Stem Cell Replacement Strategies For Parkinson's Disease   abstract
Advisor:  Carol Lin

The Role of Focal Adhesion Signaling in Microtubule Stabilization  abstract
Advisor:  Gregg Gunderson

Role of MAPK Pathways in Resistance to AD.MDA-7 and MDA-7/IL-24 Mediated Apoptosis  abstract
Advisor:  Paul Fisher

Reduction of DNA Double-Strand Breaks in S. cerevisiae Does Not Change Crossover Frequency and Reveals a Novel Phenomenon: Crossover Homeostasis   abstract
Advisor:  Scott Keeney

Bright Summer: Rapid Drug Discovery Biodefenses  abstract
Advisor:  Geoffrey Zubay

Improving the Accuracy of a Transformatoinal-Based Learner for Dependency Grammars Through Template Design  abstract
Advisor:  Steven Johnson

The Analysis of the Partial Neural Transcriptome of Aplysia californica by Sequencing 6,034 ESTs Generated from Normalized cDNA Library from the Pleural-Pedal Ganglia  abstract
Advisor:  Jingyue Ju

Analysis of Adult Mammary Glands in Mice Heterozygous for the Tbx3 Gene  abstract
Advisor:  Virginia Papaioannou

Interferon Inducible MX Genes on Chromosome 21 and Their Possible Role as Tumor Suppressors  abstract
Advisor:  Ben Tycko

SN38, a Topoisomerase 1 Inhibitor, Blocks Activation of Cyclooxygenase-2 in Human Colon Cancer Cells  abstract
Advisor:  Andrew Dannenberg

Analysis of a Pharmaceutical Compound's Degradant by HPLC  abstract
Advisor:  Kerry Cecere

Genetic Analysis of ARA70 gene:  Implications in Prostate Cancer  abstract
Advisor:  Jonathan Melamed

Laying the Foundation for Molecular Studies of Learning and Memory Using Aplysia californica: Sequencing Neuronal Soma- and Process-Specific cDNA Libraries and Transcriptome Analysis  abstract
Advisor:  Jingyue Ju

A Review of the Hypothetical Pathogenesis of Multiple Sclerosis and the Exploration of the Developments of more Targeted Therapeutics  abstract
Advisor:  Carol Lin

The Genetic Voice of Autism  abstract
Advisor:  T. Conrad Gilliam

Molecular Characterization of Platelet Apoptosis  abstract
Advisor:  David Diuguid

Experimental and Therapeutic Applications of RNA Interference   abstract
Advisor:  Daniel Weinstein

Full-length Sin Overexpression is Associated with Mistargeting of Zap-70 Following TCR Stimulation    abstract
Advisor:  Konstantina Alexandropoulos

The Neural Mechanisms Underlying Learning and Memory  abstract
Advisor:  Carol Lin

Gene Therapy for Sickle Cell Disease  abstract
Advisor:  Robert Pollack

The Odorant Receptor Protein is Localized on Both the Axon and the Dendrite of the Olfactory Sensory Neuron  abstract
Advisor:  Richard Axel

The LKB1 Tumor Suppressor and Peutz-Jeghers Syndrome  abstract
Advisor:  Lili Yamasaki

Establishment of an Improved Retroviral Packaging Cell Line that Efficiently Transduces Human Hematopoietic Cells  abstract
Advisor:  Arthur Bank

Biological activity of cyclic peptides derived from the G domain of mouse laminin α4 chain  abstract
Advisor:  Hynda Kleinman

Beginning in the late twentieth century, methods to understand our mind have been radically changed to take advantage of molecular and cellular biology based approaches by characterizing underlying processes in memory formation through utilization of Aplysia californica. Aplysia's sensitization training has provided a basis for the groundbreaking work of delineating the molecular framework of short- and long-term synaptic plasticity. In an effort to further our understanding of the molecular machinery of memory formation, single cell soma and process cDNA libraries from a single normal metacerebral (MCC) motor neuron from Aplysia californica have been created. Sequencing non-normalized cDNA libraries of wild type neuronal soma and processes has enabled us to take snapshots of soma and process specific transcripts and their expression levels. Analysis of differential gene expression between MCC soma and process showed that many up-regulated genes in soma, such as reductase, aldehyde dehydrogenases, and alpha- and beta-tubulin, are involved in standard cell functions. Higher expression of these genes in cell soma supports the current theory that only upon repetitive external stimulation, does localized protein syntheses occur in distal neuronal processes by activation of translationally dormant transcripts. Discovery of a putative transcription factor asymmetrically expressed in MCC processes further supports this notion. Similar expression levels of ribosomal proteins and many housekeeping genes were observed in neuronal soma and processes. Several novel soma and process specific up-regulated transcripts have been identified. Normal and tissue specific transcriptomes will serve as a reference point for future comparative gene expression and correlation analyses between cDNA libraries from other neuronal tissues, subcellular components, and different phenotypic states of tissues or cells including different experimental learning paradigms. Our EST project has laid the ground work for pursuing further global expression analyses and has taken us one step further towards the delineation of the comprehensive molecular network of Aplysia.

