Thesis (Ph. D.)--West Virginia University, 2005. / Title from document title page. Document formatted into pages; contains vii, 107 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

Thesis (M.S.)--University of Michigan, 1994.

Thesis (Ph.D.)--University of Nevada, Reno, 2004. / Includes bibliographical references. Online version available on the World Wide Web.

Thesis (Ph. D.)--West Virginia University, 2006. / Title from document title page. Document formatted into pages; contains xi, 180 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

Thermal scavenging is a unique behavior restricted to a few desert ant genera. Workers are among the most thermotolerant land animals known to this day, being able to survive body temperatures of sometimes more than 50°C for several minutes. Making use of their remarkable heat-hardiness, they search for food in plain day, a feat that other desert creatures cannot accomplish. They mostly feed on the corpses of heat-stricken, less tolerant arthropods that were unable to survive the blazing sun of the midday desert. Thermal scavenging has evolved independently at least three times in distantly related genera, geographical well segregated inside the different deserts of the world. First, the Cataglyphis genus ranges from the Sahara Desert and extends its distribution to reach minor Asia through the Mediterranean Basin. Second the Ocymyrmex genus can be found in the Namib and Karoo deserts of southern Africa, extending its range to eastern Africa savanna plains. Finally, the Melophorus genus can be found in Australia, with thermal scavenging species distributed in the central desert of the outback region.While this impressive behavior was already well-described by the start of this PhD project, little was known about the mechanisms supporting the remarkable heat-tolerance of workers. Using biophysical and physiological approaches in Cataglyphis and Ocymyrmex, we’ve been able to pinpoint key aspects underlying stress tolerance in those genera. First, from a biophysical standpoint, the Sahara silver ant Cataglyphis bombycina is covered with a unique and dense array of prismatic hairs reflecting visible wavelengths by total internal reflection. This allows reflection of up to 50% of the incident sunlight energy, thus shifting down the ant’s thermal equilibrium and sparing its body a few critical degrees. Second, in a comparative framework, we found numerous genes involved with critical cellular processes to be constitutively expressed or strongly up-regulated to heat in thermal scavenging ants, while their orthologs were not in mesophilic species. Those processes, such as molecular chaperoning, cell-cycle regulation, energy metabolism and muscular functions are keys that allow those ants to meet the higher requirement needed to scavenge for food at both stunning speed and under extreme heat-pressure. Overall, this work investigates the physiological and biophysical basis enabling thermal scavenging ants to survive extreme heat conditions. It provides a deeper understanding of cellular heat-tolerance pathways in non-model animals and contribute to our knowledge of life’s adaptation to extreme conditions. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

In order to survive, cells must be able to cope with a variety of environmental stressors. The heat shock response (HSR) is a pro-survival mechanism employed by cells in response to protein denaturing stress, such as heat. Since its discovery in 1960, the heat shock response has been found to be regulated by the transcription factor heat shock factor 1 (HSF1). During periods of increased stress, HSF1 undergoes a multi-step process of activation that involves homotrimerization, DNA-binding, and post-translational regulatory modifications, all of which ultimately function to control the transcription of chaperone genes. These chaperone genes encode molecular chaperone proteins which function to promote survival during stress by restoring protein homeostasis to the cell. Although HSF1 is classically studied for its role in regulating the HSR, HSF1 also has roles in regulating metabolism, development, and longevity. Studies in the nematode Caenorhabditis elegans demonstrate the HSF1 homolog, HSF-1, as a global regulator of gene expression that has both stress-dependent and -independent functions. Modulating HSF1 activity therefore has implications beyond stress-induced processes, and has been suggested as a promising therapeutic target for diseases of aging and protein dysfunction. We were interested in determining regulators of the HSR using C. elegans as a model to test for effects on proteostasis and longevity. In these studies, we observed the effects of compound treatment (Chapters 1 and 2), genetic manipulation (Chapters 3 and 4), and environmental stimuli (Chapters 5 and 6), on the HSR in C. elegans. In Chapters 1 and 2, we describe our findings that treatment with the DNA synthesis inhibitor Fluorodeoxyuridine, and treatment with coffee and caffeine, enhance the heat shock response and improve proteostasis in aging worms in an HSF-1-dependent manner. In Chapters 3 and 4, we uncovered that negative regulation of the HSR by the cell cycle and apoptosis regulator CCAR2 is conserved in C. elegans, and is mediated by the CCAR2 ortholog, LST-3. We also uncovered that negative regulation of the HSR by LST-3 requires the SIRT1 homolog Sir-2.1, and knockdown of LST-3 via lst-3 RNAi works through Sir-2.1 to enhance stress-resistance, fitness, proteostasis and longevity. In Chapters 5 and 6, we describe the global impact of HSF-1 in regulating transcriptional processes during a heat stress. The profiling of global HSF-1 mRNA and miRNA targets has allowed us to uncover a heat-dependent and -independent role for HSF-1 in regulating gene expression to impact stress-resistance, proteostasis, and longevity. Altogether, these studies demonstrate the impact of compound treatment, genetic manipulation, and environmental stimuli on the heat shock response, while also uncovering global stress-dependent and -independent roles for HSF-1. This work therefore provides insight into various methods of activating the HSR by modulating HSF-1 activity, and uncovering global HSF-1 target genes, which may be useful for designing therapeutic treatment strategies for diseases of protein dysfunction.

