Ana Carranco

Impact of Exercise on Stress Responses in an Undergraduate Student Population

When the body perceives a stressor, the hypothalamic-pituitary-adrenal (HPA) axis is activated leading to synthesis and release of cortisol from the adrenal cortex. Exercise is a physical stressor that increases cortisol levels in the body. Studies have considered exercise as a self-regulatory strategy to adapt to stressful events. Performance athletes have previously been shown to have higher baseline cortisol levels. The effects of exercise on cortisol levels in the general population is less clear. The aim of this study was to examine the relationship between exercise and physiological responses to stress in college students.
Stress was induced in the undergraduates (with the approval of the Union College Human Subjects in Research Committee) using the Trier Social Stress Test (TSST). Saliva samples were taken four times during the study; pre TSST, immediately post TSST, and 5 min and 30 min after the TSST. Salivary cortisol was measured using an enzyme-linked immunosorbent assay. In addition to physiological measures, participants completed the brief mood introspection scale, the revised undergraduate hassles scale, and a self-reported exercise survey describing the frequency and intensity of their exercise habits. Students also completed a perceived functional ability questionnaire (PFA) rating their ability to jog, walk or run over a given distance which reflects participants’ physical activity and fitness.
Participants were divided into two groups based on the mean PFA score (7.5). The results showed that participants with a PFA score above the mean had lower baseline cortisol on average, compared to participants with a PFA score below the mean. The results from this study suggest that physically active participants have lower baseline cortisol levels when compared to their peers who are less physically active. Together these data indicate the need for specific stress-reduction programs for college students that include physical activity as one aspect of the intervention.

Justin Fleischer

Mutation of Follicle Stimulating Hormone Receptor Putative Caveolin Binding Motif Results in Altered Signaling

The human follicle stimulating hormone receptor (hFSHR) is a glycoprotein hormone receptor belonging to the g protein-coupled receptor family. It is important in both male and female reproductive processes; defects in hFSHR can lead to infertility, delayed puberty, reduced muscle bulk, and osteoporosis. Work in other labs has shown that GPCRs can be localized to microdomains located within the cell membrane called lipid rafts. These regions are highly resistant to detergents because of the high concentration of sphingolipids and cholesterol. Also present within these domains is an intracellular protein, caveolin, that is essential for the function of the microdomain. Our lab has shown that hFSHR, like many other GPCRs, localizes to lipid raft domains. We hypothesize that interaction with caveolin is important in the raft residency of hFSHR through a putative caveolin binding motif (CBM). The interaction between caveolin and hFSHR in these microdomains may regulate intracellular pathways activated by the receptor.
To test our hypothesis, we have constructed CBM mutants and are in the process of creating stable cell lines expressing the mutant receptors to study the effects of mutating the key phenylalanine residues of the CBM. Interestingly, wild type hFSHR presents as a doublet on a western blot while the F479L mutant only has a single band. We hypothesize that absence of the second band for the F479L mutant is the result of altered glycosylation of the receptor. To compare the trafficking and function of the hFSHR F479L mutant we tested the activation of the protein kinase A (PKA) and p44-MAP kinase (MAPK) pathways. The mutated receptors qualitatively showed normal p44-MAPK signaling in response to hFSH, indicating that they are reaching the cell surface and are capable of binding hormone and coupling to signaling partners. We have observed a subtle difference in the phosphorylation of downstream targets of PKA between the wild type and mutant receptors suggesting that there might be a difference in the ability to activate these downstream targets. Studying the interaction between hFSHR and caveolin has the potential to develop new contraceptive antagonists and alternative agonists to regulate fertility.

Greg Geisel

Quantification of hFSHR Signaling to Determine Dependence of Lipid Raft Residency

Human follicle stimulating hormone (hFSH) is a gonadotropin responsible for regulating reproductive systems by stimulation of Sertoli cells in males and granulosa cells in females. The hFSH receptor (hFSHR) is a seven transmembrane receptor that belongs to the G protein coupled receptor family. The receptor is functionally connected to a G protein on the inside of the cell. Once FSH activates its receptor, a cascade of signaling begins, resulting in the activation of adenylyl cyclase which increases the intracellular levels of cAMP. In addition, hFSHR stimulation also activates the p44/42 MAP kinase. The spike in cAMP activates the enzyme protein kinase A (PKA) which triggers a series of downstream effectors resulting in follicular stimulation and gametogenesis.
Previous work in the Cohen Lab has shown that hFSHR is located in cholesterol-rich, detergent-resistant microdomains known as lipid rafts. In an HEK293 cell line stably expressing hFSHR, disruption of lipid rafts by the cholesterol chelator methyl beta-cyclodextrin (MβCD) interferes with PKA activation. Current research is focused on the relevance of hFSHR lipid raft residency in the human granulosa cell line hGrC1; focusing in particular on the activation of signal transduction pathways by hFSHR. The goal was to develop an enzyme-based, quantitative, non-radioactive assay for cAMP stimulation that could be used to study the effects of lipid raft disruption by MβCD on hFSHR signaling in hGrC1 cells. The β-galactosidase assay showed quantitative dose-dependent responses to hFSH which indicated that it should be useful for testing MβCD to further determine lipid raft dependence of hFSHR signaling. Studying the regulation of signaling by hFSHR provides more insight into the receptor function and potentially represents new approaches to contraception or treatment of infertility.

