Thesis (M.S. in Naval Architecture and Marine Engineering and M.S. in Civil and Environmental Engineering)--Massachusetts Institute of Technology, 2002. / Includes bibliographical references (leaves 64-65). Also available online.

The heat shock response (HSR) is the cell's molecular reaction to protein damaging stress and is critical in the management of denatured proteins. Activation of HSF1, the master transcriptional regulator of the HSR, results in the induction of molecular chaperones called heat shock proteins (HSPs). Transcription of hsp genes is promoted by the hyperphosphorylation of HSF1, while the attenuation of the HSR is regulated by a dual mechanism involving negative feedback inhibition from HSPs and acetylation at a critical lysine residue within the DNA binding domain of HSF1, which results in a loss of affinity for DNA. SIRT1 is a NAD+-dependent histone deacetylase that has been reported to deacetylate HSF1, thus promoting stress-induced HSF1 DNA binding ability and increasing HSP expression (Westerheide, Anckar et al. 2009). While an abundance of research is aimed to investigate SIRT1 substrate regulation, the mechanism in which SIRT1 itself is regulated is less understood (Haigis and Sinclair 2010). Positive and negative modulators of SIRT1 include AROS and DBC1, respectively, and have yet to be investigated in relation to SIRT1-dependent regulation of the HSR. In addition, metabolic stress such as caloric restriction has been shown to modulate SIRT1 activity in yeast (Rahat, Maoz et al. 2011), but the effect of caloric restriction on the HSR is unknown. Using cell-based assays, we have investigated how the HSR may be controlled by factors influencing SIRT1 activity. We found that heat shock results in an increase in the cellular NAD+/NADH ratio and an increase in recruitment of SIRT1 to the hsp70 promoter. Furthermore, we found that the SIRT1 modulators, AROS and DBC1, impact hsp70 transcription, HSF1 acetylation status, and HSF1 recruitment to the hsp70 promoter. The nematode Caenorhabditis elegans is a useful model organism for testing the relationship between the HSR and metabolism, as these animals can easily be calorically-restricted via bacterial limitation and possess the mammalian SIRT1 homolog, Sir2.1. Using C. elegans, we demonstrate that caloric restriction and heat shock have a synergistic effect on the HSR in a sir2.1-dependent manner. We show that caloric restriction increases the ability of heat shock to promote thermotolerance and fitness in wild-type animals and to preserve movement in a polyglutamine toxicity neurodegenerative disease model and that this effect is dependent on sir2.1. These studies provide insight into SIRT1-dependent regulation of the HSR and the impact of metabolism on this response. We highlight the SIRT1 modulators AROS and DBC1 as two new targets available for therapeutic regulation of the HSR and add caloric restriction as another HSR activator that can synergize with heat shock.

Apoptosis is a conserved program of cell death that promotes organism homeostasis in all stages of life. Two main pathways activate caspases, which are cysteinyl-aspartate proteases that execute apoptosis. The extrinsic pathway is initiated by cell surface death receptors, while the intrinsic pathway is initiated by intracellular signals that cause permeabilization of the outer mitochondrial membrane (MOMP). The Bcl-2 protein family regulates MOMP, which causes the release of several pro-apoptotic proteins (such as cytochrome c, Smac) into the cytosol. Bcl-2 proteins share homology in up to four "BH" domains and are subdivided into three subgroups. Pro-apoptotic Bax and Bak catalyze pore formation in the mitochondria, while anti-apoptotic members (Bcl-2, Mcl-1) inhibit MOMP. The third subgroup, termed BH3-only, promotes MOMP by either antagonizing Bcl-2 proteins or by directly activating Bax/Bak, and initiate apoptosis in response to various stressors, including heat shock (HS). Hyperthermia or acute HS reportedly induces apoptosis through caspase-2-mediated cleavage of BID, engaging the intrinsic pathway. However, additional evidence suggests that this pathway could represent an amplification loop. Thus we hypothesized that during HS, another BH3-only protein such as BIM, that does not require cleavage, could engage MOMP. Herein, we report that BIM mediates an alternative HS-induced apoptosis pathway. Cells lacking BIM are resistant to HS and exhibit better short and long-term survival than either Bid[superscript -/-] or Bax[superscript -/-]Bak[superscript -/-]. Moreover, caspase-2 induces apoptosis in Bim[superscript -/-] but not Bid[superscript -/-] cells, implying that caspase-2 kills exclusively through BID. Interestingly, Bim[superscript -/-] and Bax[superscript -/-]Bak[superscript -/-] cells are entirely resistant to MOMP, but the Bax[superscript -/-]Bak[superscript -/-] cells still undergo caspase-3 activation and remain partially sensitive to HS, indicating that BIM triggers caspase-3 activation upstream of mitochondria. Thus, BIM plays an important role in HS-induced apoptosis. Hyperthermia has clinical applications for the treatment of solid tumors. Unfortunately, a practical limitation is the development of thermotolerance, which confers resistance not only to subsequent HS but also to radiotherapy and chemotherapy. Therefore, a better understanding of the molecular mechanisms involved both in heat-induced apoptosis and thermotolerance could lead to new therapeutic interventions. Here we also show evidence for a putative role for the stress kinase JNK signaling pathway in the regulation of thermotolerance. / text

