Olga Davydenko
Quantum Dots as FRET Donors in Molecular Beacons
Quantum dots (QD) are fluorescent semiconductor nanostructures that have the ability to emit light through the visible and infrared regions. Due to various coatings of quantum dots, they can be conjugated to macromolecules such as peptides, DNA or RNA. Oligonucleotides with dyes can be used for such conjugations to achieve fluorescence resonance energy transfer (FRET) between the QD and the dye when excited by UV light; this is due to the hairpin sequences encoded into the nucleotides, which bring the dye within the Foster radius of the QD. In order to utilize these molecular beacons as gene expression monitors, reversibility of FRET is necessary. This was achieved by incubation of beacons with oligonucleotides of complimentary sequence and allowing the strands to denature fully and anneal. An increase in the distance between the fluorescent dye and the quantum dot in beacons hybridized to complimentary oligonucleotides brought the dye out of the Foster radius and resulted in FRET reversal. FRET reversal was observed using steady state fluorescence analysis and fluorescent lifetime measurement. As a result of this study, a successful molecular beacon conjugation protocol was established and reversible FRET was achieved through complementation.
Jen Libous
Lipid Rafts play a role in normal human follicle stimulating hormone receptor (hFSHR) signaling
The hFSHR is a member of the family of g protein-coupled receptors that has been shown to signal through both g protein linked and MAP kinase pathways. Little is known about the importance of the membrane microenvironment in normal hFSHR signaling. To study the role of lipid rafts in hFSHR signaling, cholesterol depletion experiments were carried out with methyl-β-cyclodextrin (MBCD) in HEK293 cells expressing hFSHR. Cells were treated with MBCD prior to stimulation with FSH. Cells were harvested and activation of MAP kinase enzymes (p38 and p44/42) was measured by western blot. MBCD treatment resulted in hormone independent activation of p38 MAP kinase and abolishment of normal FSH stimulated activation. There was no effect of MBCD or FSH on p44/42 MAP kinase activation. In parallel experiments, cells were treated with MBCD prior to FSH stimulation to measure cAMP accumulation. cAMP production in MBCD treated cells was almost completely eliminated, although hormone binding remained normal. One interpretation of the data suggests that the disruption of lipid rafts by depleting cholesterol disturbs the membrane microevironment sufficiently to disturb hFSHR signaling. Currently, a cholesterol add-back experiment is being carried out to follow up on the cholesterol depletion to see if signaling is restored.
Stephanie Mason
Coritsol Levels as a Result of Academic Stress
Depression and anxiety have increasingly become a problem on college campuses and may be linked to increases of cortisol levels in response to stress. The goal of this study was to determine if there is a positive correlation between academic stress and cortisol levels in the human body. High levels of this hormone, which is often triggered by stressful situations, is associated with many negative effects such as increased blood pressure, diabetes, weight gain, and most important to my study, the onset of depression. Our hypothesis was that an increase in academic stress during the term would lead to increased cortisol levels that may correlate with increased depression and/or anxiety. Cortisol was measured in saliva samples from upperclass female students over the course of the winter term using an enzyme linked immunosorbant assay (ELISA). The subjects also completed the Beck Depression Inventory and the State/Trait Anxiety Inventory, which were then analyzed in conjunction with their cortisol levels. A weak correlation was observed between anxiety and cortisol levels. Overall, it is important to understand the biological and psychological effects of stress on the human body in order to help students who suffer from anxiety and depression brought on by academic stress.
Jessica Rosenblum
Probing human follicle stimulating hormone receptor interactions with caveolin
The human follicle stimulating hormone receptor (hFSHR) is a g protein coupled receptor located in the ovaries and testes. This receptor is necessary for normal follicular development in females and sperm production in males. The main question we are asking is does the FSHR interact with caveolin, a lipid raft associated protein. We are asking this question to determine if FSHR is located in a lipid raft. If it is located in a lipid raft it will give us more information regarding the function of FSHR and how to modulate it. Many molecu les that interact with caveolin contain a caveolin recognition sequence (FXFXXXXFXXF, where F is a large hydrophobic amino acid like phenylalanine). FSHR has one of these recognition sequences located on the 4th transmembrane domain. Therefore, we predicted that the receptor interacts with caveolin and is located on lipid rafts. To test our hypothesis plasmid DNA was produced encoding the wild-type caveolin sequence as well as 2 mutants which block normal caveolin function. Plasmids will be transfected into cells expressing the hFSHR and co-immunoprecipitation experiments will be performed to determine if the receptor and caveolins interact, and how the mutants affect that interaction.
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