Brianna Godlewski

The Linkage Between 11β-hydroxysteroid Dehydrogenase Type 1 Enzyme Hyperactivity Polymorphisms & Metabolic Syndrome

A diagnosis of Metabolic Syndrome (MSX) requires patients to present with three or more of the following symptoms: elevated fasting blood glucose levels, elevated serum triglyceride levels, low serum HDL levels, elevated blood pressure, and truncal obesity. This pathology shares many similarities with Cushing’s Syndrome (CS) but a diagnosis of CS requires hypercortisolemia. This similarity has led our lab and others to hypothesize that MSX may be a Cushingoid-like state caused by hyperactivity of the cortisol reductase enzyme, also know as 11β-hydroxysteroid dehydrogenase, type 1 (11β-HSD1) . The 11β-HSD1 enzyme is responsible for the conversion of inactive cortisone to cortisol, and works primarily in the adipose tissue. Using an allele specific PCR assay for previously identified single nucleotide polymorphisms (SNP) within the 11β-HSD1 enzyme (rs12086634 and rs846910), we have observed these polymorphisms present at a higher frequency in a population of patients seeking bariatric (weight-loss) surgery when compared to a control population or reported frequencies. In our patient population, the frequency of heterozygosity for the rs12086634 SNP was nearly 50% higher than the reported frequency in the general population, suggesting that this polymorphism may contribute to increased obesity and the MSX metabolic profile by increasing the localized cortisol in adipose tissue. Greater understanding of the interplay between these and related single nucleotide polymorphisms can help physicians and patients make more informed decisions about treatment options for obesity and metabolic syndrome.

Justine Monthony

Fitness and Exercise as Linked to Overall Stress and Physiological Stress Responses

Exercise has been shown to correlate with physiological response reduction to stress, but there is not much research on short-term stress hormones for this response. For this thesis, I hypothesized that exercise and fitness levels would be negatively correlated with perceived stress and with physiological stress from a standard stressor. In the study, undergraduate college participants answered questions about their chronic stress, current mood, past week’s physical activity (amount and intensity), perceived physical fitness, and demographic characteristics. In addition, participants engaged in a social stress task, where they were given five minutes to prepare a five minute speech with no notice or resources that they then gave in front of peer who they were told was analyzing and recording them. Then they were asked to do a mental math task aloud for five minutes. To measure physiological reactions, participants’ blood pressure, heart rate, and salivary amylase levels were assessed at baseline, immediately after the stressor, ten minutes after the stressor, and thirty minutes after the stressor. Participants who had a higher perceived fitness reported significantly lower stress on a measure of everyday student hassles. Participants were found to be significantly emotionally stressed from the stress-inducing task. The correlations between exercise engagement, perceived fitness, and amount of induced stress were analyzed, but participants who exercised more or had higher perceived fitness did not show significantly lower amylase, heart rate, or diastolic blood pressure responses to the stressful task. Participants who exercised more did show significantly higher systolic blood pressure than participants who did not exercise as much, which prompts a call for further research. When comparing those high and low in perceived fitness, there was a pattern in which the amylase response was lower in the high-fitness groups; however these results should be interpreted with caution. These findings reinforce the theory that exercise reduces stress, but questions remain as to the effect of exercise and fitness on the physical response.

Shiraz Mumtaz

Does Exercise Help Improve Recognition Memory After Stressor Exposure?

The purpose of this study was to investigate how stress affects cortisol levels and memory performance, and whether it affects them differently for those who exercise more. Participants first learned a list of items. The next day, they were placed in two groups: The control group (recognition memory test given before the Trier Social Stress Test) and Stressor Group (recognition memory test given after the Trier Social Stress Test). The impact of the stressor was measured via samples of cortisol levels taken during various points during the course of the study. I am most interested in analyzing the change in concentration from sample 1 to sample 2, as sample 2 was taken after the exposure to the stressor. This analysis will help us determine if participants were convincingly stressed. Regarding memory performance, it was hypothesized that participants who exercised more would respond better to the stressor, and therefore perform better on the memory test compared to those who didn’t exercise as much. The results and their relevance to the general college student population will be discussed.