The goal of this PhD research project is to devise a robust interatomic potential for large scale molecular dynamics simulations of carbon materials under extreme conditions. This screened-environment dependent reactive empirical bond order potential (SED-REBO) is specifically designed to describe carbon materials under extreme compressive or tensile stresses. Based on the original REBO potential by Brenner and co workers, SED-REBO includes reparametrized pairwise interaction terms and a new screening term, which serves the role of a variable cutoff. The SED-REBO potential overcomes the deficiencies found with the most commonly used interatomic potentials for carbon: the appearance of artificial forces due to short cutoff that are known to create erroneous phenomena including ductile fracture of graphene and carbon nanotubes, which contradicts the experimentally observed brittle character of these materials. SED-REBO was applied in large scale molecular dynamics simulations of nanoindentation of graphene membranes and shock-induced compression of diamond. It was shown in the first computational experiment that graphene membranes exhibit a non-linear response to large magnitude of indentation, followed by a brittle fracture in agreement with experiments. The strength of graphene was determined using the kinetic theory of fracture, and the crack propagation mechanisms in the material were identified. It was found in large-scale shock simulations that SED-REBO improves the predictive power of MD simulations of carbon materials at extreme conditions.

Understanding material behavior under extreme conditions is an important area of research in physics and material science. One method to study the behavior of materials under these conditions is to drive a strong shock wave through a material and watch its response. In many cases the material response is complicated by phase transitions such as lattice restructuring (Barker 1975; Mabire and Hereil 2000; Swift, Tierney et al. 2005) and melting (Asay 1975; Elias, Chapron et al. 1988; Werdiger, Eliezer et al. 1999; Mabire and Hereil 2000; Swift, Tierney et al. 2005). To study these dynamics we are using lasers in high time resolution pump-probe experiments to develop a real time diagnostic on the phase of a shocked material. This technique enables probing of the entire phase history of a material as it shock compresses and releases. In addition to linear reflectivity and ultra-fast 2D displacement interferometry, we developed a melting diagnostics based on the non-linear optical technique of third harmonic generation (THG) using a circularly polarized laser pulse. This diagnostic resolves the less than 300 fs melting transition of laser excited Si and GaAs, and it also detects a response in shock compressed silicon. Our results show that Si remains crystalline during compression of an elastic 100 kbar shock wave. Results from Si shocked to higher pressures (> 300 kbar) indicate a decrease in THG, suggesting some level of disordering or unexplained phase change. / text

<p>Proteotoxic stress is implicated in numerous human diseases including neurodegeneration, cancer, and diabetes. Unfortunately, our mechanistic understanding of the cellular response to proteotoxic stress is limited. A critical feature of the cellular stress response is the activation of Heat Shock Transcription Factors (HSFs) that regulate the expression of numerous genes involved in protein folding, protein degradation, and cellular survival. The studies presented here utilize a diverse array of techniques including yeast genetics, recombinant protein expression and purification, biochemical analysis of protein-DNA interactions, x-ray crystallography, in vitro post-translational modification, and mammalian cell culture to illuminate novel aspects of HSF biology. Critical findings include understanding key principles of HSF-DNA interactions, identification of a novel negative regulator of HSF activity, and identification of structural features of HSF paralogs that enable precise combinatorial regulation. These unique insights lay the foundation for a greater understanding of HSF in specific cellular contexts and disease states.</p> / Dissertation

