Thesis (M.S.P.H.)--University of Michigan, 1941.

Thesis (Ph. D.)--University of Wisconsin, 1954. / Typescript (carbon copy). eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 78-85).

Thesis (Ph. D.)--Illinois State University, 1990. / Title from title page screen, viewed November 29, 2005. Dissertation Committee: Arlan Richardson (chair), Marjorie A. Jones, Lynne A. Lucher, Anthony J. Otsuka, Brian J. Wilkinson. Includes bibliographical references (leaves 168-187) and abstract. Also available in print.

Heat shock response (HSR) is an ancient and highly conserved signaling pathway in cells that regulates the expression of heat shock proteins (HSPs) in the presence of thermal and other environmental stresses. HSPs function to prevent the formation of non-specific protein aggregates and to assist proteins in acquiring their native structures. Although HSR has been extensively studied, key aspects of this pathway remain a mystery. In particular, how HSR is activated and regulated by the master transcription factor HSF1 is not well understood. The broad goal of this thesis is to develop a quantitative framework aimed at elucidating the HSF1-mediated activation of HSR in yeast cells. Understanding this process has important implications for development, physiology and disease. Indeed, HSF1 is conserved from yeast to human, has been shown to play an important role in stress resistance, health and disease, and is a therapeutic target for neurodegenerative diseases. Broadly, there are two putative (not mutually exclusive) models for activation in response to heat shock: (1) HSF1 dissociation from chaperone proteins and (2) hyper-phosphorylation and the subsequent activation of HSF1. However the relative contribution of each of these events in the activation process is not characterized. Thus far, there is no direct evidence linking either of these two events to activation, and the relative contribution of each mechanism to the activation process has not been quantitatively characterized. To address these issues, we develop a quantitative model of HSR in yeast cells. We use the model to make a series of quantitative predictions and, in a collaborative effort, experimentally test these predictions in a yeast model of HSR. Critically, we provide the first direct evidence for chaperone dissociation of HSF1 in response to heat shock. Moreover, we find that HSF1 phosphorylation is dispensable for activation of HSR, but is able to modulate its activity. Taken together, our work leads to a model for two “orthogonal” mechanisms regulating HSR in yeast, in which chaperone dissociation acts as an ON/OFF switch, whereas phosphorylation functions to tune the gain of the response. Finally, to complement and further test this quantitative model, we develop a novel microfluidic system to explore in more depth the behavior of individual cells in the presence of heat shock inputs. This includes (1) a microfluidic device with microscale on-chip heaters enabling programmable thermal perturbations and (2) a custom image analysis platform to follow single cells through heat shock time courses. In preliminary single-cell studies, we find a relationship between HSF1 phosphorylation state and cell-to-cell variability in HSR activation level (as measured by a transcriptional reporter). These preliminary results suggest that HSF1 phosphorylation may be generating and tuning noise in the HSR in order to promote phenotypic plasticity and increased survivability of a cell population in the face of stress.

