Human epidemiological studies have provided compelling evidence that prenatal environmental events are associated with significantly increased risks of developing neuropsychiatric disorders in adulthood. This phenomenon has been studied and known as ‘fetal programming of adult diseases’. According to the theory, environmental insults occurring at critical periods of pregnancy can permanently alter the structure and physiology of the developing organs, and may lead to adverse functional consequences that manifest later in life. It has been suggested that increased maternal glucocorticoids may be one common mechanism through which various environmental insults can affect on the developing fetus. Therefore, in this thesis, I studied the long-term prenatal glucocorticoid overexposure effects on the functioning of the brain and investigated possible molecular mechanisms underlying the programmed effects. By using a rat model of prenatal dexamethasone (100μg/kg per day) administered to pregnant dams during the last week of gestation, I found that prenatal dexamethasone overexposure significantly reduced birth weight in both male and female offspring. I also assessed the consequences of prenatal dexamethasone treatment on stress response and cognition in the adult male offspring by using a number of physiological and behavioural measures. Overall I found no evidence of prenatal dexamethasone treatment effect on the hypothalamic-pituitaryadrenal (HPA) axis activity and behavioural responsivities to an acute stress in adult male offspring; however, prenatal dexamethasone exposure affected hippocampus and prefrontal cortex related cognition in the adult offspring, including contextual fear conditioning and behavioural flexibility on water maze tasks. I further explored possible molecular mechanisms that underlie the behavioural phenotypes of the prenatal dexamethasone rat model, and found altered brain gene expression with possible implications in synaptic plasticity and retrotransposon activities. Collectively, my current study suggests that prenatal dexamethasone exposure induces long-term programming effects on adult behaviour, associated with altered gene expression profile of the brain in the rat offspring. These results provide further evidence that prenatal glucocorticoid exposure may affect the development of the brain and its influence may endure into adulthood.

Lipids are the main constituents of the cell membrane and maintain its fluidity. Plants undergo various changes in lipids under environmental stresses and alter the membrane fluidity and permeability. Membrane lipids mostly contain a polar or neutral head group and fatty acid tails that vary in length and degree of unsaturation. The composition of membrane affects its physicochemical properties and ability to tolerate stress. The moss Physcomitrium patens is an early land plant with unique ability to tolerate stressors like cold and dehydration. During its life cycle, for the most part, mosses remain as gametophytes, multiplying asexually. The period from germination of spores into filamentous protonema, which give rise to gametophyte is transient. They enter reproductive sporophytes stage only under cold temperatures. Because of the diverse roles of these developmental stages and the time span they are exposed to the environment, we hypothesized that these stages share distinct lipid content and composition. To this extent, using LC-MS/MS methods we carried out lipidomic analyses of five developmental stages of the moss. We identified and quantified the major and minor lipid classes (types) and their acyl composition of protonema, early, mid and late gametophyte and sporophyte tissues. Galactolipids, which typically occur in the plastid were predominant in green tissues and thus most abundant in the vegetative tissues but not in sporophytes. Throughout the life cycle, among the phospholipids, phosphatidylcholine was the abundant lipid, a feature that is typical of plant membranes. Sporophyte tissues, however, were distinct from gametophyte and protonema and also other vascular plants with high amounts of phosphatidic acid (PA). In plants, PA typically accumulates in response to stress; it is likely that the low temperature cue necessary for sporophyte formation is associated with spike in PA and needs further investigation. In comparing the acyl composition of the various lipid classes, we identified that in addition to 34C and 36C lipids, moss lipids also contain 38C and 40C, which are not represented in vascular plants. We predict that the occurrence of long-chain, highly unsaturated lipids might contribute to the dynamic nature of the membrane and stability under stress. This study serves as a primary resource to further investigate the role of specific lipids and acyl groups in maintaining membrane properties. Overall, it aids to our understanding of the evolution of stress tolerance in early land plants that coped through harsh environmental conditions during their transition from water to land.

