Jeffrey Achampong Stony Brook University Junior Year Biology Major Faculty Advisor: Dr. MPeter Brink, Chair Department of Physiology & Biophysics
Different Connexins affect mice eyeball contractions induced by light absorbance Jeffrey Achampong(1), Diane Trapani(1) and Peter Brink(1) Gap junctions are intercellular channels that permit the free passage of ions and small molecules between cells. These channels are formed by proteins called connexins. We set out to investigate if light induced contraction of the papillary sphincter muscle was affected by the presence or absence of different connexins. The papillary sphincter muscle defines the diameter of the iris. It is composed of smooth muscles that normally contain connexin43 (Cx43). The mice were dark adapted for 24 hours and then anaesthetized with chloroform.Bimolecular fluorescence complementation (BiFC) is a method of viewing protein interactions in cells. While unconscious, the mice were killed and their eyeballs were dissected. The eyeballs were soaked in a saline solution and then pinned down in a cell culture dish on a light microscope stage. The cornea was removed by microsurgery under red light and white light was flashed on the iris at various time intervals. Contraction amplitude and duration determined in response to defined light flashes of varied duration (see Krivoshik and Barr, 2000: AJP 279:C274). The light stimulus consisted of white light filter to permit the transmission of wavelengths between 450 and 530 nm. At wavelengths below 450 or above 530 no contractions are generated regardless of the mouse model used. We used three knockout mouse models to determine if connexin connecting the smooth muscle cells might affect sphincter contraction. The first was a wild type mouse sphincter that contains Cx43. It was our standard to compare against a knockout mouse for Cx40. Since Cx40 is not present in the papillary muscle it represents an internal control that demonstrates the knock out procedure does not affect contraction. We also tested mice where Cx43 was knocked out and replaced with another connexin, Cx32 (Cx43-KICx32). Cx32 differs from Cx 43 in their relative permeability of connexins to a variety of solutes including second messengers. Interestingly, Cx43-KICx32 pupillary sphincters did not respond to light stimulus. This suggests that the difference in permeability of connexins to second messengers is critical to normal function of the papillary sphincter. This result represents the basis for in situ early detection of various diseases involving the mutations of connexin. A number of diseases states have been associated with mutations of connexins. Occulodentodigital dysplasia or ODDD and accompanying neurological complications and congenital heart disorders have been attributed to mutations of Cx43 (Paznekas et al, 2003) while Cx40 mutations correlate with atrial fibrillation and other arrhythmias (Gollob et al., 2006) and neurological conditions like Charcot-Marie-Tooth syndrome are associated with Cx32 mutations. (1)Stony Brook University, Stony Brook, NY, USA
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	Hendri Chauca St. Francis College Junior Year Chemistry Major Faculty Advisor: Dr. Dale Drueckhammer, Professor Department of Chemistry
Progress on the synthesis of a prospective glucose receptor, containing boronic acid groups Hendri P. Chauca(1)(2), Dale G. Drueckhammer(2) The frequent measurement of blood glucose levels is essential for the proper management of diabetes. The ability to continuously monitor glucose levels will provide tighter diabetes management. In prior work by the Drueckhammer group, the computer program CAVEAT was used to design potential synthetic glucose receptors, having a pair of boronic acid groups attached to a computer designed backbone structure (Figures A and B). Here we show progress in the synthesis of one such prospective glucose receptor. The work has involved synthesizing candidate receptor structures from available starting materials. Chromatography techniques were used to isolate and purify the desired reaction products, and analytical methods were taken to confirm the structure of the products. Further studies would include the testing of the glucose receptor’s affinity to glucose, and the addition of a fluorescent tag to the receptor. A fluorescent-based glucose receptor could potentially be a useful application in continuous glucose monitoring. Figure A: Potential synthetic lucose receptor with boronic acid groups. Figure B: Potential synthetic glucose receptor with boronic acid groups, projected with the use of CAVEAT. (1)Stony Brook University, Stony Brook, NY (2)St. Francis College, Brooklyn, NY