<p>Inducible Heat shock protein (Hsp70i) is a protein chaperone that is utilized during tumorigenesis and viral infections for efficient propagation. Overexpression of Hsp70i is observed in a wide spectrum of human tumors, and this overexpression correlates with metastasis, poor outcomes, and resistance to chemotherapy in patients. Hsp70i aids in cancer cell propagation through regulation of anti-apoptotic and cell survival pathways. Furthermore, Hsp70i is induced following infection for several viruses and aids viral propagation, in part through regulation of anti-apoptotic pathways as well as promoting the folding of newly synthesized proteins. Due to the parallel role of Hsp70i in both cancer and viral pathogenesis, identification of small-molecule inhibitors selective for Hsp70i could provide tools for the development of novel therapeutics and further elucidate the role of Hsp70i in both cancer and viral infections.</p><p>To date, few Hsp70 inhibitors have been identified and characterized, and their efficacy in clinical settings is unknown. Through the fluorescence-linked enzyme chemoproteomic strategy (FLECS) screen, an allosteric inhibitor selective for Hsp70i was identified, called HS-72. We show that HS-72 is highly selective for Hsp70i, over the broader purinome and other Hsp70 family members, in particular the closely related constitutively active Hsp70 family member, Hsc70. Additionally, HS-72 acts as an allosteric inhibitor to induce a conformational change and inhibit Hsp70i activity. HS-72 displays hallmarks of Hsp70i inhibition in vitro by promoting Hsp70i substrate protein degradation, protein aggregation, and selective growth inhibition of cancer cells. In wild type mice HS-72 is well tolerated and a limited PK study shows HS-72 is bioavailable. Furthermore, in a MMTV-neu breast cancer mouse model, HS-72 shows efficacy to inhibit tumor growth and promote survival.</p><p>Due to the similar utilization of Hsp70i in cancer and viral pathogenesis, this suggests the potential for HS-72 as an antiviral agent. Dengue virus (DENV) is of great public health importance due to estimates of up to 400 million infections per year, coupled with the geographic distribution of the virus, which is now endemic in over 100 countries worldwide. There is also a pressing need for DENV interventions, owing to the lack of approved vaccines or antiviral therapies. DENV is reliant on host factors throughout the viral life cycle and Hsp70i has been implicated as a host factor in DENV pathogenesis. Additionally, the complete role of Hsp70i in DENV pathogenesis remains to be elucidated, highlighting a unique opportunity to use HS-72 as a tool to specifically probe Hsp70i function. In monocytes, Hsp70i is expressed at low levels preceding DENV infection, but Hsp70i expression is induced upon DENV infection. Furthermore, inducing Hsp70i expression prior to infection, correlates with an increase in DENV infection. Targeting Hsp70i with HS-72, results in a dose dependent reduction in DENV infected monocytes, while cell viability was maintained, through inhibiting the entry stage of the viral life cycle. Following infection, Hsp70i localizes to the cell surface and interacts with the DENV receptor complex to mediate viral entry. While, HS-72 treatment results in a disruption of the interaction of Hsp70i with the DENV receptor complex, yielding a reduction in infected cells. </p><p>Collectively this work further supports Hsp70i as an anticancer and anti-dengue virus target, and identifies HS-72, a chemical scaffold that is amenable to resynthesis and iteration, as an ideal starting point for a new generation of therapeutics targeting Hsp70i.</p> / Dissertation

In response to osmotic shock in Saccharomyces cerevisiae the MAP kinase Hog1 coordinates a large-scale transcriptional stress response, rapidly producing hundreds of copies of specified transcripts. Many of the most highly induced genes are bound and regulated by a transcription factor, Sko1, but lack the canonical binding site for this factor. We use ChIP-seq to demonstrate a stress-specific binding mode of Sko1. In stress, Sko1 binds to promoters in close proximity to Hog1, and another Hog1-regulated transcription factor, Hot1. This mode of Sko1 binding requires the physical presence of Hog1, but not Hog1 phosphorylation of Sko1. We identify candidate Sko1 and Hot1 binding motifs that predict co-localization of Sko1, Hot1, and Hog1 at promoters. We then demonstrate a role for Sko1 and Hot1 in directing Hog1-associated RNA Pol II to target genes, where Hog1 is present with the elongating polymerase. We suggest a possible model for Hog1 reprogramming of transcription in the early stages of the osmotic stress response. We then determine the extent and structure of the Hog1 controlled transcriptional program in a related stress, damage to the cell wall. We find that Sko1 and Hot1 have different apparent thresholds for activation by Hog1. In addition, in cell wall damage, Hog1 regulates an additional transcription factor, Rlm1, that is not involved in other Hog1 regulated stress responses. This factor is activated by the coincidence of a signal from Hog1 with that of another MAP kinase, Slt2.

