role in shaping the future of science, but that these encounters can be designed to better catalyze collaborations.Comment: 8 pages and 4 figures, main text; 8 pages and 3 figures supplementary informatio

Enhancing Grant-Writing Expertise in BUILD Institutions: Building Infrastructure Leading to Diversity

Background The lack of race/ethnic and gender diversity in grants funded by the National Institutes of Health (NIH) is a persistent challenge related to career advancement and the quality and relevance of health research. We describe pilot programs at nine institutions supported by the NIH-sponsored Building Infrastructure Leading to Diversity (BUILD) program aimed at increasing diversity in biomedical research. Methods We collected data from the 2016–2017 Higher Education Research Institute survey of faculty and NIH progress reports for the first four years of the program (2015–2018). We then conducted descriptive analyses of data from the nine BUILD institutions that had collected data and evaluated which activities were associated with research productivity. We used Poisson regression and rate ratios of the numbers of BUILD pilots funded, students included, abstracts, presentations, publications, and submitted and funded grant proposals. Results Teaching workshops were associated with more abstracts (RR 4.04, 95% CI 2.21–8.09). Workshops on grant writing were associated with more publications (RR 2.64, 95% CI 1.64–4.34) and marginally with marginally more presentations. Incentives to develop courses were associated with more abstracts published (RR 4.33, 95% CI 2.56–7.75). Workshops on research skills and other incentives were not associated with any positive effects. Conclusions Pilot interventions show promise in supporting diversity in NIH-level research. Longitudinal modeling that considers time lags in career development in moving from project development to grants submissions can provide more direction for future diversity pilot interventions

Aligning Practice to Policies: Changing the Culture to Recognize and Reward Teaching at Research Universities

Recent calls for improvement in undergraduate education within STEM (science, technology, engineering, and mathematics) disciplines are hampered by the methods used to evaluate teaching effectiveness. Faculty members at research universities are commonly assessed and promoted mainly on the basis of research success. To improve the quality of undergraduate teaching across all disciplines, not only STEM fields, requires creating an environment wherein continuous improvement of teaching is valued, assessed, and rewarded at various stages of a faculty member’s career. This requires consistent application of policies that reflect well-established best practices for evaluating teaching at the department, college, and university levels. Evidence shows most teaching evaluation practices do not reflect stated policies, even when the policies specifically espouse teaching as a value. Thus, alignment of practice to policy is a major barrier to establishing a culture in which teaching is valued. Situated in the context of current national efforts to improve undergraduate STEM education, including the Association of American Universities Undergraduate STEM Education Initiative, this essay discusses four guiding principles for aligning practice with stated priorities in formal policies: 1) enhancing the role of deans and chairs; 2) effectively using the hiring process; 3) improving communication; and 4) improving the understanding of teaching as a scholarly activity. In addition, three specific examples of efforts to improve the practice of evaluating teaching are presented as examples: 1) Three Bucket Model of merit review at the University of California, Irvine; (2) Evaluation of Teaching Rubric, University of Kansas; and (3) Teaching Quality Framework, University of Colorado, Boulder. These examples provide flexible criteria to holistically evaluate and improve the quality of teaching across the diverse institutions comprising modern higher education

Quantum Computing and Quantum Simulation with Group-II Atoms

Recent experimental progress in controlling neutral group-II atoms for optical clocks, and in the production of degenerate gases with group-II atoms has given rise to novel opportunities to address challenges in quantum computing and quantum simulation. In these systems, it is possible to encode qubits in nuclear spin states, which are decoupled from the electronic state in the $^1$S$_0$ ground state and the long-lived $^3$P$_0$ metastable state on the clock transition. This leads to quantum computing scenarios where qubits are stored in long lived nuclear spin states, while electronic states can be accessed independently, for cooling of the atoms, as well as manipulation and readout of the qubits. The high nuclear spin in some fermionic isotopes also offers opportunities for the encoding of multiple qubits on a single atom, as well as providing an opportunity for studying many-body physics in systems with a high spin symmetry. Here we review recent experimental and theoretical progress in these areas, and summarise the advantages and challenges for quantum computing and quantum simulation with group-II atoms.Comment: 11 pages, 7 figures, review for special issue of "Quantum Information Processing" on "Quantum Information with Neutral Particles

Short-term pacing in the mouse alters cardiac expression of connexin43

Background: Cardiac insults such as ischemia, infarction, hypertrophy and dilatation are often accompanied by altered abundance and/or localization of the connexin43 gap junction protein, which may predispose towards arrhythmic complications. Models of chronic dyssynchronous cardiac activation have also been shown to result in redistribution of connexin43 in cardiomyocytes. We hypothesized that alterations in connexin43 expression and localization in the mouse heart might be induced by ventricular pacing over a short period of time. Results: The subdiaphragmatic approach was used to pace a series of wild type mice for six hours before the hearts were removed for analysis. Mice were paced at 10–15% above their average anesthetized sinus rate and monitored to ensure 1:1 capture. Short-term pacing resulted in a significant reduction in connexin43 mRNA abundance, a partial redistribution of connexin43 from the sarcolemma to a non-sarcolemmal fraction, and accumulation of ubiquitinated connexin43 without a significant change in overall connexin43 protein levels. These early pacing-induced changes in connexin43 expression were not accompanied by decreased cardiac function, prolonged refractoriness or increased inducibility into sustained arrhythmias. Conclusion: Our data suggest that short-term pacing is associated with incipient changes in the expression of the connexin43 gap junction, possibly including decreased production and a slowed rate of degradation. This murine model may facilitate the study of early molecular changes induced by pacing and may ultimately assist in the development of strategies to prevent gap junction remodeling and the associated arrhythmic complications of cardiac disease

Biophysical and electrochemical studies of protein-nucleic acid interactions

This review is devoted to biophysical and electrochemical methods used for studying protein-nucleic acid (NA) interactions. The importance of NA structure and protein-NA recognition for essential cellular processes, such as replication or transcription, is discussed to provide background for description of a range of biophysical chemistry methods that are applied to study a wide scope of protein-DNA and protein-RNA complexes. These techniques employ different detection principles with specific advantages and limitations and are often combined as mutually complementary approaches to provide a complete description of the interactions. Electrochemical methods have proven to be of great utility in such studies because they provide sensitive measurements and can be combined with other approaches that facilitate the protein-NA interactions. Recent applications of electrochemical methods in studies of protein-NA interactions are discussed in detail