A diapause upregulated cDNA clone was isolated from a cDNA library generated from brain mRNA of diapausing Sarcophaga crassipalpis pupae. The clone hybridized to a 1600 bp transcript on a northern blot. The insert is 823 bp in length, has a tentative open reading frame of 615 bp, and codes for a 23 kDa protein. The clone has a high level of identity at the amino acid level with the four small heat shock proteins of Drosophila melanogaster. Northern analysis revealed no detectable expression of the transcript in diapause- or nondiapause-programmed wandering larvae, and only trace expression in nondiapausing pupae. But, the transcript was highly expressed beginning at the onset of diapause and continuing throughout diapause. Expression promptly decreased when diapause was terminated. In nondiapausing individuals the transcript was highly expressed in response to cold shock or heat shock, but temperature stress did not cause greater expression in diapausing pupae. The results imply that expression of this small heat shock protein, a response elicited by temperature stress in nondiapausing individuals, is a normal component of the diapause syndrome. The upregulation of this gene during diapause suggests that it plays an essential role during this overwintering developmental arrest.

Introduction: Myocardial Infarction (MI) is the leading cause of premature death worldwide. During MI-induced ischemia, the release of heat shock proteins (HSPs), a classic damage-associated molecular pattern (DAMP), by severely injured cells leads to prolonged inflammation through their activation of innate pattern recognition receptors, fibrosis, and subsequent contractile dysfunction. The regulation of HSPs is orchestrated by its master transcription factor, Heat Shock Factor 1 (HSF1). However, it is unknown if HSF1 is a potential integrated functional target to improve MI outcomes. We addressed this question by asking if the coordinated modulation of HSPs via genetic deletion of Hsf1 can be beneficial in MI. Hypothesis: We hypothesized that genetic deletion of Hsf1 can lead to improved survival and left ventricle (LV) remodeling through reduction of pro-inflammatory pathway activation in a murine model of MI-induced coronary artery ligation. Methods and Results: Eleven to thirteen-week-old male Hsf1-/- mice and Hsf1+/+ littermate controls were subjected to MI by left anterior descending (LAD) coronary artery ligation or sham operation. Hsf1-/- mice subjected to induced-MI had a significant higher survival rate (74%) at 28 days than WT mice post-MI in the same time frame (34%, p<0.001). Echocardiography at 3, 7, and 28 days post-MI; however, did not identify any difference in LV function between Hsf1+/+ and Hsf1-/- mice. Masson Trichrome and Picro Sirius Red staining of heart tissue sections following 7 days of sham or MI-operation indicated that MI-operated Hsf1-/- hearts had a significant smaller infarct size than Hsf1+/+ hearts at 19% compared to 32% (p<0.05), respectively; and less collagen deposition when compared to WT littermates. Cardiac expression of heat shock proteins was significantly lowered in the Hsf1-/- hearts compared to Hsf1+/+ hearts following 3 and 7 days of MI. However, no significant difference was observed in number of immune cells, cardiac gene expression of pro-inflammatory cytokines and chemokines, cardiac protein expression of NF-κB and MAPK-ERK1/2 signaling proteins, and serum IL-6 concentration between Hsf1+/+ and Hsf1-/- mice 3 days post-MI. Following 7 days of MI, there is a significant increase in the gene expression of pro-inflammatory cytokines, such as Il1b, and chemokines, such as Ccl2, in Hsf1-/- hearts than Hsf1+/+ hearts. Conclusion & Future Directions: Overall, the loss of Hsf1 improved survival and reduced infarct size following MI. However, its deletion did not affect inflammatory processes until 7 days post-MI or improved cardiac function in our specific murine MI model.