A Review of the Hypothetical Pathogenesis of Multiple Sclerosis and the Exploration of the Developments of more Targeted Therapeutics

Eti Mezei, M.A. 2003

This paper serves to explore the different pathogenic hypotheses surrounding Multiple Sclerosis and ways in which future treatments can be developed to inhibit clinical degeneration.  Even though the histology of Multiple Sclerosis was described over a century ago, the full understanding of the cellular mechanisms that contribute to the disease process are not completely understood. Multiple Sclerosis is generally viewed as an autoimmune Th1 mediated disease, which have lead to the development of therapies that specifically target the inflammatory response. This view of the disease is hypothetical, because all that is known for certain about the pathogenesis of MS is that demyelination and axonal damage occur in the presence of immune cells and elevated levels of their cytokine products.  Thus far, treatments targeted at suppressing the inflammatory response have produced disappointing results insofar as preventing further neurological degeneration.  A new view on the mechanisms behind what causes MS and leads to axonal degeneration, coupled with the benefit of the newest technological advancements could lead to the development of more effective, targeted, and preventative therapeutics.

The Genetic Voice of Autism

Miriam Nadoff, M.A. 2003

Autism is a chronic brain-based developmental disorder with behavioral symptoms that include impaired social interaction, defective communication skills, and restricted and repetitive interests and behaviors, all of which typically emerge before the age of three.  Autism is diagnosed more often in males than females with a ratio of 4:1, is typically more severe in females, and is seen in all racial, ethnic and social groups of people.  There is currently no known cure for autism, however early diagnosis and intervention programs have proven beneficial in many cases.  The marked concordance of autism in genetically identical versus fraternal twins, and the increased risk of autism among relatives as compared to the general population, both point toward a genetic disorder.  In fact, while acknowledging the involvement of environmental factors, autism is considered one of the most heritable childhood neuropsychiatric disorders.  The high concordance ratio of identical to fraternal twin pairs gave false hopes that it would be a relatively easy task to determine the genetic etiology of autism.  Yet, as the story unfolds it is becoming increasingly obvious that autism does not result from the effects of a single major gene.  Rather, researchers are faced with the challenge of identifying the set of genes, their complex interactions and the underlying mechanism and environmental influences, which, in concert, lead to this multigenic disorder

Molecular Characterization of Platelet Apoptosis

Michelle A. Ross, M.A. 2003

Apoptosis in anucleate platelets was first reported in 1997 by Vanags et al.  Over the past six years platelet apoptosis has been described in several experimental, editorial and review publications.  However, due to the fact that platelets are naturally anucleate cells, whether they undergo apoptosis has been a highly controversial area. 

 A number of apoptosis markers of nucleate cells have been recognized in platelets.  These include markers of extrinsic and intrinsic pathways, and executioners of apoptosis, suggesting that anucleate platelets undergo an apoptotic program and can be used as a physiological model of nucleus-independent cytoplasmic apoptosis.  Platelets, similar to nucleated cells, have been found to contain the proteins necessary for the apoptotic process.  Although platelets lack a nucleus and nuclear DNA, they do contain mitochondria and mitochondrial DNA, metabolically stable mRNA, and are capable of protein synthesis.  Therefore, it is possible to conclude that platelets are also designed to have a pre-programmed mechanism of cell death.

Understanding of platelet apoptosis and its role in storage for transfusion is an exciting challenge and future research is likely to provide us with further insight into this field.  Evaluation of the mechanism of apoptosis in platelets may provide a basis for developing novel strategies to enhance platelet viability during storage as well as creating models of apoptosis for other anucleate cells. 

