Cells die not merely as a consequence of catastrophic failure of homeostasis but when programmed cell death is activated. The existence of non-apoptotic modes of regulated cell death is increasingly appreciated. However, the full extent and diversity of these alternative cell death mechanisms remains uncharted. In this thesis, we developed a systematic framework to discover and characterize lethal compounds that induce distinct cell death phenotypes. In the first part, we investigated the landscape of pharmacologically-accessible non-apoptotic cell death mechanisms. This effort resulted in the discovery of a novel ferroptosis inducer, FIN56. The rest of my work focused on characterizing the mechanism of action of FIN56. Technologies used here should be generally applicable for the systematic study of various cell death mechanisms. First, to globally survey pharmacologically-accessible cell death mechanisms, we used 3,169 uncharacterized lethal compounds as cell death probes. We found that 451 compounds (14%) were lethal without activating caspase activity. 56 most potent and structurally diverse compounds were more closely studied using the 'modulatory profiling' approach, which involves examining changes in potency of lethal compounds by co-treatment with chemical modulators. We discovered that caspase-independent lethals induced three types of regulated non-apoptotic cell death: metal ion-dependent cell death, necrostatin-1-dependent cell death, and ferroptosis, a regulated form of iron-dependent oxidative cell death. With further structural optimization, we discovered a specific ferroptosis inducer, FIN56. Ferroptosis is induced when the lipid repair enzyme glutathione peroxide 4 (GPX4) is inhibited or inactivated by depletion of glutathione. We found that, in contrast, FIN56 induced ferroptosis through decreasing the abundance of GPX4. Second, we developed a technology that identifies proteins responsible for cell death mechanisms of interest utilizing chemical library screening. The technology consists of three steps: (i) binding targets of each molecule in the chemical library were predicted using Similarity Ensemble Approach, a chemoinformatic ligand-based target prediction algorithm; (ii) the chemical library was screened for enhancers/suppressors of the cell death; (iii) incorporating the screening data into the prediction to make the prediction more reliable. This approach, termed `Target Enrichment Analysis', resulted in the discovery of two features of FIN56-induced ferroptosis: calcium ion influx and activation of lipoxygenases, enzymes that peroxidize fatty acids. Inhibiting either of them suppressed FIN56-induced ferroptosis. Third, we tried to capture metabolic changes induced upon FIN56 treatment that were relevant to the mechanism of action of FIN56 and identify protein targets of FIN56 using chemoproteomics. Metabolomic profiling experiments discovered that non-steroidogenic intermediates in the mevalonate pathway regulated cellular sensitivity to FIN56-induced ferroptosis. Although none of the proteins identified through target identification effort has yet been fully confirmed as responsible for induction of ferroptosis triggered by FIN56, we found through the analysis that inhibition of squalene synthase, an enzyme in the mevalonate pathway, suppressed FIN56-induced ferroptosis consistently. Finally, to define biomarkers that predict sensitivity to ferroptosis inducers including FIN56, we investigated the molecular determinants of sensitivity in the NCI60 panel and identified nicotinamide adenine dinucleotide phosphate (NADPH) levels as a global predictor of sensitivity to ferroptosis. These studies demonstrate that sensitivity to ferroptosis is regulated by metabolic pathways, suggesting that it may be a relevant form of cell death in cases of dysregulated metabolism. This systematic approach using a combination of modulatory profiling and cell line selectivity analysis is an effective means to explore, discover and characterize cellular phenotypes induced by unknown small molecules.

