A Current Review of Bioanalytical Services Aiding Drug Discovery  abstract
Advisor:  Carol Lin

Dominant Negative Serum Response Factor Transgene Expression Affects the Regulation of Activity-Dependent Early Response Genes Coding for Cell Signaling Proteins, Transcription Factors, Structural Proteins, and Enzymes in Primary Neuronal Cultures   abstract
Advisor:  Amit Etkin

Expression and Production of Recombinant Heat Shock Protein for Eliciting an Immune Response in the Therapeutic and prophylactic Treatments of Cancer  abstract
Advisor:  Michael Yamin

Stem Cell Transplantation for Repair Following Traumatic Spinal Cord Injury  abstract
Advisor:  Saadi Ghatan

The Scientific Evolution of Genetically Engineered Plants: Current Applications and Future Implications  abstract
Advisor:  Amer Beg

Mann Sheuan Pamela Chao, M.A.

Alternative Splicing, Disease and the New Age of Antisense Therapeutics  abstract
Advisor:  Lawrence Chasin

Jing Chen, M.A.

Frontiers in Alzheimer’s Disease – Advances and Controversies  abstract
Advisor: Daniel Kalderon

The History and Nature of Applied Cloning and the Ethics and Laws Governing Its Use  abstract
Advisor:  John Loike

Transgenic Approaches to Seasonal Grass Allergies  abstract
Advisor:  Carol Lin

A Look into the Pathology and Treatments of Alzheimer ’s Disease  abstract
Advisor:  Mark Albers

An Abbreviated Tour of the Path Taken While Unraveling the Mysteries of Autoimmunity & Systemic Lupus Erythematosus  abstract
Advisor:  Amer Beg

Damara T. Gebauer, M.A.

 MAPK Scaffold: Kinase Suppressor of Ras  abstract
 Advisor: Liang Tong

Directed Evolution: A Review of Evolutionary Approaches to Molecular Design  abstract
Advisor:  Daniel Kalderon

Automated Primer Design for Speciation of Closely Related Taxa  abstract
Advisor:  Ian Lipkin

Jennifer M. Jolivet, M.A.

Current Research in Prion Diseases: Understanding the Molecular Biology of PrP Proteins  abstract
Advisor:  Stuart Firestein

Therapeutic Targets for Resistant Gastrointestinal Stromal Tumors (GIST)  abstract
Advisor:  Daniel Kalderon

Use of Statistical Analysis Methods on Hippocampal Multivariate Data to Avoid Acceptance of False Positives  abstract
Advisor:  Paul Pavlidis

Alzheimer's Disease: Drugs in Development  abstract
Advisor:  Carol Lin

Development of Methodology for Performing Sequence-Based Typing of Psoriatic Arthritis Patients in a Large Cohort of Irish Families, Using the HLA-B Locus  abstract
Advisor:  Robert Winchester

Sheila Ramgopal, M.A.

Pre-Erythrocytic Malaria Vaccines: Identifying and Developing a Candidate Vaccine that will Induce Sterile   Protective Immunity  abstract
Advisor:  Amer Beg

Jeannette Richmond

The Role of Fas-Mediated Apoptosis in the Development of Hashimoto’s Thyroiditis  abstract
Advisor:  Amer Beg

 Nima Shah, M.A.

Evidence the Spliceosome Is A Ribozyme  abstract
Advisor:  James Manley 

 Timothy J. Smith, M.A.

RNA Interference: A Revolutionary Approach to Defining Gene Function  abstract
Advisor:  Ron Prywes

Gene Therapy for Hepatocellular Carcinoma  abstract
Advisor:  Howard Worman

Advances in the Treatment of Acromegaly  abstract
Advisor:  Pamela Freda

Human Papillomavirus: The Silent Predator  abstract
Advisor:  Thomas Wright

Dominant Negative Serum Response Factor Transgene Expression Affects the Regulation of Activity-Dependent Early Response Genes Coding for Cell Signaling Proteins, Transcription Factors, Structural Proteins, and Enzymes in Primary Neuronal Cultures

Analysis of early response genes (IEGs) is currently at the forefront of neurobiological research because the protein products from these genes may be directly involved in processes underlying the physiology of learning and memory. Two classes of IEGs are known: one group codes for transcription factors regulating late response or effector genes, while another group encodes directly for effector proteins.  IEGs involved in learning and memory may be regulated by the serum response element (SRE), yet very little is known about the SRE’s specific role in these processes or what genes are affected downstream of this regulatory element.

