NOVEL THERAPEUTIC TARGETS IDENTIFIED BY HIGH-THROUGHPUT TECHNOLOGIES FOR TRIPLE-NEGATIVE BREAST CANCER

Triple-negative breast cancers (TNBC) are characterized by the lack of or reduced expression of the estrogen and progesterone receptors, and normal expression of the human epidermal growth factor receptor 2. The lack of a well-characterized target for treatment leaves only systemic chemotherapy as the mainstay of treatment. Approximately 60-70% of patients are chemosensitive, while the remaining majority does not respond. Targeted therapies that take advantage of the unique molecular perturbations found in triple-negative breast cancer are needed. The genes that are frequently amplified or overexpressed represent potential therapeutic targets for triple-negative breast cancer. The purpose of this study was to identify and validate novel therapeutic targets for triple-negative breast cancers. 681 genes showed consistent and highly significant overexpression in TNBC compared to receptor-positive cancers in 2 data sets. For two genes, 3 of the 4 siRNAs showed preferential growth inhibition in TNBC cells. These two genes were the low density lipoprotein receptor-related protein 8 (LRP8) and very low-density lipoprotein receptor (VLDLR). Exposure to their cognate ligands, reelin and apolipoprotein E isoform 4 (ApoE4), stimulated the growth of TNBC cells in vitro. Suppression of the expression of either LRP8 or VLDLR or exposure to RAP (an inhibitor of ligand binding to LRP8 and VLDLR) abolished this ligand-induced proliferation. High-throughput protein and metabolic arrays revealed that ApoE4 stimulation rescued TNBC cells from serum-starvation induced up-regulation of genes involved in lipid biosynthesis, increased protein expression of oncogenes involved in the MAPK/ERK and DNA repair pathways, and reduced the serum-starvation induction of biochemicals involved in oxidative stress response and glycolytic metabolism. shLRP8 MDA-MB-231 xenografts had reduced tumor volume, in comparison to parental and shCON xenografts. These results indicate that LRP8-APOE signaling confers survival advantages to TNBC tumors under reduced nutrient conditions and during cellular environmental stress. We revealed that the LRP8-APOE receptor-ligand system is overexpressed in human TNBC. We also demonstrated that this receptor system mediates a strong growth promoting and survival function in TNBC cells in vitro and helps to sustain the growth of MDA-MD-231 xenografts. We propose that inhibitors of LRP8-APOE signaling may be clinically useful therapeutic agents for triple-negative breast cancer.

2-methylthio-N$\sp6$-dimethylallyl modification of adenosine 37 of tRNAs in {\it Escherichia coli\/} K-12