Experimental and Therapeutic Applications of RNA Interference

The discovery of the structure of DNA fifty years ago heralded a new age of molecular biology.  Prior to the elucidation of the double helix, the elements of genetic determinants were completely unknown.  In just half a century the mode of the genetic transmission of DNA between cells via transcription and translation has been discovered as well as the different structural modes in which DNA is organized within the cell.  The underpinnings of molecular biology have been discovered quite rapidly culminating in the Human Genome Project which has sequenced the entire human genome.  There were great hopes that the sequencing of the human genome would yield an exponential number of new drug targets and the possibility of personalized genetic treatments. The results of the Genome Project raised more questions than answers when only 30,000 out of what was believed to be over 100,000 genes were identified.  The linear gene theory of genetic determination that DNA encodes RNA which in turn encodes proteins does not seem to adequately explain the complexity of human beings in light of the sequenced genome.  Epigenetic mechanisms, modifications of gene structure, and the role of non-coding small regulatory RNAs are three areas being investigated to fill in the gaps of the linear gene theory.  RNA interference is one such mechanism that is involved in epigenetic regulation.  Believed to be conserved through evolution as an antiviral and transposon defense mechanism, RNAi is evolving into an exciting and lucrative experimental tool and potential therapeutic.  The application of double stranded RNA into a cell results in the degradation of its homologous mRNA. The mechanism of RNAi is reviewed as a means of reverse genetics and functional genomics.  Additionally, RNAi has broad implications as a therapeutic mechanism.  The potential to effectively knock out any protein by an endogenous mechanism was not known to exist hitherto.  Researchers are exploring ways to use RNAi to knock out oncogenes, genes involved in HIV infection and many others.  The ways of expressing RNAi in mammalian cells and the limitations in creating RNAi drugs are also explored.

Full-length Sin Overexpression is Associated with Mistargeting of Zap-70 Following TCR Stimulation

Adaptors are important components of signal transduction pathways which contain no known enzymatic activity. Rather, these contain modular protein-interaction domains allowing the formation of multi-protein signaling complexes. Our group is studying the novel adaptor Src-interacting protein (Sin), which is a family member of p130Cas and CasL. Sin contains an SH3 domain, multiple proline- and tyrosine-rich motifs, as well as a potential consensus immunoreceptor tyrosine-based activation motif (ITAM). The putative Sin ITAM is most homologous to that of CD3-e, which is known to interact with the tyrosine kinase Zap-70. Previous data from transgenic mice expressing a truncated Sin-mutant, SinDC, suggests that Sin-isoforms act as negative regulators of T cell receptor (TCR) signaling and T cell development. In this study, we use Jurkat T cells which stably overexpress Fl-Sin as a model system to study the function of Sin in TCR signaling. Specifically, we address if Fl-Sin overexpression effects Zap-70 signaling. We demonstrate that both Fl-Sin and Zap-70 rest in the cytosol, and that Zap-70 translocation to the membrane following TCR engagement is inhibited in the presence of overexpressed Fl-Sin. However, we were not able to show specific and direct interaction of Fl-Sin and Zap-70. Therefore, it appears that Fl-Sin indirectly inhibits Zap-70 function. Finally, CD3-z phosphorylation was not reduced, suggesting that this does not participate in the reduction of Zap-70 translocation. Thus, although the mechanism remains undefined, we demonstrate that TCR-induced Zap-70 activation is inhibited by overexpressed Fl-Sin.

The Neural Mechanisms Underlying Learning and Memory

Scientists have spent centuries trying to understand the biology of the brain. Of particular interest are the processes of learning and memory. How does the brain function to learn new information and how is that information retained in the form of memories? The first documented studies on learning and memory were performed 200 years ago when Herman Ebbinghaus forced himself to memorize long lists of nonsense syllables and then tested his own recall. Ebbinghaus determined that there were least two phases of memory: a transient short term phase and a more stable, long term phase. In the 1950's William Scoville and colleagues determined that the temporal lobe was the main structure responsible for the acquisition and retention of new information. Since the 1950's much of the work on learning and memory has focused on the hippocampus, as lesions of this area cause anterograde amnesia for explicit memory. Today it is known that long term memory storage requires both the transcription and translation of new genes. According to current models, constitutively expressed transcription factors are activated, leading to the transcription of target genes, some of which are also transcription factors, which then continue this signaling cascade, resulting in a long term enhancement of synaptic efficacy and the consolidation of new memory. What follows is a review of what is known about learning and memory as well as an overview of the neural mechanisms underlying the processes of learning and memory.

Gene Therapy for Sickle Cell Disease

As ethical commentary should be grounded in science, I present here an attempt to give a scientific overview of the prospect for gene therapy in sickle-cell disease as a grounding for future ethical commentary.  The primary issues I present here are: (1) who should be subjects for gene therapy--children or adults, and (2) whether gene therapy for sickle cell disease is warranted, both from scientific and social perspectives.  The heterogeneity of African-Americans as a group makes them less likely to find matches for stem cell transplantation, the only current curative method of sickle cell treatment.  A review of recent literature about gene therapy for sickle cell disease is presented, looking at both viral and non-viral techniques.   In addition, the known risks of gene therapy as discovered in  X-linked SCID are assessed in terms of their relevance to prospects for sickle cell disease.  Such information can help interpret the risks and potential benefits of sickle cell gene therapy in children and adults, but only when looked at in a broader social, ethical, and possibly religious context, can the questions I raise be answered more completely.