Background: The obesity epidemic is the greatest public health problem of our time, and exerts an enormous health and economic burden by acting as a risk factor for multiple disorders and all-cause mortality. While environmental and social factors certainly contribute to the complex etiology of obesity, there is strong evidence of a substantial genetic component. The majority of obesity genes are involved the leptin-melanocortin receptor pathway governing energy homeostasis, but mutations affecting this circuit are often untreatable and rare, and an improved understanding of other genetic risk factors could aid in the development of novel therapies. In this thesis I study two obesity candidate genes with unclear direct relevance to disease: 1) rare structural variation at the 16p11.2 BP4-BP5 locus and 2) common variation in the Fat Mass and Obesity-Associated (FTO) gene. Methods: 1) I analyzed disinhibited eating measurements from families with 16p11.2 copy number variation (CNV) carriers, to test whether eating in the absence of hunger (EAH) and loss of control (LOC) eating behaviors mediate the dosage-dependent CNV-BMI relationship. 2) Using association data from a study of over 20,000 African Americans and 1,145 functional annotations from the Encyclopedia of Non-coding Elements (ENCODE) and Roadmap Epigenomics projects, I statistically fine-mapped the FTO locus to identify the SNP(s) and cellular contexts underlying the association between FTO and obesity. Results: 1) EAH due to external triggers mediates over 30% of the 16p11.2 deletion’s effect on obesity, while other EAH and LOC behaviors were not significant mediators. This result was independent of IQ deficits and autism related to the CNV, as well as parents’ feeding behaviors and practices. 2) Given 51 FTO SNPs’ association statistics, correlation, and overlap with functional annotations, rs9927317 and rs62033405 had the highest posterior probability of association with obesity. Obesity-associated SNPs may regulate expression of FTO and/or nearby genes through the activity of enhancers and 5’ ends of transcribed genes in the substantia nigra of the brain, bone chondrocytes, and white adipose. Conclusions: These results may help pinpoint the specific genes, regulatory elements, and cellular contexts through which the 16p11.2 and FTO loci exert their effects on obesity.

Recentemente, tem sido reconhecido que a água pode ser ao mesmo tempo uma fonte vital para os organismos e um veículo para eliminação de poluentes. Esse paradoxo pode ser considerado como parte da crise do presente ambiente que está inserida no conflito entre natureza e tecnologia, sendo acompanhado pelo emprego de biomarcadores, que consistem em indicadores bioquímicos e celulares da presença de contaminantes através da análise de fluidos corpóreos bem como células ou tecidos. O principal objetivo deste trabalho foi o de verificar quais os possíveis efeitos do cobre na homeostase do caranguejo de mangue Ucides cordatus expostos ao cobre na água (CuSO4) por diferentes períodos de tempo, através de teste agudo com exposição durante 24h e 96h e teste crônico com duração de 15 dias. Após a exposição dos animais à agua contaminada, estes foram crio-anestesiados pra dessensibilização e então foram retiradas alíquotas de hemolinfa e urina para determinação de concentrações de sódio, potássio, magnésio e cálcio, bem como as concentrações de glicose e lactato, além de brânquias e hepatopâncreas para a dosagem de enzimas como Na+/K+-ATPase, anidrase carbônica e a concentração da proteína metalotioneína, não esquecendo o músculo da quela que foi utilizado para determinação da concentração de glicogênio (assim como para o hepatopâncreas). Para complementar o amplo trabalho, foi determinado o transporte de cobre na membrana celular de brânquias e hepatopâncreas, através do qual foi possível verificar que a entrada do cobre pela membrana plasmática pode ocorrer através de diversas vias, podendo ser elas dependentes ou independentes de cálcio, porém é estritamente dependente da concentração de sódio no meio extra e intracelular, demonstrando interação entre transportadores para manutenção da homeostase. Além disso, o cobre se apresenta como um íon competidor com outros cátions como magnésio e potássio, além, claro, de alterar atividades enzimáticas como da Na+/K+-ATPase e anidrase carbônica. No entanto é válido notar que, principalmente para as brânquias anteriores, que são predominante respiratórias, houve aumento da síntese de metalotioneína, proteína esta induzida na presença de altas concentrações de cobre, que dentre outras funções, é principal componente da hemocianina, pigmento respiratório dos crustáceos / Recently, it has been recognized that water can be both a vital source for the organisms and a vehicle for disposal of pollutants. This paradox may be considered as part of the environment crisis that is inserted between nature and technology conflict, accompanied by the use of biomarkers that consists of cellular and biochemical indicators of contamination by analysis of body fluids as well as cells or tissues. The main objective of this study was to determine what the possible effects of copper homeostasis mangrove crab Ucides cordatus exposed to copper in water (CuSO4) for different periods of time, by testing acute exposure for 24h and 96h and chronic test, lasting 15 days. After exposure of the animals to contaminated water, they were cryo-anesthetized for desensitization and then aliquots of haemolymph and urine were collect to determine hemolymph and urine concentration of sodium, potassium, magnesium and calcium, and the concentrations of glucose and lactate as well as gills and hepatopancreas for measure enzymes such as Na+/K+-ATPase, carbonic anhydrase and metallothionein protein concentration, not forgetting the muscle which was used for the determination of glycogen concentration (as for the hepatopancreas). To complement the extensive study, it was determined copper transport in gills and hepatopancreas epithelial cells, whereby it was verified that the input of copper by plasma membrane can occur through various routes, which can be dependent or independent of calcium, but it is strictly dependent on the sodim concentration in intracellular and extracellular medium, showing interaction between transport proteins for maintaining homeostasis. Furthermore, copper is presented as a competitive with other cations such as magnesium and potassium, in addition of course to alter enzymatic activity as Na+/K+-ATPase and carbonic anhydrase. However it is worth noting that, especially prior to the anterior gill cells, which are predominant respiratory, that there was increased synthesis of metallothionein, which is a protein induced in the presence of high concentrations of copper, which among other functions, is the main component of hemocyanin, the respiratory pigment crustaceans