The hypermodified, hydrophobic 2-methylthio-N$\sp6$-(dimethylallyl)-adenosine (ms${2{\cdot}6}\atop1$A) residue occurs $3\sp\prime$ to the anticodon in tRNA species that read codons beginning with U. The first step (i$\sp6$A37 formation) of this modification is catalyzed by dimethylallyl diphosphate:tRNA dimethyallyltransferase (EC 2.5.1.8), which is the product of the miaA gene. Subsequent steps were proposed to be catalyzed by MiaB and MiaC enzymes to complete the ms${2{\cdot}6}\atop1$A37 modification. The study of functions of the ms${2{\cdot}6}\atop1$A37 is very important because this modified base is one of the best candidates for a role in global control in response to environmental stress. This dissertation describes the further delineation of functions of the ms${2{\cdot}6}\atop1$A37 modification in E. coli K-12 cells. This work provides significant information on functions of tRNA modifications in E. coli cells to adapt to stressful environmental conditions. Three hypotheses were tested in this work.^ The first hypothesis tested was that non-optimal translation processes cause increased spontaneous mutagenesis by the induction of SOS response in starving cells. To test this hypothesis, I measured spontaneous mutation rates of wild type cells and various mutant strains which are defective in tRNA modification, SOS response, or oxidative damage repair. I found that the miaA mutation acts as a mutator that increased Lac$\sp+$ reversion rates and Trp$\sp+$ reversion frequencies of the wild-type cells in starving conditions. However, the lexA3(Ind)(which abolishes the induction of SOS response) mutation abolished the mutator phenotype of the miaA mutant. The recA430 mutation, not other identified SOS genes, decreased the Lac$\sp+$ reversion to a less extent than that of the lexA3(Ind) mutation. These results suggest that RecA together with another unidentified SOS gene product are responsible for the process.^ The second hypothesis tested was that MiaA protein binds to full-length tRNA$\sp{\rm Phe}$ molecules in form of a protein dimer. To test this hypothesis, three versions of the MiaA protein and seven species of tRNA substrates were purified. Binding studies by gel mobility shift assays, filter binding assays and gel filtration shift assays support the hypothesis that MiaA protein binds to full-length tRNA$\sp{\rm Phe}$ as a protein dimer but as a monomer to the anticodon stem-and-loop. These results were further supported by using steady state enzyme kinetic studies.^ The third hypothesis tested in this work was that the miaB gene in E. coli exists and is clonable. The miaB::Tn10dCm insertion mutation of Salmonella typhimurium was transduced to E. coli K-12 cells by using P$\sb1$ and P$\sb{22}$ bacteriophages. The insertion was confirmed by HPLC analyses of nucleotide profiles of miaB mutants of E. coli. The insertion mutation was cloned and DNA sequences adjacent to the transposon were sequenced. These DNA sequences were 86% identical to the f474 gene at 14.97 min chromosome of E. coli. The f474 gene was then cloned by PCR from the wild-type chromosome of E. coli. The recombinant plasmid complemented the mutant phenotype of the miaB mutant of E. coli. These results support the hypothesis that the miaB gene of E. coli exists and is clonable. In summary, functions of the ms${2{\cdot}6}\atop1$A37 modification in E. coli cells are further delineated in this work in perspectives of adaptation to stressful environmental conditions and protein:tRNA interaction. (Abstract shortened by UMI.) ^

Stress-induced targeting of molecular chaperones in the yeast Saccharomyces cerevisiae

The eukaryotic stress response is an essential mechanism that helps protect cells from a variety of environmental stresses. Cell death can result if cells are not able to properly adapt and protect themselves against adverse stress conditions. Failure to properly deal with stress has implications in human diseases including neurodegenerative disorders and distinct cancers, emphasizing the importance of understanding the eukaryotic stress response in detail. As part of this response, expression of a battery of heat shock proteins (HSP) is induced, which act as molecular chaperones to assist in the repair or triage of unfolded proteins. The 90-kDa HSP (Hsp90) operates in the context of a multi-chaperone complex to promote the maturation of nuclear and cytoplasmic clients. I have discovered that Hsp90 and the co-chaperone Sba1 accumulate in the nucleus of quiescent Saccharomyces cerevisiae cells in a karyopherin-dependent manner. I isolated nuclear accumulation- defective HSP82 mutant alleles to probe the nature of this targeting event and identified a mutant with a single amino acid substitution (I578F) sufficient to prevent nuclear accumulation of Hsp90 in quiescent cells. Diploid hsp82-I578F cells exhibited pronounced defects in spore wall construction and maturation, resulting in catastrophic sporulation. The mislocalization and sporulation phenotypes were shared by another previously identified HSP82 mutant allele, further linking localization to Hsp90 functional status. Pharmacological inhibition of Hsp90 with macbecin in sporulating diploid cells also blocked spore formation, underscoring the importance of this chaperone in this developmental program. The yeast molecular chaperone Hsp104 is a member of the Hsp100 superfamily of AAA+ ATPases. Unlike the Hsp90 family of chaperones, Hsp104 is not restricted to a specific set of client proteins, but rather assists in reactivating stress-denatured proteins by solubilizing protein aggregates. I have discovered that Hsp104, along with the Hsp70 chaperone, Ssa1, and the sHSP Hsp26 accumulate into RNA processing bodies (P- bodies) and stress granules, sites of mRNA metabolism. I found that Hsp104 recruits both Ssa1 and Hsp26 to P-bodies and that these three chaperones are required for stress granule formation. These findings suggest a possible role for chaperones in mRNA metabolism by aiding in the assembly, disassembly or conversion of these enigmatic mRNP complexes. Taken together, the work presented in this dissertation serves to better understand the eukaryotic stress response by illustrating the importance of subcellular-chaperone localization in key biological processes.

