Aalia Al Barwani |
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Research Advisor & Department
Jun (Kelly) Liu - Molecular Biology and Genetics
Name of Project:
Genetic Mapping of Suppressor Mutations of sma-9, a Gene Involved in Patterning of the M Lineage in C. elegans Abstract:
Our lab is using the post-embryonic mesodermal lineage, the M lineage of C. elegans to study the mechanisms underlying the diversification and cell fate specification of muscle development. The M lineage is ideal for investigating mesodermal development for several reasons, the most important of which is that the lineage has been mapped and it is not important for viability. In addition to this, the C. elegans genome has been sequenced and it is a genetically amenable organism. The M lineage is derived from a single precursor cell which asymmetrically divides to give rise to distinct dorsal and ventral cell fates. Specifically, the dorsal daughter of M gives rise to coelomocytes (CCs), whereas the ventral daughter gives rise to sex myoblasts (SMs). We have identified a gene, sma-9 as a factor known to participate in the mechanisms involved in this asymmetry. Mutations in sma-9 alter M lineage polarity by a cell fate transformation of dorsal fates to ventral fates, resulting in extra SMs and loss of M derived CCs. To better understand the role that sma-9 plays in this dorsal/ventral asymmetry a suppressor screen was carried out. In this screen, seven potential sma-9 suppressor mutations were isolated. Described here is the characterization of two of these potential suppressors; jj4 and jj7. They are being mapped via snip-SNP mapping, first to a specific chromosome and then to a more defined region on the chromosome. jj7 has been preliminarily mapped to chromosome two and jj4 is yet to be mapped. Our goal is to identify the genes carrying the suppressor mutations.
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Suehyb Al Khatib |
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Research Advisor & Department
Rob Thorne - Molecular and Cell Biology
Name of Project:
Cryopreservation of Sperm Abstract:
It is known that at low temperatures metabolic and biological processes’ rates slow down. This concept is used in the cryopreservation of living cells, particularly, sperm; cooling the sperm down to temperatures such that the biological processes in the sperm stop completely. The frozen sperm can then be thawed at a later time and used. Complications arise due to the lattice structure of ice. When water is cooled down at a relatively slow rate (as in freezers), it forms an opaque crystalline ice. This form of ice is harmful to membrane structures, as the water expands to form the hexagonical lattice, a sperm cell would erupt. Alternatively, if a cell culture were cooled at a slightly slower rate, the ice lattice structures will form outside the cell faster than inside, this will cause a gradient making water flow out of the cell subsequently making the cell membrane flaccid. This reduces the damage caused to the membrane of cells. The current model for the cryopreservation of sperm uses this concept and involves a multitude of steps whose mechanisms are poorly understood, it is expensive and complex as well. Research is being carried out to determine a more reliable and better understood method for the freezing of sperm. If water is cooled down at fast enough rates a transparent amorphous ice can be produced. Ice formed in this way will not damage membrane structure. However, no practical method exists for cooling ice down at rates fast enough to achieve the amorphous state. Cryoprotectants are added to water to help break up the lattice formation of ice, and thus amorphous ice can be prepared at lower cooling rates. Examples of cryoprotectants include glycerol, methanol, and PEG (polyethylene glycol). With a high enough concentration of cryoprotectant, water can be frozen into amorphous ice simply by submerging it into liquid nitrogen. The aim of this project is to look at the effects of increasing the surface area of the liquid being frozen by freezing it in tubes. Tubes of different diameters and wall thickness will be tested, the results will then be compared with those obtained by freezing droplets of different volumes. The diameters will be in the order of 1/8" and smaller. When liquids are passed through a small diameter they experience a shearing force. It is thought that the shear is damaging to sperm. Experiments will be carried out to test this hypothesis.