The purpose of this study was to analyze the role of SRE in the regulation seventy-nine IEGs that have been previously shown to increase in response to stimulus-dependent gene expression.  Serum Response Factor (SRF) can be exploited for the purpose of this analysis because it is a factor that interacts with SRE directly to control transcription.  Primary neuronal cultures made from E18 rat embryos were transfected by engineered sindbis viruses containing an RNA template for dominant-negative SRF. A virus containing a template for EGFP acted as a control. Radiolabeled cDNA probes were made from the cell extracts of the infected cultures. The extracts were hybridized to dot-blots for quantification by radiography and phosphoimaging. 

The expression of transgenic dominant negative SRF was found to affect the regulation of a variety of activity-dependent IEGs in primary neuronal cultures. The transcription factors were: c-fos, CREM, SRF, AML-1, STAT3, and DNA pol beta.  Cell signaling genes included: E4BP4, Rheb, Homer, BDNF, and seven in absentia homolog (SIAH). The enzymes affected were BSM-1, Cox-1, and TIMP.  The structural proteins were Arc/Arg 3.1 and ABGP. The alteration in activity-dependent expression of these IEGs in the presence of dominant negative SRF implies that the SRE may control specific cellular functions relating to synaptic plasticity, either as the major regulatory element in IEG transcription, or in cooperation with other regulatory elements such as the cAMP-Responsive Element (CRE). However, further research is needed in vitro and in vivo to elucidate the roles of these genes in specific molecular mechanisms that underlie learning and memory.

Expression and production of recombinant Heat Shock Protein for eliciting an immune response in the therapeutic and prophylactic treatments of cancer

George Christopher Angelos, M.A. 2004

The involvement of heat shock proteins (HSP) in the antigen-delivery arm of the immune response is well established in the literature. Thus, it has been the goal of several groups to exploit these proteins for their known epitope-ferrying abilities and develop a new class of immunovaccines. Mojave Therapeutics, Inc., a privately held biopharmaceutical company, has two proprietary HSP-based approaches designed to enhance immune responsiveness against both malignant neoplasms and chronic viral infections, where CTL responses are essential to disease resolution. One is a gene therapy platform for transfecting the diseased target cells with a recombinant HSP encoding gene, selectively enhancing antigenic peptide/HSP association in vivo following HSP overexpression. As first step in the continued process of developing that goal, I successfully created an mHSP70/pcDNA3 vector showing murine HSP70 overexpression in transfected Cos-1 cells (also performed for mBiP, the endoplasmic mHSP70 homolog). The other approach has been using in vitro complexes of recombinant HSP70 associated with antigenic peptides that have been synthesized in tandem with high-affinity, HSP-binding (JavelinTM) sequences. Thus, to provide a pure source of recombinant protein, I implemented a commercially available chitin-bead based purification system (NEB) exploiting the self-catalytic properties of intein as a self-cleavable fusion tag. In addition, I successfully optimized this purification scheme through Triton-washing, minimizing endotoxin contamination (a pyrogenic artifact of bacterial fermentation) while maintaining biochemical and functional integrities of the expressed HSP70 protein; this system, however, was not yet found to be amendable for corresponding production of murine BiP. Finally, using HSP70 made per this protocol, I observed positive CTL response against murine and human epitopes in mice immunized with peptide/HSP complexes (directly and indirectly measured). Finally, I studied functional responses by following tumor progression in mice challenged with EG7 tumor cells following prophylactic HSP/peptide immunization.

Stem Cell Transplantation for Repair Following Traumatic Spinal Cord Injury

Traumatic spinal cord injury (SCI) is a devastating condition that exacts a lifelong physical, psychological, and financial toll from its sufferers. Complex secondary pathologic processes and the limited regenerative capacity of the central nervous system hinder repair following SCI. Recently, stem cells have received significant attention as possible therapeutic modalities in SCI due both to their ability for self-renewal and multi-lineage differentiation, and the ease in which they may be expanded and manipulated in culture. Stem cells have been isolated from a variety of embryonic and adult sources both within and outside the nervous system. In various animal models, it has been demonstrated that these cells can survive, migrate and differentiate following transplantation into injured or damaged spinal cords.  Strategies for stem cell transplant therapy in SCI include remyelination, neuron replacement and axonal regrowth. Problems remain concerning the identity of stem cells, the timing of transplantation, and the appropriate differentiation of stem cells both in vitro and in vivo. Research from developmental neuroscience will necessarily accompany efforts aimed at stem cell therapy for SCI.