Generalized resistance resources of informal caregivers: A study of demands for care, competing demands, perception of burden and cognitive stress

This study examines the relationship among psychological resources (generalized resistance resources), care demands (demands for care, competing demands, perception of burden) and cognitive stress in a selected population of primary family caregivers. The study utilizes Antonovsky's Salutogenic Model of Health, specifically the concept of generalized resistance resources (GRRs), to analyze the relative effect of these resources on mediating cognitive stress, controlling for other care demands. The study is based on a sample of 784 eligible caregivers who (1) were relatives, (2) had the main responsibility for care, defined as a primary caregiver, and (3) provided a scaled stress score for the amount of overall care given to the care recipient (family member). The sample was drawn from the 1982 National Long-Term Care Survey (NLTCS) of individuals who assisted a given NLTCS sample person with ADL limitations.^ The study tests the following hypotheses: (a) There will be a negative relationship between generalized resistance resources (GRRs) and cognitive stress controlling for care demands (demands for care, competing demands, and perceptions of burden); (b) of the specific GRRs (material, cognitive, social, cultural-environmental) the social domain will represent the most significant factor predicting a decrease in cognitive stress; and (c) the social domain will be more significant for the female than the male primary family caregiver in decreasing cognitive stress.^ The study found that GRRs had a statistically significant mediating effect on cognitive stress, but the GRRs were a less significant predictor of stress than perception of burden and demands for care. Thus, although the analysis supported the underlying hypothesis, the specific hypothesis regarding GRRs' greater significance in buffering cognitive stress was not supported. Second, the results did not demonstrate the statistical significance or differences among the GRR domains. The hypothesis that the social GRR domain was most significant in mediating stress of family caregivers was not supported. Finally, the results confirmed that there are differences in the importance of social support help in mediating stress based on gender. It was found that gender and social support help were related to cognitive stress and gender had a statistically significant interaction effect with social support help. Implications for clinical practice, public health policy, and research are discussed. ^

Compounds that bind to the gamma-aminobutyric acid benzodiazepine ionophore complex modulate the cellular response to oxidant stress

The γ-aminobutyric acid benzodiazepine (GABAA /BZDR) ionophore complex has been widely studied in the central nervous system (CNS) and it regulates Cl− ion movement across the plasma membrane. The complex has been found in the distal tubule and the thick ascending limb of the kidney. The goal of this study was to see if modulation of this complex by agonists or antagonists could affect the way Madin-Darby Canine Kidney (MDCK) cells responded to an oxidant stress induced by menadione. When compared to cells incubated with menadione alone, preincubation with lindane, a nonspecific GABAA antagonist, coincubation with bicuculline, a specific GABAA antagonist, and coincubation with FG7142, an inverse agonist for the BZDR, protected cells from menadione cytotoxicity. Preincubation of cells in media containing PK11195 had no effect on menadione cytotoxicity. Coincubation with flurazepam, a BZDR agonist, exacerbated menadione cytotoxicity. This suggests that modulation of the GABAA/BZDR ionophore complex within MDCK cells with agonists and antagonists can alter the cellular responsiveness to an oxidant-induced injury. These responses via agonists and antagonists may be due to alterations of Cl− ion influx during late stage necrotic cell death. ^

A comprehensive assessment of environmental ultraviolet radiation induced DNA damage in marine microorganisms