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Lisa Allen |
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Robert Gilmour - Biomedical Sciences
Name of Project:
Ventricular Fibrillation and Sudden Cardiac Death Abstract:
Ventricular fibrillation (VF) is one of the leading causes of death around the world. There are several hypothesis about the electrical signals in the heart that lead to fibrillation. One of them is the restitution hypothesis, which states that VF results if the slope of the restitution curve, that is the plot of Action potential duration versus diastolic interval, is greater than 1. Our project includes collecting data from purkinje fibers and ventricular muscle, excised from canine hearts. The fibers are stimulated using bipolar electrodes to model heart rhythms before, during and after VF and the resulting action potentials are recorded using micro-electrodes. The collected data is then analyzed using a computer program that helps in constructing restitution curves. The analysis of the data is being used to construct useful models of the heart that will enable us to discover treatments to prevent VF.
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Brundha Balaraman |
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George Hess - Molecular Biology and Genetics
Name of Project:
Transient Kinetic Studies of the Inhibition of the N-Methyl-D-Aspartate (NMDA) Receptor by Cocaine Abstract:
Glutamate receptors are important excitatory receptors in the central nervous system. Two types of glutamate receptors are currently known: ionotropic (excitation via flow of ions), and metabotropic (activation via G proteins and second messengers). The N-methyl-D-aspartate receptor (NMDAR) is one type of ionotropic glutamate receptor. Excessive excitation of the NMDAR with agonists will set in motion a cascade of events that ultimately lead to neurodegeneration. Neurodegeneration is associated with several neuropathological diseases. (i.e. Alzheimer’s disease, epilepsy, etc). In previous studies, cocaine has been shown to inhibit the NMDAR, the mechanism of which is unknown. Several variants of the NMDAR are found in the body. Each variant responds differently to agonists and antagonists. It is necessary to transiently transfect HEK-293 (human embryonic kidney) cells in order to express the receptor of interest and control the otherwise highly dynamic environment that is found within biological systems. In order to determine whether the receptor of interest is expressed, it is necessary to perform whole-cell current recordings. My research project will focus on the transient kinetics of cocaine interaction with NMDARs in order to determine the mechanism of inhibition and to explore potential agents that would help alleviate this inhibition. Whole-cell recordings in combination with laser-pulse photolysis will be used to study the kinetics of inhibition of NMDARs. Since the effects of ligands occur almost instantaneously, it is necessary to uniformly and rapidly introduce these ligands to the cell. In ordinary cell-flow techniques, it is difficult to ensure simultaneous delivery of ligands to all surfaces of a cell (spherical entity) due to hydrodynamic hindrances. In effect, data may be an inaccurate representation of what actually occurs on the microsecond timescale. Presently, it is possible to use an inactive photolabile precursor of the ligand (caged ligand) to resolve this problem. In the techniques developed by this laboratory, a caged ligand solution is equilibrated with the cell surface. Subsequent photolysis causes the caged compounds to break and release the active form of the ligand. In this way, the ligand is uniformly and rapidly applied to all areas of the cell surface in microseconds. In similar investigations with other receptors by this laboratory, understanding the mechanism of receptor dysfunction has led to an approach to alleviate the dysfunction.
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Rajat Bansal |
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Research Advisor & Department
Tudorita Tumbar - Molecular Biology and Genetics
Name of Project:
Characterizing the Proliferation Potential of Slow Cycling Stem Cells in the Hair Follicle Bulge Abstract:
Hair follicles in the epidermis contain a stem cell niche in an area called the bulge. A distinctive character of these stem cells is infrequent division, also referred to as "slow cell-cycling (SC)." Their low mitotic activity has been used as a labeling tool, so these SC cells can be tracked in the tissue or isolated for in vitro characterization. We use transgenic mice that have been engineered to express a histone binding green fluorescent protein (H2B-GFP) in epidermal cells and the hair follicle. The expression of this gene can later be shut off by external controls, after which the brightness of the GFP label depends on the cell cycling characteristics of a particular cell — the slower a cell divides the longer it will retain the label. We have found that not all the SC cells in the bulge have similar characteristics, but rather there are distinct fractions that retain the label at different levels of brightness. One particular fraction retains the GFP label for an exceptionally long time, and thus we believe it to be especially quiescent. In fact, it may be terminally differentiated and may have completely withdrawn from the cell cycle. We tested this hypothesis using mouse as a model organism. To determine whether the brightly labeled fraction was capable of cell-division, or if it had in fact withdrawn from the cell-cycle, we induced cell proliferation in the skin using TPA (a strong tumor promoter). During the treatment, the mice were also injected with BrdU, which is a label that incorporates itself into dividing cells during DNA synthesis. Afterwards, we sacrificed the mice and observed which bulge cells proliferated and to what extent. We also plan to characterize some of the biochemical differences between the differentially labeled cell fractions by running a microarray and observing how gene expression differs.