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	Angelica Delgado St. John’s University Sophomore Year Biology Major Faculty Advisor: Dr. Michael Hayman, Professor Department of Molecular Genetics & Microbiology) Other Research Mentor: Kimberly Gottfredsen, Ph.D. Candidate Genetics Irene Ischenko, Sr. Research Support Specialist
Effects of Co-expression of MET and RON on migration induced by Hepatocyte Growth Factor in MCF-10A Cells Angelica Delgado(1) , Kim Gottfredsen(2), Irene Ischenko(2), and Dr. Michael Hayman(2) RON and MET belong to the subfamily of tyrosine kinase receptors. MET, the prototype of the oncogenic receptor family, codes for the Hepatocyte Growth Factor/Scatter Factor 1 (HGF/SF1) receptor and RON codes for a hepatocyte growth factor-like protein, called Macrophage Stimulating Protein/Scatter Factor2 (MSP/SF2) receptor. Both receptors can induce invasive cell growth, cell-cell dissociation (scattering), proliferation, and motility. In carcinomas, RON and MET when co-expressed appear to induce a more aggressive phenotype and poor prognosis in patients compared to patients with tumors that express either RON or MET alone. To study the co-expression of RON and MET, we used MCF-10A cells; epithelial cells derived from normal human breast tissue. In this study, MCF-10A cells, which expressed MET endogenously, were engineered to express RON as well. Then MCF-10A cells, either expressing RON or not, were activated specifically by the MET ligand, HGF. We tested both cell types for migratory behavior through Transwell filters. We predict that when RON and MET are co-expressed, this will induce a more invasive metastatic phenotype in vitro, which will result in increased migration through the Transwell filters. (1)St. John’s University, Queens, NY (2)Stony Brook University, Stony Brook, NY, USA
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	Trichelle Harris Grambling State University Junior Year Mathematics Major Faculty Advisor: Dr. Stephen Finch, Professor Department of Applied Math & Statistics Other Research Mentor: Rose Saint Fleur, Ph.D. Candidate
Phosphatase roles in regulation of fyn tyrosine kinase activity are required for oligodendrocyte differentiation Trichelle Harris(1), Rose Saint Fleur(2), Stephen J. Finch(2) DNA sequence variations such as singlenucleotide polymorphisms (SNPs) were initially assumed to account for the major differences among people. Recently, the discovery of DNA copy-number polymorphisms (CNPs) has documented a previously unexpected type of genomic variation in humans. CNPs can be simple deletions or replications leading to a gain or loss of one copy of a DNA segment, or can lead to copy number changes at several locations in the human genome. Although the exact density of CNPs in humans has not yet been established, recent studies have suggested that CNPs may be a crucial element in some genetic diseases. One tool for determining the number of CNPs is the Bayesian Information Criterion (BIC). The BIC is a statistical model that is based on the likelihood function. We studied the BIC using a factorial design applied to simulated data. This design was based on four factors: sample size, number of CNPs, probability distribution of CNPs, and separation. We then conducted a simulation study which generated samples with different design combinations of the factors to determine how accurately the BIC determines the correct number of CNPs. Based on the analysis of the data, we found that, of the four factors, separation is the most important, accounting for about 72% of the variation. As expected, CNPs that have large separation are identified with greater accuracy. In addition, sample size played a minor role in the amount of CNPs chosen. (1)Department of Mathematics, Grambling State University, Grambling, LA (2)2Department of Applied Mathematics and Statistics, Stony Brook University, Stony Brook, NY
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	Rey Phillip Llenes Stony Brook University Sophomore Year Biology Major Faculty Advisor: Dr. Suzanne Scarlata, Professor Department of Physiology & Biophysics