There is evidence that ultraviolet radiation (UVR) is increasing over certain locations on the Earth's surface. Of primary concern is the annual pattern of ozone depletion over Antarctica and the Southern Ocean. Reduction of ozone concentration selectively limits absorption of solar UV-B (290–320 nm), resulting in higher irradiance at the Earth's surface. The effects of ozone depletion on the human population and natural ecosystems, particularly the marine environment, are a matter of considerable concern. Indeed, marine plankton may serve as sensitive indicators of ozone depletion and UV-B fluctuations. Direct biological effects of UVR result from absorption of UV-B by DNA. Once absorbed, energy is dissipated by a variety of pathways, including covalent chemical reactions leading to the formation of photoproducts. The major types of photoproduct formed are cyclobutyl pyrimidine dimer (CPD) and pyrimidine(6-4)pyrimidone dimer [(6-4)PD]. Marine plankton repair these photoproducts using light-dependent photoenzymatic repair or nucleotide excision repair. The studies here show that fluctuations in CPD concentrations in the marine environment at Palmer Station, Antarctica correlate well with ozone concentration and UV-B irradiance at the Earth's surface. A comparison of photoproduct levels in marine plankton and DNA dosimeters show that bacterioplankton display higher resistance to solar UVR than phytoplankton in an ozone depleted environment. DNA damage in marine microorganisms was investigated during two separate latitudinal transects which covered a total range of 140°. We observed the same pattern of change in DNA damage levels in dosimeters and marine plankton as measured using two distinct quantitative techniques. Results from the transects show that differences in photosensitivity exist in marine plankton collected under varying UVR environments. Laboratory studies of Antarctic bacterial isolates confirm that marine bacterioplankton possess differences in survival, DNA damage induction, and repair following exposure to UVR. Results from DNA damage measurements during ozone season, along a latitudinal gradient, and in marine bacterial isolates suggest that changes in environmental UVR correlate with changes in UV-B induced DNA damage in marine microorganisms. Differences in the ability to tolerate UVR stress under different environmental conditions may determine the composition of the microbial communities inhabiting those environments. ^

The eukaryotic cellular stress response: Biochemical and genetic analyses in Saccharomyces cerevisiae

All cells must have the ability to deal with a variety of environmental stresses. Failure to correctly adapt to and/or protect against adverse stress conditions can lead to cell death. In humans, stress response defects have been linked to a number of neurodegenerative diseases and cancer, underscoring the importance of developing a fundamental understanding of the eukaryotic stress response.^ In an effort to characterize cellular response to high temperature stress, I identified and described one member of a novel gene family— RTR1. I show that the RTR1 gene and its protein product genetically and biochemically interact with core subunits of the RNA polymerase II enzyme. Appropriately, loss of RTR1 results in defective transcription from multiple promoters. These data provide evidence that Rtr1, which is essential under stress conditions, acts as a key regulator of transcription.^ In addition to transcriptional regulation, cells deal with many stressors by inducing molecular chaperones. Molecular chaperones are ubiquitous in all living cells and bind unfolded or damaged proteins and catalyze refolding or degradation. Hsp90 is a unique chaperone because it targets specific clients—typically signaling proteins—for maturation. While it has been shown that Sse1, the yeast Hsp110, is a critical regulator of the Hsp90 chaperone cycle, this work describes the molecular basis for that regulation. I show that Sse1 modulates Hsp90 function through regulation of Hsp70 nucleotide exchange. Further, Hsp110-type nucleotide exchange factors (NEFs) appear to have a specific role in modulating Hsp90 function in this manner. Finally, in addition to Hsp110, the eukaryotic cytosol contains two other types of Hsp70 NEF: Snl1 (BAG-domain protein) and Fes1 (HspBP1-like protein). I investigated the cellular roles of these NEFs to better understand the reason that eukaryotic cells contain three distinct protein families that perform the same biochemical function. I show that while cytsolic Hsp70 NEFs have some degree of functional overlap, they also exhibit striking divergence. Taken together, the work presented in this dissertation provides a more detailed understanding of the eukaryotic stress response. ^

Differential stress response and susceptibility to oxidative injury in alveolar macrophages from C57BL/6J and C3H/HeJ mice following oxidant exposure