Chlamydomonas priscuii is a psychrophilic green alga found 17 m below the permanently ice-covered surface of the Antarctic Lake Bonney, where it experiences a myriad of extreme environmental conditions, including low temperature, low light, and high salinity. While this habitat is extreme, it is also very stable, and this alga rarely experiences changes in its environment. Heat shock proteins (HSPs) are a ubiquitous family of chaperone proteins that perform important housekeeping and stress-related roles. In most organisms, including the model green alga Chlamydomonas reinhardtii, HSP expression is induced during abiotic stress to regain protein homeostasis – a process regulated by heat shock transcription factors (HSFs). This work shows that C. priscuii constitutively accumulates high protein levels of HSPs in steady-state conditions but fails to induce additional HSP accumulation during heat and low temperature, high and low salt, high light, and with canavanine treatment. In this study, a single HSF was identified in the C. priscuii genome. Comparative sequence analysis revealed that most domains characteristic of a functional HSF are conserved, but the expression of a full length HSF1 transcript could not be detected in the cell. Furthermore, the promoters of many C. priscuii HSPs lack binding sites for HSF. This work has shown that C. priscuii has a diminished ability to regulate HSP expression under stressful conditions, which we hypothesize is a result of life in an extreme but very stable environment. This is the first demonstration of a loss of HSP accumulation in green algae, which carries implications on the ability of psychrophiles to survive in the face of climate change.

Small heat shock proteins (sHSPs) are a ubiquitous family of molecular chaperones that play a vital role in maintaining protein homeostasis in cells. They are the first line of defence against the detrimental effects of cellular stress conditions like fluctuations in temperature, pH, oxidative and osmotic potentials, heavy metal toxicity, drought and anoxia. Many sHSPs are also constitutively expressed during developmental stages of different plant tissues. Members of this family are ATP-independent chaperones, with monomeric masses varying from 12-40 kDa. A characteristic feature of sHSPs is their ability to assemble into large oligomers, ranging from dimers to 48-mers. Under stress conditions, these oligomers dissociate and/or undergo drastic conformational changes to facilitate their binding to misfolded substrate proteins in the cell. This interaction prevents the substrate from aggregating during stress. When physiological conditions are restored, the substrates are transferred to other ATP-dependent heat shock proteins for refolding. Thus sHSPs do not refold their substrates, but instead prevent them from aggregating and maintain them in a „folding-competent‟ state. The clientele of sHSPs includes proteins with a wide range of molecular masses, secondary structures and pIs. This promiscuity has led to sHSPs occupying key positions in the protein quality control network. As molecular chaperones that protect proteins, sHSPs prevent disease. Concomitantly, mutations in sHSPs have also been linked to various human diseases. Till date, high resolution crystal structures are available only for 3 sHSP oligomers. This insufficiency of structural information has hindered our understanding of the mechanism of chaperone function, the link between the oligomeric status and chaperone activity, identification of substrate binding sites and the role of the flexible terminal segments in mediating both the oligomerization and chaperone function. We undertook structural and functional characterization of plant and bacterial sHSPs in order to address some of these questions. Chapter 1 of this thesis gives an overview of the sHSP family, with special emphasis on the oligomeric assemblies of sHSPs of known structures. We highlight what we know about this family through mutational studies, what is as yet unknown, and why it is important to study this family. Chapter 2 describes our efforts at structural and functional characterization of 5 sHSPS in rice, each targeted to a different organelle. We probed the role played by the N-terminal region in mediating oligomer assembly and in the chaperone activity of the protein. Rice sHSPs displayed a wide range of hydrodynamic radii, from 4 nm to 14 nm, suggesting that their oligomeric assemblies are likely to be diverse. In chapter 3, we discuss our attempts at the structural characterization of a bacterial sHSP, Aggregation suppressing protein A, or AgsA from Salmonella enterica. We obtained a high resolution crystal structure of the dimer of the core sHSP domain. We compared this dimer with other known sHSP dimers, reported the deviations that we observed and analysed the structure to account for these differences. We used this dimer structure to successfully obtain solutions for low resolution X-ray diffraction data for oligomers of different truncated constructs of AgsA. We observed that a C-terminal truncated construct formed an octahedral 24¬mer (4.5 Å resolution), whereas a construct truncated at both termini formed a triangular bipyramidal 18-mer (7.7 Å resolution), an assembly hitherto unobserved for any sHSP. A similar 18-mer was obtained when the C-terminal truncated construct was incubated with a dipeptide prior to crystallisation (6.7 Å resolution). The cryo-EM map of the wild type protein (12 Å resolution) could be fitted with a different 18-mer. The low resolution of the data pre-empted an atomic-level description of the interfaces of the assemblies. However, our work highlights the structural plasticity of this protein and probes the sensitivity of the oligomeric assembly to minor differences in construct length.