The Scientific Evolution of Genetically Engineered Plants: Current Applications and Future Implications

In recent years plant biotechnology has dramatically evolved, resulting in novel applications, increased productivity and diversification while developing a more sustainable agricultural industry.  Through the use of transgenic crops also known as Genetically Modified Organisms (GMOs) scientists are able to manipulate plant genes to enhance their desired end-product.  These plants are primarily created by biolistic techniques or through vector-mediated Agrobacterium tumefaciens bacteria transformation.  Transgenic plants were initially designed for specific agronomic benefits; however, their applications have broadened over time.  First utilized to complement existing methods in insect maintenance, the next generation of crops modified “output” traits to create plants with improved nutritional characteristics.  The third generation of crops offered new alternatives to drug development.  In this capacity, transgenic plants are utilized as a vehicle for pharmaceutical production.  Over time, the role of plant biotechnology will become more pervasive and influential in our daily lives.  Although genetically engineered plants possess great promise, they have been met with staunch debate.  Public concern regarding safety and environmental risk requires immediate attention from scientific authorities to promote clear empirical data that conveys the purpose and benefit of such technologies.  Field studies have demonstrated the potential environmental risks posed by Bacillus thuringiensis insect resistant transgenic plants.  However, the social benefits incurred by the adoption of Vitamin-A enriched plants for those malnourished, shed light on the humanitarian facets of transgenic crops.  Therefore, a benefit/risk approach is proposed to evaluate the key components needed to properly assess the environmental safety, scientific validity and utility of transgenic plants.

Alternative splicing, disease and the new age of antisense therapeutics

Mann Sheuan Pamela Chao, M.A. 2004

 Alternative splicing is an important mechanism that leads to the diversity of protein in humans. A mutation that causes a disruption in an alternative splicing pattern may have adverse effect to the human body. In this review, we will give an overview of the mechanism behind alternative splicing, as well as examine the human diseases that are related to defective alternative splicing, such as Frasier syndrome. Next, we will take a look at a few current therapeutic options, specific and none specific, for disease related to alternative splicing disruption. Finally, we will introduce the idea of antisense technology, a method that utilizes antisense oligonucleotide to block or destabilize mRNA transcripts, as a possible future therapeutic option for these diseases.

Frontiers in Alzheimer’s Disease –Advances and Controversies

 Jing Chen, M.A. 2004

              As the population of our current society ages, the occurrence of Alzheimer’s disease (AD) becomes more prevalent. It becomes imperative to understand the pathogenesis of this devastating disease and look for better treatment. Studies have established that genetics contributes to this disease. Decades of efforts and recent advances in searching for the genetic, biochemical, and pathological etiology have resulted in several hypotheses: the Aβ hypothesis, the Tau hypothesis, the mitochondrial dysfunction model, the two hit model, and the apoptosis hypothesis. Though some of these might be more convincing than others, there are considerable controversies surrounding them. While stressing the controversies on the etiology and genetic ambiguity of Alzheimer’s disease, this thesis will also review its clinical manifestation, pathology, genetics, current treatments, as well as new discoveries, and recent progress in this field.

        Currently many therapeutic strategies for AD are proposed or being developed based on the Aβ theory. In order to test this theory and to test whether these therapeutic strategies might be effective, a brief proposal at the end of this thesis will outline the engineering of temporal and tissue specific expression of a mutant human APP gene in a mouse model to ask whether the phenotype of poor memory in the aged AD mice model can be improved by shutting off the synthesis of the mutant human APP gene product  after Aβ plaques formation.

The History and Nature of Applied Cloning and the Ethics and Laws Governing Its Use

Cloning has a long and colorful history both within nature and within the lab.  An ancient survival tool used by a plethora of species in their drive to reproduce, cloning has only relatively recently found its way into the laboratory.  However, once scientists were able to successfully begin cloning animals they opened a Pandora’s Box with which humanity has yet to successfully deal with.  Issues such as politics, the social status quo, religion and ethics have and will continue to a say in the way this new science plays out.  It is the purpose of this paper to review those topics to date so as to provide a more thorough understanding of the issues for those going forward.  

Transgenic approaches to seasonal grass allergies

Allergies are among the most common conditions in the world where almost 500 million people suffer from them. One of the most common causes of allergies is grass pollen allergy. Current treatment for allergy includes antihistamines, corticosteroids and decongestants. But now, biotechnology may offer a cure for this disease: by genetically modifying the source of allergies.  Researchers have now found the genes responsible for inducing allergies and have silenced them by knocking-out the genes. Transgenic grass with hypoallergenic properties has been produced. But, far from been in the public in the near future, transgenic plants take a long time to commercialized due to public fear and government laws.