Studies have demonstrated a variable response to ozone among individuals and animal species and strains. For instance, C57BL/6J mice have a greater inflammatory response to ozone exposure than C3H/HeJ mice. In these studies, I utilized these strain differences in an effort to derive a mechanistic explanation to the variable strain sensitivity to ozone exposure. Therefore, alveolar macrophages (AM) from C57BL/6J and C3H/HeJ mice were exposed in vitro to hydrogen peroxide ($\rm H\sb2O\sb2$), heat and acetyl ceramide or in vivo to ozone. Necrosis and DNA fragmentation in macrophages from the two murine strains were determined to assess cytotoxicity following these treatments. In addition, synthesis and expression of the stress proteins, stress protein 72 (SP72) and heme oxygenase (HO-1), were examined following treatments. The in vitro experiments were conducted to eliminate the possibility of in vivo confounders (i.e., differences in breathing rates in the two strains) and thus directly implicate some inherent difference between cells from the two murine strains. $\rm H\sb2O\sb2$ and heat caused greater cytotoxicity in AM from C57BL/6J than C3H/HeJ mice and DNA fragmentation was a particularly sensitive indicator of cell injury. Similarly, AM from C57BL/6J mice were more sensitive to ozone exposure than cells from C3H/HeJ mice. Exposure to either 1 or 0.4 ppm ozone caused greater cytotoxicity in macrophages from C57BL/6J mice compared to macrophages from C3H/HeJ mice. The increased sensitivity of AM to injury was associated with decreased synthesis and expression of stress proteins. AM from C57BL/6J mice synthesized and expressed significantly less stress proteins in response to heat and ozone than AM from C3H/HeJ mice. Heat treatment resulted in greater synthesis and expression of SP72. In addition, macrophages from C57BL/6J mice expressed lower amounts of HO-1 than macrophages from C3H/HeJ mice following 0.4 ppm ozone exposure. Therefore, AM from C57BL/6J mice are more susceptible to oxidative injury than AM from C3H/HeJ mice which might be due to differential expression of stress proteins in these cells. ^

THE EFFECT OF PERSON-ENVIRONMENT FIT ON THE PSYCHOLOGICAL WELL-BEING OF PSYCHIATRIC AIDES WORKING IN A STATE HOSPITAL (COPING RESOURCES, SOCIAL SUPPORT, OCCUPATIONAL STRESS, LOCUS-OF-CONTROL)

This study addressed two purposes: (1) to determine the effect of person-environment fit on the psychological well-being of psychiatric aides and (2) to determine what role the coping resources of social support and control have on the above relationship. Two hundred and ten psychiatric aides working in a state hospital in Texas responded to a questionnaire pertaining to these issues.^ Person-environment fit, as a measure of occupational stress, was assessed through a modified version of the Work Environment Scale (WES). The WES subscales used in this study were: involvement, autonomy, job pressure, job clarity, and physical comfort. Psychological well-being was measured with the General Well-Being Schedule which was developed by the National Center for Health Statistics. Co-worker and supervisor support were measured through the WES and finally, control was assessed through Rotter's Locus of Control Scale.^ The results of this study were as follows: (1) all person-environment (p-e) dimensions appeared to have linear relationships with psychological well-being; (2) the p-e fit - well-being relationship did not appear to be confounded by demographic factors; (3) all p-e fit dimensions were significantly related to well-being except for autonomy; (4) p-e fit was more strongly related to well-being than the environmental measure alone; (5) supervisor support and non-work related support were found to have additive effects on the relationship between p-e fit and well-being, however no interaction or buffering effects were observed; (6) locus of control was found to have additive effects in the prediction of well-being and showed interactive effects with work pressure, involvement and physical comfort; and (7) the testing of the overall study model which included many of the components mentioned above yielded an R('2) = .27.^ Implications of these findings are discussed, future research suggested and applications proposed. ^

Recommendations for dementia caregiver stress interventions based on Intervention Mapping guidelines

Stress can affect a person's psychological and physical health and cause a variety of conditions including depression, immune system changes, and hypertension (Alzheimer's Association, 2010; Aschbacher et al., 2009; Fredman et al., 2010; Long et al., 2004; Mills et al., 2009; von Känel et al., 2008). The severity and consequences of these conditions can vary based on the duration, amount, and sources of stress experienced by the individual (Black & Hyer, 2010; Coen et al., 1997; Conde-Sala et al., 2010; Pinquart & Sörensen, 2007). Caregivers of people with dementia have an elevated risk for stress and its related health problems because they experience more negative interactions with, and provide more emotional support for, their care recipients than other caregivers. ^ This paper uses a systematic program planning process of Intervention Mapping to organize evidence from literature, qualitative research and theory to develop recommendations for a theory- and evidence-based intervention to improve outcomes for caregivers of people with dementia. A needs assessment was conducted to identify specific dementia caregiver stress influences and a logic model of dementia caregiver stress was developed using the PRECEDE Model. Necessary behavior and environmental outcomes are identified for dementia caregiver stress reduction and performance objectives for each were combined with selected determinants to produce change objectives. Planning matrices were then designed to inform effective theory-based methods and practical applications for recommended intervention delivery. Recommendations for program components, their scope and sequence, the completed program materials, and the program protocols are delineated along with ways to insure that the program is adopted and implemented after it is shown to be effective.^