Homeostatic plasticity is important in neurobiology for stabilising neuronal networks in the face of Hebbian forms of synaptic plasticity that are thought to mediate memory storage. Impairment of homeostatic plasticity has also been implicated in neurological diseases such as Rett syndrome and fragile X syndrome. Homeostatic plasticity can be achieved through scaling of the strength of synaptic connections between neurones or by changes in intrinsic excitability. While homeostatic plasticity has been studied mainly using in vitro preparations, it is for the most part not known whether changes of neural activity in vivo induce homeostatic changes. The molecular pathway responsible for homeostatic plasticity still remains unclear. In this thesis, I have used stereotaxic surgery to over express Kir2.1, an inwardly rectifying potassium channel, in vivo in the brains of adult mice. I show that the expression of Kir2.1 through adeno-associated virus (AAV) does not cause any adverse effects in the dentate gyrus nor the CA1 of the mouse hippocampus. I go on to use slice patch clamp methods to measure the change in electrical properties of granule cells in the dentate gyrus and pyramidal cells in CA1 caused by expression of Kir2.1. I show that the excitability of neurones expressing Kir2.1 was reduced compared to control neurones. By 2 weeks after virus injection the neurones showed homeostatic plasticity in response to Kir2.1 over expression. Interestingly, the mechanism of adaptation was different in different types of cells; dentate gyrus granule cells adapted through change in their intrinsic excitability, whereas CA1 pyramidal cells adapted by modifying the strength of their synaptic inputs. To establish whether induction of homeostatic plasticity is associated with changes in gene expression I used fluorescent activated cell sorting (FACs) to isolate pure population of neurones infected with viruses. I then sequenced RNA extracted from neurones expressing Kir2.1 and control neurones. Analysis of the RNAseq data revealed molecular candidates involved in homeostatic plasticity. In summary, I show that Kir2.1 over expression causes change in excitability and subsequent homeostatic plasticity in vivo. The mechanism of adaptation differs between cell types. RNAseq results identify novel candidates for future investigation.

To experience the world, we depend on the ability of our brains to process information. Problems can occur when communication between neurons is not regulated, and a significant enough loss of stability could lead to conditions such as migraine and epilepsy. Homeostatic plasticity is thought to constrain activity within physiologically useful ranges. Our lab uses the fruit fly neuromuscular junction as a model synapse to study homeostatic plasticity. Homeostatic potentiation and homeostatic depression are two forms of homeostatic synaptic plasticity. Expression of a dominant negative glutamate receptor subunit in the muscle impairs its sensitivity to glutamate and triggers an increase in the number of vesicles released per evoked potential, or quantal content. This increase in quantal content is called homeostatic potentiation. We found that homeostatic potentiation is a reversible process: quantal content returns to normal levels when expression of the dominant negative ceases. We additionally found that homeostatic potentiation can be ablated at high temperature. Overexpression of the Vesicular Glutamate transporter (VGlut) causes an increase in the amplitude of spontaneous events, leading to a corresponding decrease in quantal content, called homeostatic depression. It is unknown to what degree homeostatic potentiation and homeostatic depression may share regulatory machinery. We screened genes required for homeostatic potentiation in the neuron for additional roles in homeostatic depression. We found that certain genes involved in calcium regulation, such as the IP3 receptor and ryanodine receptor, showed a substantial decrease in evoked potential amplitude in a VGlut overexpression background.