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Karlyn Beer |
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Research Advisor & Department
Kathryn Boor - Food Science
Name of Project:
The role of the LisRK two-component regulatory system in Listeria monocytogenes virulence gene regulation Abstract:
Listeria monocytogenes is a gram-positive, food-borne bacterial pathogen responsible for the disease listeriosis, a malady characterized by gastrointestinal upset and in serious cases, septicemia, nervous system damage, and death. L. monocytogenes is able to thrive under stressful environmental conditions including refrigeration, acidity, salinity, heat and exposure to alcohol and bile salts. Our laboratory has previously demonstrated that the alternative sigma factor, sB, contributes to the ability of L. monocytogenes to survive under a variety of stressful environmental conditions. Moreover, the stress-response mechanisms activated by sB contribute to virulence in L. monocytogenes. The goal of my project is to understand the mechanism by which harsh environmental conditions lead to the activation of sB and subsequent transcription of stress-response and virulence genes; external stress stimuli must somehow be transferred across the cell wall to result in the transcription of these genes. We hypothesize that the two-component regulatory system lisRK is involved in signal transmissions that result in sB activation. Testing the hypothesis involves two major phases: mutant creation and characterization of stress tolerance. I have previously created a knockout mutant lacking the entire LisRK system and am in the process of creating another mutant lacking only the response regulator, LisR. Characterization of the existing mutant has begun and involves subjecting the cells to a variety of environmental stresses, including acid media and oxidative stress. By comparing the survival of the mutants under these conditions to that of the wild type and sB knockout mutant, I will be able to assess the relative contribution of the LisRK regulatory system to virulence and stress response in Listeria.
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Jennifer Brady |
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Research Advisor & Department
Greg Martin - Plant Pathology
Name of Project:
Characterization of Genes Involved in Tomato Immune Response Using Virus-induced Gene Silencing Abstract:
When Pseudomonas syringae pv. tomato invades susceptible tomato plants, it causes bacterial speck disease. Resistant plants invoke an immune response accompanied by programmed cell death localized to the area of infection, known as the hypersensitive response (HR). Resistant tomato plants possess the Pto gene which recognizes AvrPto and AvrPtoB, effector proteins secreted by Pseudomonas, setting off a signal transduction cascade leading to the HR. Approximately 150 genes have been previously identified that compromise resistance to P. s. pv. tomato or contribute to the HR. One of these genes, a MAP kinase kinase kinase (MAPKKK), has been identified as part of the signaling pathway required for inducement of the HR. Interestingly, MAPKKK was found to be a positive cell death regulator involved in both plant immunity and disease susceptibility. This summer I will use virus-induced gene silencing to conduct a series of epistasis experiments. My goal is to place each of the previously identified 150 genes with respect to MAPKKK in the signal transduction cascade leading to the HR. On each silenced plant, I will overexpress genes known to cause cell death that lie upstream and downstream of MAPKKK, as well as MAPKKK itself. The presence or absence of an HR will enable me to determine where the silenced gene lies in the MAPKKK-mediated cell death pathway. Placement and characterization of these genes will contribute to our understanding of plant defense mechanisms and the signal transduction involved in tomato immune response.
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