An assessment of post-disaster psychological stress in hazardous waste operations and emergency response (HAZWOPER) workers

Personnel involved in natural or man-made disaster response and recovery efforts may be exposed to a wide variety of physical and mental stressors that can exhibit long-lasting and detrimental psychopathological outcomes. In a disaster situation, huge numbers of "secondary" responders can be involved in contaminant clean-up and debris removal and can be at risk of developing stress-related mental health outcomes. The Occupational Safety and Health Administration (OSHA) worker training hierarchy typically required for response workers, known as "Hazardous Waste Operations and Emergency Response" (HAZWOPER), does not address the mental health and safety concerns of workers. This study focused on the prevalence of traumatic stress experienced by secondary responders that had received or expressed interest in receiving HAZWOPER training through the National Institute of Environmental Health Sciences Worker Education and Training Program (NIEHS WETP). ^ The study involved the modification of two preexisting and validated survey tools to assess secondary responder awareness of physical, mental, and traumatic stressors on mental health and sought to determine if a need existed to include traumatic stress-related mental health education in the current HAZWOPER training regimen. The study evaluated post-traumatic stress disorder (PTSD), resiliency, mental distress, and negative effects within a secondary responder population of 176 respondents. Elevated PTSD levels were seen in the study population as compared to a general responder population (32.9% positive vs. 8%-22.5% positive). Results indicated that HAZWOPER-trained disaster responders were likely to test positive for PTSD, whereas, untrained responders with no disaster experience and responders who possessed either training or disaster experience only were likely to test PTSD negative. A majority (68.75%) of the population tested below the mean resiliency to cope score (80.4) of the average worker population. Results indicated that those who were trained only or who possessed both training and disaster work experience were more likely to have lower resiliency scores than those with no training or experience. There were direct correlations between being PTSD positive and having worked at a disaster site and experiencing mental distress and negative effects. However, HAZWOPER training status does not significantly correlate with mental distress or negative effect. ^ The survey indicated clear support (91% of respondents) for mental health education. The development of a pre- and post-deployment training module is recommended. Such training could provide responders with the necessary knowledge and skills to recognize the symptomology of PTSD, mental stressors, and physical and traumatic stressors, thus empowering them to employ protective strategies or seek professional help if needed. It is further recommended that pre-deployment mental health education be included in the current HAZWOPER 24- and 40-hour course curriculums, as well as, consideration be given towards integrating a stand-alone post-deployment mental health education training course into the current HAZWOPER hierarchy.^

Effects of salinity, temperature and cadmium stress on cadmium -binding protein in the grass shrimp, {\it Palaemonetes pugio}

The combined effects of salinity, temperature and cadmium stress on survival and adaptation through cadmium-binding protein (CdBP) accumulation were studied in the grass shrimp, Palaemonetes pugio. In 96-hour bioassays, shrimp were exposed to zero or one of three levels of cadmium, under one of six different salinity (15, 25, or 35$\perthous$) and temperature (20 or 30$\sp\circ$C) regimes. CdBP concentrations were quantified in survivors from the 24 exposure groups. Salinity and temperature did not affect survivorship unless the shrimp were also exposed to cadmium. Grass shrimp were most sensitive to cadmium at low salinity-high temperature, and least sensitive at high salinity-low temperature. The incidence of cadmium-associated black lesions in gill tissue was influenced by salinity and temperature stress. P. pugio produced a 10,000 dalton metallothionein-like CdBP when exposed to at least 0.1 mg Cd$\sp{2+}$/L for 96 hours. Accumulation of CdBP was increased with increases in the exposure cadmium level, increases in temperature and decreases in salinity, independently and in conjunction with one another. Maximum CdBP concentrations occurred in grass shrimp that survived the salinity-temperature-cadmium conditions creating maximum stress as measured by highest mortality, not necessarily in shrimp exposed to the highest cadmium levels. The potential utility of this method as a monitor of physiological stress in estuarine biota inhabiting metal-polluted environments is discussed. ^