Identification of Residues on the Pleckstrin Homology Domain of Phospholipase CI2 are Critical for Binding and Activation with Gßy Subunits Rey Phillip Llenes(1), Urszula Golebiewska(1), And Suzanne Scarlata(1) Phospholipase Cß2 (PLCßy) signaling enzymes hydrolyze the membrane-bound phospholipid phosphatidylinositol 4,5 bisphosphate [PI (4,5) P2] to trigger the release of secondary messengers 1,4,5 – inositol trisphosphate (IP3) and diacylglycerol (DAG) leading to the activation of various cellular activities. PLCß2 is activated when Gßy protein regulating subunits bind to its N-terminal pleckstrin homology (PH) domain. It is currently known that a region on the PH domain is the binding site but the individual residues that physically dock with GßM are unknown. To test the possible binding sites, we mutated three residues believed to be critical based on our docking model. The effects of a triple point mutation (K71A, P90I, D91G) were tested by mutating these residues on the PLCß2-PH domain. Altering these residues should effectively reduce PLCI2 activation, thereby indicating that K71, P90, and D91 are the important docking sites on the PH domain. Activity assays performed using Gßy and the mutated PLCß2 showed a noticeable reduction in PIP2 hydrolysis compared to the wild type, indicating less activity. These results support our docking model, suggesting that K71, P90, and D91 are the three critical residues for PLCß2-Gßy binding and activation. (1)Stony Brook University, Stony Brook, NY, USA
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	Tahirah Sylvester Daniel Medgar Evers College Junior Year Biology Major Faculty Advisor: Dr. Holly Colognato, Asst. Professor Department of Pharmacology
Csk: A Key Regulator of Fyn During Oligodendrocyte Myelination Tahirah Sylvester Daniel-Wilson(1), Iva D. Tzvetanova(2), Holly Colognato(2) In the Central Nervous System, myelination is essential for normal brain development and function. Oligodendrocytes arise from progenitor cells that differentiate and proliferate into mature myelin-making oligodendrocytes. If myelin production or stability is compromised, then an array of neurodegenerative diseases, such as Multiple Sclerosis, may arise. The precise mechanisms involved in myelination are not fully understood. However, it is known that a Src family kinase, Fyn, is a necessary regulatory molecule in this process. Fyn can be inactivated when cytosolic C-terminal kinase (Csk) is recruited by the Csk binding protein (Cbp), which ultimately directs Csk to lipid rafts. Fyn is located in lipid rafts, and therefore if Csk is transported by Cbp to the lipid raft, Fyn will be deactivated, resulting in a reduction of myelin production. Our research was conducted in order to investigate the mechanisms involved in Fyn regulation and function during myelination. This was done by testing whether Csk and its kinase activity are necessary to deactivate Fyn during oligodendrocyte development. Several DNA constructs were made expressing Csk wild type, myristolated Csk, Kinase dead Csk, and Kinase dead- myristolated Csk, and the control pECFP empty vector. We utilized assays involving cellular proliferation, BrdU incorporation, and programmed cell death assay, TUNEL, in conjunction with GFPlabeling immunocytochemistry on cultured cells. Preliminary data suggests that the transfection process was successful, as many cells incorporated the vector and expressed the Csk proteins. This was concluded because the immunofluorescent tag associated with the Csk fusion protein allows cells that have expressed the plasmid DNA to be fluorescent. Our prediction is that Csk may be one of the proteins involved in myelin production and we are working to establish its role in cell proliferation and /or cell death. (1)Medgar Evers College, Brooklyn, NY (2)Stony Brook University, Stony Brook, NY
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	Cindy Thomas Stony Brook University Junior Year Biology Major Faculty Advisor: Dr. Anne Savitt, Research Assistant Professor Department of Molecular Genetics & Microbiology
Binding and inhibition/enhancement of binding and uptake of Francisella tularensis LVS by murine bone marrow derived macrophages Cindy Thomas(1), Anne Savitt(1) Francisella tularensis is a gram-negative coccobacilli, the causative agent of tularemia. In addition, F. tularensis is considered a potential agent for biological terrorism. In these experiments, the effects of binding complement proteins or antibodies against bacterial and cellular antigens on the binding and uptake of Francisella tularensis Live Vaccine Strain (LVS) by murine bone marrow macrophages is investigated. Bone marrow macrophages derived from the femurs of one C3H/HeN mouse were infected with Francisella tularensis LVS in the absence or presence of these antibodies or proteins. Fluorescence microscopy and Colony Forming Unit (CFU) assays were used to assess the binding and uptake of the bacteria by the macrophage cells. Our data suggests that preincubation of the bacteria with complement proteins and some of the antibodies alters binding and uptake of F. tularensis LVS by murine macrophages. (1)Stony Brook University, Stony Brook, NY