The Odorant Receptor Protein is Localized on Both the Axon and the Dendrite of the Olfactory Sensory Neuron

In this study, we focused on the SP1 receptor, which is one of several olfactory receptors being examined in our laboratory. We generated antibodies to both the intracellular and the extracellular epitopes of the SP1 receptor. The specificity of these antibodies to the receptor was demonstrated using a variety of methods. In the process, we were also able to show that the olfactory receptor could be found on the dendrite as well as the axon terminal of the olfactory neuron. This is an important observation since the location of the olfactory receptor enables it to participate in a variety of cellular activities, such as axonal guidance during neuronal development. Based on this result, we are considering other experiments to further study the olfactory receptor protein and its activity.

The LKB1 Tumor Suppressor and Peutz-Jeghers Syndrome

Peutz-Jeghers Syndrome (PJS) is a dominantly inherited disease described by a predisposition to hamartomatous polyps, pigmentations of the mucous membranes, and an increased risk of cancer. Mutations in LKB1, a serine/threonine kinase located at 19p13.3 has been found to be responsible for PJS syndrome. A majority of the mutations found in LKB1 patients are loss-of-function mutations, and disrupt LKB1 enzymatic activity. LKB1 is suggested to act as a tumor suppressor gene in this syndrome because hamartoma formation in PJS patients carrying an germline mutation is usually associated with somatic loss of the remaining wild-type LKB1 allele. However, it has also been observed that biallelic inactivation is not necessary for the initiation of the hamartoma development. The gene responsible for the syndrome has been conserved during evolution and homologues have been found in Xenopus, C. elegans, Drosophila, Mouse, and Raja erinacea. The gene plays a crucial role in normal development in the embryonic stage. Recent studies have began to elucidate that LKB1 is involved in regulating cell proliferation, p53-dependent apoptosis, and VEGF dependent angiogenesis. Nevertheless, the complete mechanism of how LKB1 mutations predispose Peutz-Jeghers syndrome patients to cancer has not been deciphered. This thesis will attempt to present an updated summary of the research conducted on LKB1 and provide a rationale for the increased risk in cancer development.

Establishment of an Improved Retroviral Packaging Cell Line that Efficiently Transduces Human Hematopoietic Cells

Genetically engineered retroviruses have proven to be a safe and efficient tool for gene therapy because of their unique mechanism for integrating DNA into primary cells. However, several barriers exist to high efficiency transfer of therapeutic genes into human hematopoietic stem cells (HSCs) using complex oncoretroviral vectors. One is low level expression of the target receptor on human HSCs and another is the ability to produce high titer viral supernatants. Using an alternative viral envelope, I have constructed a stable murine fibroblast based viral packaging cell line which produces murine oncoretrovirus pseudotyped to the feline endogenous viral envelope (RD114). Viral particles from this cell line were concentrated up to 100-fold (105 viral particles/ml to107 viral particles/ml) by ultracentrifugation. Human hematopoietic progenitors from cord blood and sickle cell CD34+ cells were efficiently transduced with a NeoR gene containing retroviral vector after a single exposure to concentrated RD114-pseudotyped virus produced from this cell line. Up to 78% of progenitors from transduced cord blood CD34+ cells and 51% of progenitors from sickle cell CD34+ cells expressed the NeoR gene. Previous reports demonstrate efficient transfer and express human b-globin in murine HSC using a human b-globin containing oncoretrovirus. Using this same vector I was able to show transfer of a human b-globin gene into progenitor cells from clinically relevant sickle cell hematopoietic progenitor cells with this new RD114 stable packaging system.

Biological activity of cyclic peptides derived from the G domain of mouse laminin α4 chain

Laminins, multifunctional glycoproteins in the basement membrane, consist of three distinct chains, α, β and γ.  So far, five α chains have been identified.  The α chains contain a C-terminal globular domain (G domain) consisting of five tandem modules (LG1-LG5), and are tissue- and/or developmental stage-specifically expressed.  Previously, A4G82 sequence, located on the LG4 module of the α4 chain G domain, was shown to have strong cell attachment activity.  Recently, the crystal structure of the α2 LG5 module was reported, revealing that it consists of a 14-stranded β-sheet (A-N) sandwich structure.  Based on structure-based sequence alignment, the A4G82 peptide is located between β-strands E and F in the α4 chain LG4 and forms a connecting loop structure in the native protein.  In order to show the structural importance of the loop region, I prepared cyclic A4G82 peptides and evaluated the activities.  As a result, cyclic form of the A4G82 peptides showed enhanced biological activities compared to the linear form.  These results suggest that cyclic form of the A4G82 peptide mimic the E-F loop structure and that the E-F loop region in the α4LG4 module plays a crucial role in various biological functions of the laminin.