Regulation of the heat shock response by thiol-reactive compounds in the yeast Saccharomyces cerevisiae

Cells govern their activities and modulate their interactions with the environment to achieve homeostasis. The heat shock response (HSR) is one of the most well studied fundamental cellular responses to environmental and physiological challenges, resulting in rapid synthesis of heat shock proteins (HSPs), which serve to protect cellular constituents from the deleterious effects of stress. In addition to its role in cytoprotection, the HSR also influences lifespan and is associated with a variety of human diseases including cancer, aging and neurodegenerative disorders. In most eukaryotes, the HSR is primarily mediated by the highly conserved transcription factor HSF1, which recognizes target hsp genes by binding to heat shock elements (HSEs) in their promoters. In recent years, significant efforts have been made to identify small molecules as potential pharmacological activators of HSF1 that could be used for therapeutic benefit in the treatment of human diseases relevant to protein conformation. However, the detailed mechanisms through which these molecules drive HSR activation remain unclear. In this work, I utilized the baker's yeast Saccharomyces cerevisiae as a model system to identify a group of thiol-reactive molecules including oxidants, transition metals and metalloids, and electrophiles, as potent activators of yeast Hsf1. Using an artificial HSE-lacZ reporter and the glucocorticoid receptor system (GR), these diverse thiol-reactive compounds are shown to activate Hsf1 and inhibit Hsp90 chaperone complex activity in a reciprocal, dose-dependent manner. To further understand whether cells sense these reactive compounds through accumulation of unfolded proteins, the proline analog azetidine-2-carboxylic acid (AZC) and protein cross-linker dithiobis(succinimidyl propionate) (DSP) were used to force misfolding of nascent polypeptides and existing cytosolic proteins, respectively. Both unfolding reagents display kinetic HSP induction profiles dissimilar to those generated by thiol-reactive compounds. Moreover, AZC treatment leads to significant cytotoxicity, which is not observed in the presence of the thiol-reactive compounds at the concentrations sufficient to induce Hsf1. Additionally, DSP treatment has little to no effect on Hsp90 functions. Together with the ultracentrifugation analysis of cell lysates that detected no insoluble protein aggregates, my data suggest that at concentrations sufficient to induce Hsf1, thiol-reactive compounds do not induce the HSR via a mechanism based on accumulation of unfolded cytosolic proteins. Another possibility is that thiol-reactive compounds may influence aspects of the protein quality control system such as the ubiquitin-proteasome system (UPS). To address this hypothesis, β-galactosidase reporter fusions were used as model substrates to demonstrate that thiol-reactive compounds do not inhibit ubiquitin activating enzymes (E1) or proteasome activity. Therefore, thiol-reactive compounds do not activate the HSR by inhibiting UPS-dependent protein degradation. I therefore hypothesized that these molecules may directly inactivate protein chaperones, known as repressors of Hsf1. To address this possibility, a thiol-reactive biotin probe was used to demonstrate in vitro that the yeast cytosolic Hsp70 Ssa1, which partners with Hsp90 to repress Hsf1, is specifically modified. Strikingly, mutation of conserved cysteine residues in Ssa1 renders cells insensitive to Hsf1 activation by cadmium and celastrol but not by heat shock. Conversely, substitution with the sulfinic acid and steric bulk mimic aspartic acid led to constitutive activation of Hsf1. Cysteine 303, located in the nucleotide-binding/ATPase domain of Ssa1, was shown to be modified in vivo by a model organic electrophile using Click chemistry technology, verifying that Ssa1 is a direct target for thiol-reactive compounds through adduct formation. Consistently, cadmium pretreatment promoted cells thermotolerance, which is abolished in cells carrying SSA1 cysteine mutant alleles. Taken together, these findings demonstrate that Hsp70 acts as a sensor to induce the cytoprotective heat shock response in response to environmental or endogenously produced thiol-reactive molecules and can discriminate between two distinct environmental stressors.

Quality in healthcare organizations: Its meaning and measurement

This dissertation explores two important aspects of quality in healthcare: its meaning and its measurement. For a better understanding of what quality means, the history of quality in the manufacturing and service industries is reviewed. Concepts that are similar are pointed out as are concepts that are different. The definition introduced by the Institute of Medicine (IOM) for quality in healthcare and the six IOM aims of safety, timeliness, patient-centeredness, effectiveness, efficiency, and equitableness for a high quality healthcare system are adopted. The current activities by various organizations that proclaim improvement in quality or measurement of quality as their goal are reviewed. This is followed by examining what is offered by these organizations in terms of how many of IOM aims they address.^ This dissertation ends by offering a quality measurement framework that satisfies all IOM aims. Operational aspects of the measurement framework are discussed. Future areas of research are also discussed.^

A comparative analysis of hospital -based physician monitoring systems

The purpose of the multiple case-study was to determine how hospital subsystems (such as physician monitoring and credentialing; quality assurance; risk management; and peer review) were supporting the monitoring of physicians? Three large metropolitan hospitals in Texas were studied and designated as hospitals #1, #2, and #3. Realizing that hospital subsystems are a unique entity and part of a larger system, conclusions were made on the premises of a quality control system, in relation to the tools of government (particularly the Health Care Quality Improvement Act (HCQIA)), and in relation to itself as a tool of a hospital.^ Three major analytical assessments were performed. First, the subsystems were analyzed as to their "completeness"; secondly, the subsystems were analyzed for "performance"; and thirdly, the subsystems were analyzed in reference to the interaction of completeness and performance.^ The physician credentialing and monitoring and the peer review subsystems as quality control systems were most complete, efficient, and effective in hospitals #1 and #3. The HCQIA did not seem to be an influencing factor in the completeness of the subsystem in hospital #1. The quality assurance and risk management subsystem in hospital #2 was not representative of completeness and performance and the HCQIA was not an influencing factor in the completeness of the Q.A. or R.M. systems in any hospital. The efficiency (computerization) of the physician credentialing, quality assurance and peer review subsystems in hospitals #1 and #3 seemed to contribute to their effectiveness (system-wide effect).^ The results indicated that the more complete, effective, and efficient subsystems were characterized by (1) all defined activities being met, (2) the HCQIA being an influencing factor, (3) a decentralized administrative structure, (4) computerization an important element, and (5) staff was sophisticated in subsystem operations. However, other variables were identified which deserve further research as to their effect on completeness and performance of subsystems. They include (1) medical staff affiliations, (2) system funding levels, (3) the system's administrative structure, and (4) the physician staff "cultural" characteristics. Perhaps by understanding other influencing factors, health care administrators may plan subsystems that will be compatible with legislative requirements and administrative objectives. ^