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	Tiffani Tumbaga University of Notre Dame Sophomore Year Psychology Major Faculty Advisor: Dr. Hoi-Chung Leung, Assistant Professor Department of Psychology Other Research Mentors: Yuji Yi, Ph.D. Candidate in Psychology
Encoding And Maintaining Multiple Stimuli in Visual Short-Term Memory Tiffani Tumbaga(1), Yuji Yi(2), Hoi-Chung Leung(2) Visual short-term memory (VSTM) allows visual information to be retained for a short period of time. There are many aspects of visual stimuli that affect retention, such as the number of features and the complexity of the object. Other important factors in VSTM include the time it takes to consolidate an image and the length of the interstimulus interval (ISI). However, the mechanism of encoding and maintaining multiple items, especially items from different categories, has not been well addressed. In this study, we investigated how multiple items are being encoded and maintained in VSTM. Using a delayed recognition paradigm, VSTM was tested on a pair of visual stimuli that are selected from either the same or across a group of color stimuli and a group of line stimuli. We chose colors and lines as stimuli because they are shown to involve different underlying neural pathways. Memory accuracy for 4 task conditions (color-color, line-line, color-line, linecolor) was recorded. The stimuli, which were presented sequentially for 100 msec, were separated by an ISI of either 250 or 1500 msec. After a 1500 msec delay, the second stimulus was followed by a probe. We hypothesized that heterogeneous conditions would have higher accuracy than homogeneous conditions, and that a recency effect would be observed in both ISI’s. We found that with a short ISI, heterogeneous conditions had higher accuracy than homogeneous conditions. Only the color-line condition showed a recency effect. We suggest that these results were due to an increased focus of attention on the line stimuli and too short of an interval to shift attention from the first stimulus to the second. With a long ISI, the color-line and line-line conditions showed a significant recency effect. This can also be attributed to an increased focus of attention on the line stimuli, as well as a sufficient duration of time to shift attention from the first stimulus to the second. There was no significant difference in heterogeneous versus homogeneous conditions. (1)1University of Notre Dame, Notre Dame, IN (2)Stony Brook University, Stony Brook, NY
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	Danielle Rosemond Stony Brook University Master’s Student Physiology & Biophysics Faculty Advisor: Dr. Carol Carter, Professor Department Molecular Genetics & Microbiology
Phosphatidylinositol 4, 5- bisphosphate (PIP2) role in HIV trafficking and viral release Danielle Rosemond(1), Carol Carter(1) The human immunodeficiency virus (HIV) is a member of the retrovirus family and contains two subtypes, HIV-1 and HIV-2. Both subtypes possess nine genes of which one, the gag gene, is responsible for targeting the viral proteins to the plasma membrane, assembly of the viral proteins at that site, and release of infectious viral particles. Gag, all by itself, can form viral-like particles (VLP) and therefore can be used to study the entire trafficking, assembly, and release process. There are proteins and lipids in the host that contribute to retroviral trafficking. In this study, we used (i) antibody against the lipid phosphatidylinositol 4, 5- bisphosphate (PIP2) and (ii) the pleckstrin homology (PH) domain from PLC∂1, which enables the PLC∂1 to target PIP2 sites on the plasma membrane, as probes to identify PIP2 sites on the plasma membrane in order to investigate whether regions containing PIP2 are sites of HIV-1 release. If PIP2 is important for release, we hypothesize that HIV Gag and PIP2 should co-localize at these sites on the plasma membrane. Cos-1 cells were (case i) transfected with HIV-1 Gag tagged with GFP protein (Gag-GFP) or (case ii) co-transfected with Gag-GFP and the PH domain tagged with CFP (PH CFP). Forty-eight hours later, the cells were either, (case i) fixed, permeabilized and stained with TRITC (red)-tagged secondary antibody (rabbit anti-mouse) that recognizes the primary mouse anti-PIP2 antibody OR, (case ii) fixed and permeabilized or left unpermeabilized. In both cases, they were examined by confocal microscopy. We found that PIP2 was easily lost from the plasma membrane and that optimal conditions for its detection in this location had to be defined. If PIP2 is important for HIV release, we expect to detect Gag-GFP co-localization with the antibody (in case i) and with PH-CFP (in case ii). (1) Stony Brook University, Stony Brook, NY