Diabetes and insulin resistance are on the rise in the United States. Early detection and deployment of therapies has allowed for the reversal of pancreatic beta cell damage. Unfortunately, not all providers can offer the support to facilitating the required life style modifications. The introduction of clinical health consultants (CHC) as supplemental care has improved patient health for a variety of chronic diseases. Missing in the literature are studies investigating the correlation between the number of CHC interactions and improvement in biomarkers. The study utilized a non-experimental, retrospective study design to evaluate the relationship between the use between the use of CHCs and the number of CHC interactions, and the mean changes in glucose, hemoglobin A1c, insulin, proinsulin, C-peptide, and 1,5-anhydroglucitol, over a one-year period for patients presented with the opportunity to participate in CHC interactions. The subjects’ follow-up results were compared to their initial results for each group using the ANCOVA and one-way t-test. A statistically significant difference was detected between the mean change in BMI and the use of CHCs (p

Passive and active heat loading causes cardiovascular strain, which can have diverse and substantial effects. Thus, cardiovascular function is integral to work and heat stress tolerance, but recent hyperthermia and exercise literature has not emphasised this relationship, instead focusing on the roles of upper �critical� core temperature or rate of heat storage as primary mechanisms of fatigue. Therefore, the aim of this thesis was to examine some potential causes and effects of cardiovascular strain under heat stress, including potential strategies for attenuating that strain. Body precooling before exercise increases heat storage capacity; the primary mechanism by which attenuations in thermal and cardiovascular strain, and improved work capacity is thought to occur. However, no precooling study has utilised realistic airflow in the laboratory, possibly inflating its purported benefits. Therefore, Study One examined the cardiovascular, thermal, psychophysical and ergogenic effects of precooling with and without airflow in the heat (30�C, 50% rh). Ten males completed four trials in balanced order, comprising 60- min immersion in thermoneutral (35�C) or cool (24�C) water before cycling at 95% ventilatory threshold with airflow (~4.8 m�s⁻�) or no airflow, until exhaustion. Heart rate and mean core, body, and skin temperatures were attenuated for 15 min into cycling after precooling. Endurance time was extended by 30 � 23 min with airflow, and 16 � 15 min with precooling relative to control (28 � 12 min) but not further extended with strategies combined (29 � 21 min). Precooling removed 784 � 223 kJ�m⁻� (calorimetrically); less than the effect of airflow alone (1323 � 1128 kJ�m⁻�). Competition for blood between tissues is pronounced during exercise in the heat: skin and gut have marked increases and decreases, respectively. Gut ischemia affects epithelial tight junction integrity, allowing lippopolysaccharide ingress and immune responses. Bovine colostrum may attenuate gut permeability. Study Two (double-blind, placebo controlled) investigated the effects of aerobic fitness (7 highly fit, 8 moderately fit) and bovine colostrum on physiological and perceived strain, and performance during mixed-mode exercise; cycle 15 min at 50% maximal heart rate range (HRR), run 30 min at 80% HRR then 30 min self-selected paced before another 15 min cycle at the same work-rate. Airflow was graded to running speed. During the last cycle, blood pressure, stroke volume and total peripheral resistance were lower, heart rate and skin blood flow increased, and skin temperature was unchanged compared to the first cycle. Indices of fever response (IL-1β, TNF-α) were not evident during exercise, nor were those of blood-brain barrier permeability (S100β) or cognitive impairment (Stroop test). Neither bovine colostrum, nor higher fitness modified these measures. Moving to upright posture is orthostatically stressful and can initially decrease cerebral perfusion. Compression garments are used to assist venous return; while their effectiveness is unknown, they could reduce heat or orthostatic-induced hypoperfusion. Study Three investigated the cardiovascular and cerebrovascular responses to orthostatic stress with and without passive heating (+0.5�C). Fifteen participants completed two trials (compression v placebo garments) in balanced order. Cerebral autoregulation was assessed via 3-min stand, and via thigh cuff inflation. All participants experienced initial orthostatic hypotension upon standing in one or more trials, with 4/15 individuals experiencing presyncopal symptoms, aborting the standing protocol. In those who "fainted", reductions in blood pressure and partial pressure of end-tidal CO₂ reduced middle cerebral artery velocity. Neither training status nor compression trousers modified the responses. Collectively, cardiovascular strain to heat stress is attenuated when realistic airflow is provided. Increased cardiovascular strain does not inevitably result in clinical outcomes to heat stress. Higher fitness does not necessarily attenuate cardiovascular responses or higher tolerance to heat stress.