Jenna Pradhuman

Use of CRISPR to Develop a Follicle Stimulating Hormone Receptor Knockout in Human Granulosa Cells to Study Lipid Raft Residency

Human follicle stimulating hormone (hFSH) is a protein hormone responsible for stimulating the gonads and is necessary for regulating reproductive systems in both females and males. The actions of hFSh are carried out by the hFSH receptor (hFSHR), a seven transmembrane receptor that belongs to the G protein-coupled receptor family. Once FSH activates its receptor, the G protein inside the cell that is associated with the receptor is activated and starts a cascade of signaling that results in activation of protein kinase A (PKA) and the p44/42 MAP kinase (MAPK). The activation of these secondary proteins is responsible for follicular stimulation in females and gametogenesis in males and females. Previous work in the Cohen Lab has shown that hFSHR is located in cholesterol and sphingolipid enriched microdomains referred to as lipid rafts. These microdomains are detergent resistant membranes that “float” in sucrose gradients. Current research is focused on the relevance of hFSHR lipid raft residency in the human granulosa cell line hGrC1; focusing in particular on the activation of signal transduction pathways by hFSHR. To study this in more detail, we are creating a human granulosa cell line where the hFSHR gene has been deleted, known as a knockout mutant. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a revolutionary gene editing technique that can create knockouts by precisely targeting a 20 nucleotide sequence of the genome using the Cas9 protein. This knockout line will enable us to study lipid raft residency and signaling of mutant hFSHR proteins in the native cell background. By studying the regulation of hFSHR signaling we hope to learn more about the receptor’s function and identify new ways to modulate the receptor for novel approaches to contraception or treatment of infertility.

Justin Salm

Investigating the effects of polymorphisms of the glucocorticoid receptor on obesity and depression

Previous studies in this lab have investigated the complex interaction between cortisol and its receptor, the glucocorticoid receptor (GR) by exploring the relationship between Cushing’s Syndrome (CS) and metabolic syndrome (MetS). While CS and MetS are phenotypically similar, CS is found in conjunction with elevated cortisol levels, while MetS occurs in the absence of elevated cortisol. We hypothesized that the similar phenotypes are a result of glucocorticoid hypersensitivity mutations in the GR. The lab identified two single-nucleotide polymorphisms (SNPs) in the GR, N363S and Bcl I, which both lead to cortisol hypersensitivity and characterized their prevalence in a population of obese patients.
The scope of the current research has expanded to also explore the relationship between depression and the GR. Studies suggest that mood disorders including bipolar disorder (BD), major depressive disorder (MDD), and unipolar depression result from hypothalamic-pituitary-adrenal (HPA) axis dysregulation, leading to both high and low cortisol levels. Both Cushing’s Syndrome, a result of hypercortisolism, and Addison’s disease, a result of hypocortisolism, are partly characterized by neuropsychiatric depressive moods. The Corticosteroid Receptor Hypothesis suggests that depression can be caused by not only by cortisol dysregulation but also by dysfunction of the glucocorticoid receptors. GRs, dependent on serum cortisol or cortisone levels, work in tandem, affecting HPA regulation, due the inhibitory nature of cortisol feedback on the hypothalamus and anterior pituitary.
In addition to the previously studied GR hypersensitivity SNPs, which included Bcl I and N363, two additional GR hypersensitivity polymorphisms (RS296 and RS770), and two GR resistance polymorphisms (TthIII1 and ER/EK) were investigated. The GR polymorphisms were investigated in populations of bariatric patients and Union College students. Based on the results of the allelic frequencies found in the bariatric and student/control populations, we can hypothesize what this same data would look like within a depressed patient population. Improving the understanding of the genotypic causes of HPA axis dysregulation in psychiatric disorders may lead to biomarkers for diagnosis and open new treatment pathways.

Research Practicum Students

Naaila Phoenix ’17

Liposome Encapsulation for Drug Delivery

Extensive research has been conducted to determine an optimal method of drug delivery. Pills are not always effective for delivering drugs due to their degradation in the stomach and premature release of medication to unintended parts of the body. To combat these issues, we tested two different anticancer drugs: Olaparib and Cordycepin. Olaparib is a poly (ADP-ribose) polymerase (PARP) inhibitor that disables DNA repair, leading to death in BRCA gene-deficient tumor cells. Cordycepin is an RNA inhibitor derived from the fungus Cordyceps militaris. Our hypothesis was that these drugs would exhibit greater efficacy after incorporation into hydrophobic liposomes. Encapsulation would allow the drugs to more easily enter the cancer cells because of the liposome’s ability to fuse with the cell membrane. In separate trials, drugs were packaged in different liposomal preparations using 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) or a mixed chain phosphatidylcholine (PC) and cell death was observed. MCF-7 breast cancer cells showed the most cell death after 24 hours of treatment before proliferation resumed, indicating that the Olaparib and Cordycepin may not be killing the cells, but delaying proliferation. HepG2 liver cancer cells exhibited virtually no cell death in DOPC trials but high cell death in PC trials, indicating PC may make a more effective liposome. Future experiments will test increased dosages as well as drug combinations encapsulated in phosphatidylcholine. This has the potential to cause much less harm to the body and possibly kill a wide range of cancers.