archives@tulane.edu / Alcoholic and nonalcoholic fatty liver disease is projected to be the most common cause of liver disease in developing countries. The main significant risk factors are obesity, diabetes mellitus type 2, cardiovascular disease, and dyslipidemia. Louisiana is ranked seventh in liver cancer diagnoses and ranked sixth in the leading cause of death. Recent findings indicated that multifaceted stress response due to the accumulation of fatty acids from the diet is the driving force of disease progression. We sought to study multifaceted integrated stress response (ISR) in liver cells cultured with saturated fatty acids. Understanding the process that ISR takes to either induce or inhibit autophagy, self-eating machinery, in strongly permissive HUH 7.5 cells is vital when treating liver abnormalities. The major protein kinase, P-EIF2 alpha, was the targeted factor contributing the most to autophagy due to its functional link to the endoplasmic reticulum, mitochondria, and cellular membrane by further assessment using the inductive drug, Sephin 1. HUH, 7.5 liver cells are treated with increasing amounts of palmitic acid for 24 hours in DMEM with 10% FBS. ISR activated after substantial cellular damage leading to autophagy impairment. The cell culture was assessed for lipid accumulation, and the expression of PKR, IRE1 alpha, PERK, ATF6, P-EIF2 alpha, HRI, MTORC1, GCN2, P62, and LC3B was achieved by immunoblot analysis. Membrane fluidity PKR, lysosomal MTORC1, and protein synthesis GCN2 activated to elicit an integral response to the ISR pathway. Endoplasmic reticulum protein kinases induced in response to UPR activation lead to an integration of the P-EIF2 alpha pathway. Mitochondrial stress heme regulated inhibitor proliferated to provoke an activation in the significant protein kinase leading to autophagy impairment. The P-EIF2 alpha kinase invoked autophagic deficiency even when dephosphorylation was prevented by Sephin 1 drug treatment. ISR constrained autophagy in the liver-derived cell line due to the accumulation of the toxic saturated fatty acid. Keywords: palmitate, autophagy, fatty liver disease, integrated stress response, Sephin 1 / 1 / Glory Ogunyinka

Indiana University-Purdue University Indianapolis (IUPUI) / The protein kinase General control non-derepressible-2 (GCN2) is a key regulator of the Integrated stress response that responds to various stress signals, including nutritional deprivation. As a result of high levels of uncharged tRNAs during amino acid depletion, GCN2 phosphorylates serine-51 of the α subunit of eukaryotic initiation factor-2 (eIF2), a translation factor that delivers initiator tRNA to ribosomes. Phosphorylation of eIF2α inhibits general translation, which conserves energy and nutrients and facilitates reprogramming of gene expression for remediation of stress damage. Phosphorylation of eIF2α also directs preferential translation of specific transcription factors, such as ATF4. ATF4 reprograms gene expression to alleviate stress damage; however, under chronic stress, ATF4 directs the transcriptional expression of CHOP, which can trigger apoptosis. Because multiple stresses can induce eIF2α phosphorylation and translational control in mammals, this pathway is referred to as the Integrated stress response. GCN2 and CHOP are subject to alternative splicing that results in multiple transcripts that differ in the 5'-end of the gene transcripts. However, the effect of the different GCN2 and CHOP isoforms on their function and regulation have not been investigated. Our data suggests that GCN2 is alternatively spliced into five different transcripts and the beta isoform of GCN2 is most abundant. Also alternative splicing of CHOP creates two CHOP transcripts with different 5'-leaders encoding inhibitory upstream open reading frames that are critical for translational control of CHOP during stress. This study suggests that alternative splicing can play an integral role in the implementation and regulation of key factors in the Integrated stress response.

SABP2-interacting protein 428(SIP428) is a SIR2-type deacetylase, also called sirtuins. The SIP428 proteins belong to a family of NAD+-dependent deacetylase enzyme that was identified in tobacco. SABP2 is an important methyl esterase enzyme that catalyzes the conversion of methyl salicylic acid (MeSA) into salicylic acid (SA) during the pathogenic challenge. Accumulation of SA induces systemic acquired resistance (SAR), a broad-spectrum defense mechanism in other uninfected distal parts of the plant. Sirtuins play diverse roles in DNA repair, apoptosis, and stress responses. Cellular proteins are known to undergo posttranslational modifications such as methylation, phosphorylation, and ubiquitination. A more recent addition to the list is acetylation. Protein acetylation is a reversible modification that plays role in regulating transcription, activation, and deactivation of certain pathways by transferring acetyl group to lysine residues. This change neutralizes the positive charge of the amino group thereby affecting the biological function of the affected proteins. Preliminary research has shown that SIP428 is a non-histone deacetylase. To understand better about the role of SIP-428 in plant physiology and how it plays a vital role in SABP2 signaling pathway we will be using transgenic tobacco plant in which the expression of SIP 428 has been silenced/knocked down.

Attentional control and attentional bias are important factors that contribute to the development and maintenance of anxiety disorders (Eysenck, Derakshan, Santos, & Calvo, 2007; Van Bockstaele et al., 2014). However, the effects of attentional control and attentional bias on the relationship between anxiety and stress response is understudied. Further, much of the research to date has relied on self-report measures of attentional control and stress response, representing a significant limitation. The current study addressed these problems and examined the relationship between attentional control, attentional bias, anxiety, and stress response. First, this study examined the relationship between self-report and performance-based measures of attentional control and stress response with anxiety. Study results found poor agreement between attentional control measures, good convergence between self-reported distress and physiological distress, and a negative association between anxiety and self-reported attentional control and stress response. Second, results showed that attentional control and attentional bias were not significant moderators of the relationship between anxiety and stress response. Explanation of study findings and future directions are discussed.