The molecular chaperone process of assisted protein folding, characteristic of members of the Heat Shock Protein 70 kDa (Hsp70) and Heat Shock Protein 40kDa (Hsp40) families, is essential for cytoprotection in stressful cellular conditions. Examples of such conditions are heat shock or invasion by pathogens. The Hsp70/Hsp40 process of assisted protein folding is dependent on ATP (governed by the intrinsic ATPase activity of Hsp70) and the ability of molecular chaperones to recognise and bind non-native protein conformations. Here, we analyse and attempt to characterise the molecular chaperone activity of an inducible, cytoplasmic Hsp70 (TcHsp70) from Trypanosoma cruzi and its interactions with its potential partner Hsp40s, Tcj 1, Tcj2, Tcj3 and Tcj4. A bioinformatic analyses of the primary sequences of the trypanosomal proteins revealed that they all contained the canonical domains that define other members of the Hsp70 and Hsp40 family. Tcj2 and Tcj4 showed deviations from the consensus sequence in their substrate binding regions, which may have implications for their substrate binding specificities. TcHsp70, Tcj 1, Tcj2, Tcj3 and Tcj4 were over-expressed recombinantly as 6xHis-tag fusion proteins in Escherichia coli. His-TcHsp70, Tcjl-His and His-Tcj2 were successfully purified by Nickel-affinity chromatography for functional analyses to assess the molecular chaperone activity of His-TcHsp70 in terms of its ATPase activity and substrate binding ability. The basal ATPase activity of His-TcHsp70 was determined as 40 nmol Pi/min/mg, significantly higher than that reported for other Hsp70s. This basal ATPase activity was stimulated to a maximal level of 60 nmol Pi/min/mg in the presence of His-Tcj2 and a model non-native substrate, reduced carboxymethylated αx-lactalbumin (RCMLA). Using native polyacrylamide gel electrophoresis and Western analysis, His-TcHsp70 was shown to form discrete complexes when in the presence of Tcj 1- His, His-Tcj2 and/or RCMLA. These complexes potentially represent His-TcHsp70 - RCMLA or His-TcHsp70 - Tcj interactions, that may be indicative of chaperone activity. In vivo complementation assays showed that Tcj2, but not Tcj3, was able to overcome the temperature sensitivity of the ydjJ mutant Saccharomyces cerevisiae strain JJ160, suggesting that Tcj2 may be functionally equivalent to the yeast Hsp40 Ydj1.

The response of porcine adipose and skeletal muscle tissues to shock compression has been investigated using the plate-impact technique in conjunction with manganin foil pressure gauge diagnostics. This approach has allowed for measurement of the levels of uniaxial stress imparted to both skeletal muscle and rendered adipose tissue by the shock. In addition, the lateral stress component generated within adipose tissue during shock loading has also been investigated. The techniques employed in this study have allowed for equation-of-state relationships to be established for the investigated materials, highlighting non-hydrodynamic behaviour in each type of tissue over the range of investigated impact conditions. While the adipose tissue selected in this work has been shown to strengthen with impact stress in a manner similar to that seen to occur in polymeric materials, the skeletal muscle tissues exhibited a ow strength, or resistance to compression, that was independent of impact stress. Both the response of the adipose material and tested skeletal muscle tissues lie in contrast with the shock response of ballistic gelatin, which has previously been shown to exhibit hydrodynamic behaviour under equivalent loading conditions. Plate-impact experiments have also been used to investigate the shock response of a homogenized variant of one of the investigated muscle tissues. In the homogenized samples, the natural structure of skeletal muscle tissue, i.e. a fibrous and anisotropic composite, was heavily disrupted and the resulting material was milled into a fine paste. Rather than matching the response of the unaltered tissues, the datapoints generated from this type of experiment were seen to collapse back on to the hydrodynamic response predicted for skeletal muscle by its linear equation-of-state (Us = 1.72 + 1.88up). This suggests that the resistance to compression apparent in the data obtained for the virgin tissues was a direct result of the interaction of the shock with the quasi-organized structure of skeletal muscle. A soft-capture system has been developed in order to facilitate post-shock analysis of skeletal muscle tissue and to ascertain the effects of shock loading upon the structure of the material. The system was designed to deliver a one-dimensional, at-topped shock pulse to the sample prior to release. The overall design of the system was aided by use of the non-linear and explicit hydrocode ANSYSR AUTODYN. Following shock compression, sections of tissue were imaged using a transmission electron microscope (TEM). Both an auxetic-like response and large-scale disruption to the I-band/Z-disk regions within the tissue's structure were observed. Notably, these mechanisms have been noted to occur as a result of hydrostatic compression of skeletal muscle within the literature.

No description available.

