Adam Bender
The Effect of Circadian Rhythm on Cortisol and Perceived Stress Correlations
Stress is a known trigger of the Hypothalamus-Pituitary-Adrenal Axis (HPA), which leads to the production and secretion of the catabolic steroid cortisol by the adrenal gland. Since cortisol production is affected by stress, it follows that a high self-perception of stress would be correlated with high blood and saliva cortisol levels. Surprisingly, the literature generally shows a gap in identifying this correlation, perhaps because of the interconnected nature of endocrine pathways. New experimental methods that control for the effects circadian rhythm have shown limited success in demonstrating this correlation. Our purpose is to continue exploring the relationship between cortisol, perceived stress, and circadian rhythm. This will be done by comparing the results of the Trier Inventory for Chronic Stress (TICS) survey, to the cortisol concentration in saliva samples taken during the cortisol awakening response (CAR). When these results are analyzed based on individual chronotype (whether someone is a morning or evening person based on the Lark or Owl Questionnaire), we hypothesize that a greater correlation between perceived stress and cortisol will be exhibited. Research to determine accurate correlations between perceived stress and cortisol is important when determining the effect of psychological stress on the body, through its association with cortisol.
Evan Leibovitz
Lack of Glucocorticoid Receptor Hypersensitivity-Related Polymorphisms in an Undergraduate Population
Cushing’s Syndrome (CS) and Metabolic Syndrome (MetS) share many phenotypic characteristics. The main diagnostic criterion for CS is elevated cortisol which is not seen in MetS. We therefore hypothesize that MetS, in some cases, represents a CS like state without elevated cortisol due to hypersensitivity of the glucocorticoid receptor (GR). Hypersensitivity of the GR correlates with polymorphisms in the receptor sequence. One such mutation abolishes the BclI restriction site in the second intron of the receptor. The purpose of this study was to investigate the presence of this polymorphism in a population of college students. Student DNA was isolated using a chelex isolation method from buccal cells and a specifically designed novel set of allele specific primers were used to detect the polymorphism using polymerase chain reaction (PCR). In a population of white, non-obese, college aged students the mutant allele frequency was 0.31, however these mutant alleles did not correlate with BMI. This finding correlates with what is seen in the general population. Future research will investigate correlations between polymorphism presence and symptom severity in a population of morbidly obese adults seeking bariatric surgery as well as the interaction of the BclI polymorphism with other hypersensitivity causing polymorphisms.
Allyson Staats
The Hormone Dependence of the Interaction Between the Human Follicle Stimulating Hormone Receptor and Caveolin
FSH is a pituitary hormone targeting granulosa cells in the ovaries to promote follicle development. In males, FSH targets the Sertoli cells in the testes to stimulate sperm development. This hormone acts through its receptor (hFSHR), a g protein-coupled receptor embedded in the cell membrane. Previous work in our lab has shown that the protein caveolin co-immunoprecipitates with hFSHR. Caveolin exists in caveolae, specialized lipid rafts within the cell membrane that are thought to play a role in cell signaling. It is unknown if the interaction between hFSHR and caveolin is hormone dependent. This question was addressed by observing co-localization through fluorescence microscopy and co-fractionation on a sucrose gradient. Transfection of cDNA for a caveolin-GFP fusion into a human granulosa cell line (hGrC1) was used to characterizeco-localization. Overlapping fluorescence signals from caveolin-GFP and hFSHR would indication that the proteins are in proximity. Sucrose gradients separate proteins by density using centrifugation. Using an HEK293 cell line expressing hFSHR, sucrose density gradient experiments were performed to determine if co-fractionation of the proteins is hormone dependent. Infromation about the colocalization of hFSHR and caveolin can be applied to practical uses such as new forms of contraception or improved assisted reproductive technologies.
Jack Steinharter
Mutagenesis of the putative Caveolin Interaction Motif in the Human FSH Receptor
Human follicle-stimulating hormone (FSH) is a product of the gonadotrophs of the anterior pituitary which plays a large role in pubertal maturation, development and reproductive processes of the body. FSH acts on a g protein-coupled receptor (GPCR) called hFSHR which has a putative caveolin binding motif (CBM). The CBM is thought to localize hFSHR to a form of lipid raft called caveolae. The hFSHR CBM is a particular pattern of four aromatic amino acids, ΦΧΦXXXXΦXXΦ, where Φ is an aromatic amino acid (phenylalanine in the case of hFSHR). Using splicing by overlap extension to create a point mutation, cDNA containing four separate mutations at each phenylalanine were synthesized. These four plasmids were transfected into human embryonic kidney cells (HEK 293) Western blot was used to detect protein expression and immunofluorescence microscopy was used to check for cell surface expression using a monoclonal antibody specific to hFSHR. FSH treatment was used to determine if the point mutations affected signal transduction pathways. By understanding the interaction between hFSHR and caveolin we will be able to further understand the effect of lipid rafts on hFSHR signalling which is so integral in human development and reproductive processes.
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Warren Thompson
Investigating the Spare Receptor Hypothesis: Ligand-Independent Phosphorylation of the Follicle Stimulating Hormone Receptor
Follicle stimulating hormone (FSH) acts through a g-protein coupled receptor, the FSH receptor (FSHR), to promote growth of reproductive organs and to stimulate reproduction. The phosphorylation of the FSHR is crucial for the regulation of this signaling pathway: here, we investigate the role of receptor phosphorylation in explaining the spare receptor hypothesis. We hypothesize that ligand-independent phosphorylation of FSHR can explain the excess of receptors found in granulosa and Sertoli cells. Wild type hFSHR contains an extracellular hormone binding domain and a transmembrane signaling domain. A mutant transmembrane-only form of hFSHR was generated and expressed in a human embryonic kidney cell line (HEK 293) stably expressing wild type FSHR. Expression of the mutant receptor was characterized by western blot and flow cytometry. Phosphorylation of the wild-type and mutant FSHR was characterized following FSH treatment. Phosphorylation of the mutant receptor, which has no hormone binding domain, signifies that phosphorylation can occur in a ligand-independent manner. This may also indicate the importance of receptor dimerization in receptor phosphorylation, and lead to new information about regulating the reproductive pathway.
Sara Zeltsman
Investigation of FSH Responsiveness in a Granulosa Cell Line
The follicle stimulating hormone receptor (FSHR) is a g protein-coupled receptor found in granulosa cells in the ovary. FSHR activation by FSH leads to proliferation and maturation of ovarian follicles towards ovulation; understanding this pathway could lead to developments in regulating fertility. Previously, our lab studied FSHR using an HEK293 cell line stably transfected with FSHR cDNA. More recently we have investigated an immortalized human granulosa cell line (hGrC1) to study FSHR signaling in a more physiologically relevant context. The stage of follicular development represented by the hGrC1 cells is currently not known. Based on previous data from our lab, the question arose of whether hGrC1 cells undergo developmental changes in culture analogous to follicular maturation in vivo. hGrC1 cells were grown in culture for a variable number of days before treatment with FSH. To measure FSH signaling the presence of phospho-CREB and phospho-p44 were determined by western blot. Both pathways were increased after cells were treated 4 days post-confluency, consistent with a developmental shift leading to more hFSHR protein the longer the cells were grown in culture. This suggests that hGrC1 cells mimic granulosa cell differentiation in vitro and may be a viable model for understanding folliculogenesis.
Scholars Research Students
- Ana Carranco
- Brittany Gay