Freshwater organisms are known to maintain hyperosmotic internal conditions despite outward diffusive loss of ions. The freshwater common pond snail Lymnaea stagnalis faces this challenge while additionally attaining the necessary ions for calcification. These are the first documented assessments of the time and mode of recovery for ions lost due to full-body withdrawal in adults of this species. Additionally, this document reports on the physiological and developmental onset of embryonic calcification and the commencement of active acquisition of shell-forming ions from the surrounding environment. The effect of water chemistry and lead (Pb) exposure on embryonic growth, development, and calcium (Ca2+) acquisition was also tested. Pharmacological and water chemistry manipulations were used to determine mechanisms for embryonic Ca2+ and HCO3-/CO32- acquisition and the sensitivity of those pathways. Lastly, L. stagnalis, was shown to have a lowest effective concentration of <1.5 µg Pb l-1 using net Ca2+ uptake, growth, and developmental endpoints in laboratory and natural waters. This is the lowest effective concentration observed for any organism to date. One of the most insightful findings reported here is the interconnectedness of the pathways for acquisition of Na+ and Ca2+ through endogenous production of H+ and HCO3- via carbonic anhydrase-catalyzed hydration of metabolic CO2. The combination of high demand for Ca2+ throughout early life stages and periodic acute demands for Na+ recovery following extracellular fluid loss apparently causes L. stagnalis to be highly sensitive to changes in water chemistry, including [Pb] in the embryos, and possibly pH. The findings reported here warn of the need to establish freshwater environmental indicators and consider raising awareness of the threat of freshwater acidification, which may be greater than that of ocean acidification.

Substantial environmental contamination has occurred from coal tar creosote and pentachlorophenol (C5P) in wood preserving solutions. The present studies focused on the characterization and remediation of these contaminants. The first objective was to delineate a sequence of biological changes caused by chlorinated phenol (CP) exposure. In Clone 9 cells, short-term exposure to 10 ?M C5P decreased pH, GJIC, and GSH, and increased ROS generation. Long-term exposure caused mitochondrial membrane depolarization (25 ?M), increased intracellular Ca2+ (50 ?M), and plasma membrane depolarization (100 ?M). Cells were affected similarly by C5P or 2,3,4,5-C4P, and similarly by 2,3,5-C3P or 3,5-C2P. Endpoints were affected by dose, time, and the number of chlorine substituents on specific congeners. Thus, this information may be used to identify and quantify unknown CPs in a mixture to be remediated. Due to the toxic effects observed due to CP exposure in vitro, the objective of the second study was to develop multi-functional sorbents to remediate CPs and other components of wood preserving waste from groundwater. Cetylpyridinium-exchanged low pH montmorillonite clay (CP-LPHM) was bonded to either sand (CP-LPHM/sand) or granular activated carbon (CP-LPHM/GAC). Laboratory studies utilizing aqueous solution derived from wood preserving waste indicated that 3:2 CP-LPHM/GAC and CP-LPHM/sand were the most effective formulations. In situ elution of oil-water separator effluent indicated that both organoclay-containing composites have a high capacity for contaminants identified in wood preserving waste, in particular high molecular weight and carcinogenic PAHs. Further, GAC did not add substantial sorptive capacity to the composite formulation. Following water remediation, the final aim of this work was to explore the safety of the parent clay minerals as potential enterosorbents for contaminants ingested in water and food. Calcium montmorillonite and sodium montmorillonite clays were added to the balanced diet of Sprague-Dawley rats throughout pregnancy. Based on evaluations of toxicity and neutron activation analysis of tissues, no significant differences were observed between animals receiving clay supplements and control animals, with the exception of slightly decreased brain Rb in animals ingesting clay. Overall, the results suggest that neither clay mineral, at relatively high dietary concentrations, influences mineral uptake or utilization in the pregnant rat.

Title from PDF of title page (University of Missouri--Columbia, viewed on Mar. 15, 2010). The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Dissertation advisor: Dr. J. David Robertson. Vita. Includes bibliographical references.