A 30.5 cm diameter, detonation driven shock tube facility has been designed, constructed and tested. The design goals of the shock tube were to reproduce free field blast wave profiles on a laboratory scale using atmospheric gaseous detonation as the energy source. Numerical simulations were utilized to explore the gas dynamic evolution inside detonation driven shock tubes and to select the optimal design parameters for the shock tube.The Friedlander profile was used to evaluate the generated pressure profiles as an approximation of free field blast waves. It has been found that the detonation driver length should be kept below 20% of the total length of the tube in order to produce Friedlander waves. Additionally, it has been found that an annular vent can be added to the shock tube to enhance the negative phase of the blast profile, more accurately reproducing real free field blast waves. The shock tube has been constructed in a modular fashion from 2.54 cm thick steel tubing. An adjustable bag type diaphragm has been employed to allow for a variable driver size and a high voltage ignition system is used to initiate detonation in the driver section. Due to the available location for the shock tube, tests using the vented configuration could not be accomplished for safety reasons. Conducted experiments produced results that agree well with corresponding numerical simulations. Overall, the shock tube design was successful in creating Friedlander blast waves. At the time of writing, a manufacturer error in correctly reporting the specifications of the clamps used on the shock tube resulted in a lower maximum pressure of operation.

The present work investigates the dynamics and stability of shock waves in granular gases. The problem was modelled for a piston propagating into a system of disks that can undergo inelastic collisions if an impact threshold is exceeded. The model was addressed numerically at the microscopic and macroscopic levels. The molecular dynamics methodology employed the Event-Driven Molecular Dynamics method, and the continuum model was formulated using the Navier-Stokes equations for granular gases with the transport terms of Jenkins and Richman and a modified cooling rate term. The inviscid steady state shock structure was derived and analyzed. The results indicated that a relaxing shock structure is expected for sufficiently strong shock waves. Beyond this limit the structure was shown to be independent of the initial energy, a finding similar to the strong shock approximation in molecular gases. One-dimensional simulations demonstrated that the molecular dynamics and continuum models yield similar evolutions and structures of the shock wave, validating the continuum description of this study. Two-dimensional results showed that sufficiently strong shock waves can exhibit multi-dimensional instability with high density non-uniformities and convective rolls within the structure, with the size of instabilities shown to scale with the relaxation length of the shock structure. Instabilities were observed with the continuum description only with the inclusion of statistical fluctuations to density mimicking the molecular model. The cases that were unstable were shown to be in a regime whereby statistical fluctuations can become important, following the description for this regime by Bird. Based on these findings, it is proposed that unstable shock behaviour can be observed for highly dissipative shock waves that yield short relaxation length scales, where fluctuations become important. The current work may shed light on unstable shock behaviour observed in dissipative gases, having implications for both granular media and molecular gases.

The effects of fin-leading-edge radius and sweep angle on peak heating rates due to shock-shock interactions were investigated in the NASA Langley Research Center 20-inch Mach 6 Air Tunnel.  The fin model leading edges, which represent cylindrical leading edges or struts on hypersonic vehicles, were varied from 0.25 inches to 0.75 inches in radius.  A 9° wedge generated a planar oblique shock at 16.7° to the flow that intersected the fin bow shock, producing a shock-shock interaction that impinged on the fin leading edge.  The fin angle of attack was varied from 0° (normal to the free-stream) to 15° and 25° swept forward.  Global temperature data was obtained from the surface of the fused silica fins through phosphor thermography.  Metal oil flow models with the same geometries as the fused silica models were used to visualize the streamline patterns for each angle of attack.  High-speed zoom-schlieren videos were recorded to show the features and temporal unsteadiness of the shock-shock interactions.  The temperature data were analyzed using one-dimensional semi-infinite as well as one- and two-dimensional finite-volume methods to determine the proper heat transfer analysis approach to minimize errors from lateral heat conduction due to the presence of strong surface temperature gradients induced by the shock interactions.  The general trends in the leading-edge heat transfer behavior were similar for the three shock-shock interactions, respectively, between the test articles with varying leading-edge radius.  The dimensional peak heat transfer coefficient augmentation increased with decreasing leading-edge radius.  The dimensional peak heat transfer output from the two-dimensional code was about 20% higher than the value from a standard, semi-infinite one-dimensional method. / Master of Science