Dopamine (DA) is a catecholamine that is involved in several neural functions such as modulation of locomotor behaviors, arousal states to appetitive aversive learning and memory. The relationships between DA, stress response and aging are unclear. This thesis examines numerous physiological, behavioral and biochemical parameters following perturbations in DA synthesis and transport in the Drosophila melanogaster model system. Intriguingly, elevated DA pools appear to confer protection, while depleted DA levels or transport increase susceptibility to oxidative insult. Resistance to oxidative stress in mutants with elevated DA levels was attributed to a significant up-regulation of glutathione S-transferase Omega-1. A sexually dimorphic response in aging and senescence characteristics was also recorded among the mutants tested, but no discernable role of DA in these characteristics was observed. Taken together, these results point to a key role played by DA in stress response, which might have implications to age-related neurodegenerative diseases.

The Crocus Sativus flower gives rise to the Saffron spice which contains five active ingredients known as crocin, crocetin, picrocrocin and safranal. The rich antioxidant containing components of Saffron have been shown to cross the blood brain barrier by passive transcellular diffusion, making them effective in relation to treating neurological disorders. In order to test the effects of Saffron on memory and anxiety, Zebrafish Danio Rerio were placed into two groups and put through a series of assessments including a three compartment memory tank and a dive tank. Zebrafish continue to be at the forefront of behavioral and pharmacological studies due to their ability to display real time effects of substances in the environment. Many strong similarities have been drawn between zebrafish’s neuroanatomy and physiology in comparison to mammals, making them a reliable tool in assessing environmental toxicity. Zebrafish have shown to be a great asset in drug screenings and preclinical trials due to their sensitivity to pharmacological stimulants and fast phenotypic responses. Saffron was administered to a test group via skin absorption, while the control group was transported directly from their habitat to the assessment tanks. The three compartment memory tank consisted of a middle, right, and left chamber separated by movable dividers. Subjects were initially placed in the center compartment and given a choice of swimming either towards the right or left sections. The two sides of the tank contained visual cues which included photos of shapes taped to the wall. The choice of swimming towards the correct compartment was rewarded by the subject being reunited with their tank mates; while making the incorrect decision led to adverse stimuli such as reduction of swim area. Subject memory was tested by their ability to promptly swim towards the intended side during each trial. It was hypothesized that subjects in the test group will learn to swim in the correct compartment significantly faster than the control group. The anxiety evaluation was conducted by individually placing subjects from the control and test groups into a dive tank. The dive tank utilizes vertical distribution in a novel environment to measure the behavioral response in Zebrafish. Once placed in the tank, the subjects will combat their inherent nature of seeking protection in an open environment. During each trial, subject movements, behavior, and activity were tracked and analyzed by the Nolds Ethovision software. It was hypothesized that Saffron dosed Zebrafish will show a reduction in anxiety by increasing their vertical swimming and exploration significantly faster than those in the control groups.

Indiana University-Purdue University Indianapolis (IUPUI) / In response to environmental and physiological stresses, cells impose translational control to reprogram adaptive gene expression and conserve energy and nutrients. A central mechanism regulating translation involves phosphorylation of the a-subunit of the eukaryotic initiation factor -2 (p-eIF2a), which reduces delivery of initiator tRNA to ribosomes and represses global protein synthesis. The pathway featuring p-eIF2a is called the integrated stress response because it involves multiple related eIF2a kinases, each responding to different stress arrangements. While p-eIF2a limits global protein synthesis, a subset of mRNAs are preferentially translated in response to p-eIF2a. Preferential translation of stress adaptive mRNAs is regulated by upstream opening reading frames (uORFs) present in the 5’-leader of these transcripts. In most cases uORFs are inhibitory in nature, but in some case uORFs can instead promote the translation of the downstream CDS. This study is focused on preferential translation of the gene Inhibitor of Bruton’s Tyrosine Kinase-alpha (IBTKa) in response to endoplasmic reticulum stress. The human IBTKa gene encodes a 1353 amino acid residue protein, along with a 5’-leader featuring predicted canonical uORFs. Among the four predicted uORFs, the 5'-proximal uORF1 and uORF2 are phylogenetically conserved among mammals and are well translated as judged by reporter assays, whereas uORF3 and uORF4 are not conserved and are poorly translated. In addition to the uORFs in the IBTKa mRNA, a phylogenetically conserved stem-loop (SL) of moderate stability is present 11 nucleotides downstream of uORF2. Using luciferase reporter assay, the uORF2 and SL were shown to function together to repress the translation of human IBTKa. In non-stressed conditions, the SL combined with uORF2 are critical for reducing ribosomes from reinitiating at the IBTKa coding sequence (CDS), thus repressing IBTKa expression. Upon ER stress and induced p-eIF2a, the more modestly translated uORF1 facilitates the bypass of the inhibitory uORF2/SL to enhance the translation of main CDS of IBTKa. This study demonstrates that uORFs in conjunction with RNA secondary structures can be critical elements that serve as a “bar code” by which scanning ribosomes decide which mRNAs are preferentially translated in the integrated stress response. / 2023-10-03

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