<p>The primary goal of this dissertation was to assess the feasibility of transforming an electromagnetic (EM) shock wave lithotripter with an acoustic lens as its focusing device from the original axisymmetric pressure distribution to a non-axisymmetric steerable acoustic field. This work was motivated by the desire to better match the distribution of effective acoustic pressure and pulse energy with the trajectory and anatomical features around renal and ureteral calculi during clinical shock wave lithotripsy (SWL). The acoustic field transformation was accomplished by the design of a fan-shaped acoustic barrier (mask) placed on top of the lithotripter acoustic lens to selectively reduce the source aperture along the direction of the barrier axis, therefore effectively broadening the beam width (<italic>BW</italic>) of the lithotripter field in this preferred direction. Moreover, the geometry of the original lens (L₁) was modified so that the acoustic focus of the new lens (L₂) at high output voltages (necessitated by the incorporation of the mask) is closely aligned with the lithotripter focus. The mask was further driven by a motor-controlled gear system to rotate around the lithotripter axis, generating a steerable and non-axisymmetric acoustic field. In this dissertation project, a linear acoustic model was first used for parametric studies to assess the effects of mask geometry (opening angle and thickness) on beam elongation and peak pressure reduction. Based on this analysis, two mask geometries (L₂+M₈₀₂₅ and L₂+M₉₀₃₀) were selected for modest and maximum beam elongation within the acceptable output range of the shock wave source. The acoustic and cavitation fields of the new lens with masks, as well as the corresponding field produced by the original lens, were characterized using fiber optical probe hydrophone measurements and stereoscopic high-speed imaging. Different output voltage settings were used for each lens configuration (i.e., 14 kV for L₁, 15.8 kV for L₂+M₈₀₂₅, and 17 kV L₂+M₉₀₃₀) to produce equivalent acoustic pulse energy of 45 mJ in all setups, measured in the lithotripter focal plane. Under this condition, L₂+M₈₀₂₅ and L₂+M₉₀₃₀ generate lower peak pressure (38.2 and 36.8 MPa) with a significantly broadened BW_y (11.4 and 14.3 mm) along the y-axis (head-to-toe direction of the patient), which is aligned with the mask axis, compared to the high peak pressure (44.1 MPa) and moderate <italic>BW</italic> (7.5 mm) of L₁. It is worth noting that L₂+M₈₀₂₅ and L₂+M₉₀₃₀ produce a <italic>BW</italic>_x (7.6 and 7.5 mm) in the orthogonal direction to the mask axis, which is also comparable to L₁. Similarly, the beam width of the cavitation field was broadened from 8.1 to 12.2 mm for L₂+M₈₀₂₅, and from 10.9 to 17.9 mm for L₂+M₉₀₃₀, compared to the range of 8.8 to 9.4 mm measured from L₁. In comparison, L₂+M₈₀₂₅ produces a denser and narrower bubble cloud along the y-axis than L₂+M₉₀₃₀. In vitro stone comminution (<italic>SC</italic>) tests in a tube holder (Diameter = 14 mm) have demonstrated that L₂+M₈₀₂₅ and L₂+M₉₀₃₀ are more effective at off-axis positions and during simulated respiratory motion along the elongated beam direction. The results of <italic>SC</italic> also confirmed the correlation between <italic>SC</italic> and the average peak pressure, p_+(avg), and effective acoustic pulse energy, E_eft, delivered to the stone, as shown in previous studies. Furthermore, a ureter model was developed and used to assess the performance of L₂+M₉₀₃₀, which has the maximally elongated <italic>BW</italic> under various static and simulated respiratory motion conditions. The results suggest that L₂+M₉₀₃₀ can produce significantly better <italic>SC</italic> than L₁ when the elongated beam is effectively aligned with the stone/fragments in the ureter or with their motion trajectory during the course of SWL treatment. Altogether, the results of this dissertation work have demonstrated <italic>in vitro</italic> that a non-axisymmetric and steerable acoustic field can significantly enhance stone comminution under clinically relevant SWL conditions. Future work is warranted to optimize the mask design and steering protocol to maximize the benefit of such an adaptable and versatile design to improve the performance and safety of clinical EM lithotripters.</p> / Dissertation

Astrophysical flows have an enormous dynamic range of relevant length scales. The physics occurring on the smallest scales often influences the physics of the largest scales, and vice versa. I present a detailed study of the multiscale and multidimensional behavior of three high-energy astrophysical flows: jet-driven supernovae, massive black hole accretion, and current-driven instabilities in gamma-ray burst external shocks. Both theory and observations of core-collapse supernovae indicate these events are not spherically-symmetric; however, the observations are often modeled assuming a spherically-symmetric explosion. I present an in-depth exploration of the effects of aspherical explosions on the observational characteristics of supernovae. This is accomplished in large part by high-resolution, multidimensional numerical simulations of jet-driven supernovae. The existence of supermassive black holes in the centers of most large galaxies is a well-established fact in observational astronomy. How such black holes came to be so massive, however, is not well established. In this work, I discuss the implications of radiative feedback and multidimensional behavior on black hole accretion. I show that the accretion rate is drastically reduced relative to the Eddington rate, making it unlikely that stellar mass black holes could grow to supermassive black holes in less than a Hubble time. Finally, I discuss a mechanism by which magnetic field strength could be enhanced behind a gamma-ray burst external shock. This mechanism relies on a current-driven instability that would cause reorganization of the pre-shock plasma into clumps. Once shocked, these clumps generate vorticity in the post-shock plasma and ultimately enhance the magnetic energy via a relativistic dynamo process. / text