A Look into the Pathology and Treatments of Alzheimer’s Disease

Alzheimer’s disease (AD) is a progressive neurological disease that results in the irreversible loss of neurons that manifest in a wide spectrum of clinical symptoms that range from declines in memory to rapid changes in mood behavior.  The disease was characterized in 1906 by Dr. Alois Alzheimer who was the first to observe, under a microscope, an accumulation of plaque-like deposits in brain tissue. The markers that he observed were extracellular senile plaques containing the peptide amyloid beta (Aβ) and neurofibrillary tangles, which are known to be the pathological hallmarks of AD.  Today, approximately 4.5 million Americans have AD, which has more than doubled since 1980.  A rise in life expectancy and aging of the “baby boomers” generation are predicted to raise prevalence of AD to approximately 16 million Americans by the year 2050. 

Prevalence curves show that AD cases occur at a rising number with age.  Early onset Alzheimer’s disease (EOAD) are cases in which symptoms occur before age 60, and late onset Alzheimer’s disease (LOAD) are cases in which symptoms occur after age 60.  Acetylcholinesterase and glutamate inhibitors are available to alleviate the symptoms of AD, but they do not stop or reverse the progression of the disease.  Scientists are now in pursuit of drugs that will attack the mechanism of AD.  Based on the amyloid hypothesis the two mechanisms believed to modify the pathogenesis of AD are the following 1.) decrease the production of Aβ peptide with β- secretase and γ-secretase inhibitors and/or 2.) increase in the clearance of Aβ peptide through vaccination, passive immunization, or small molecules to destabilize aggregates of Aβ.  Clinical trials are being conducted on the above targets, and on other drugs on the market that have shown efficacy in other disease.  Results from these clinical trials are eagerly awaited as they may provide a cure and further understanding of AD. 

An Abbreviated Tour of the Path Taken While Unraveling the Mysteries of the Autoimmunity & Systemic Lupus Erythematosus

Scientists and clinicians alike have long marveled at the remarkable specificity and efficiency that are characteristic of the human immune system.  Yet, even with the great insight and achievements of our scientific ancestors, immunology is a field still in its infancy, for until relatively recently researchers didn’t have the tools to decipher the intricate workings of the human immune system.  Thankfully, the field of immunology has experienced a plethora of discoveries in the last quarter of a century due in large part to the many advances in biotechnology and biological protocols.  For the first time in history, researchers across many of the life science disciplines have the physical means to investigate and solve some of the greatest puzzles of our time.  For many scientists it has been a long road, but one that must be traveled for each new revelation and accomplishment is history in the making and which ultimately will benefit the entire human population.

MAPK Scaffold: Kinase Suppressor of Ras

Damara Gebauer, M.A. 2004

 To further understand mitogen activated protein kinase (MAPK) scaffold assembly and mechanism I will examine the biological role and structural function of kinase suppressor of Ras (KSR).The function of KSR as a scaffolding protein in the Ras/Raf/Mek/Erk pathway is widely accepted; however, its function as a kinase remains unclear as different groups report contradicting results. I propose to establish a robust in-vitro assay that will determine if KSR is a kinase and can phosphorylate its implicated substrate Raf [25].  Moreover, I plan to study the structural mechanism of KSR as a scaffolding protein by crystallizing the Raf-binding and kinase domain of the murine ksr gene product. The NMR structure of the cysteine-rich, CA3, conserved domain of KSR has been obtained and is currently available in the protein data bank (PDB) [18].  Solving a crystal structure of the Raf-binding and kinase domain will give us insight and much needed information about how MAPK scaffold complexes are formed.  In addition, I will determine the crystal structure of the dual specific kinase, Mek, which will be the first structure of this type of enzyme.

Directed Evolution: A Review of Evolutionary Approaches to Molecular Design

Because of their structural complexity, proteins have proved difficult to modify or design. Directed molecular evolution is an approach to protein design that uses an iterative  selection process that mimics natural evolution.  This approach has proven capable of generating diverse useful modifications of existing proteins, and may ultimately become powerful enough to be used for de novo protein design.  It has also generated many new functional RNAs and served as a window into the processes of evolution itself, perhaps especially at the beginning of life when the first replicating molecules emerged.   The use of evolution as a design tool may enable a new level of flexibility and power in laboratory-engineered macromolecules.

Automated Primer Design for Speciation of Closely Related Taxa

Polymerase Chain Reaction (PCR) is a fundamental tool in the detection of pathogens. Primers determine the sensitivity and specificity of any PCR based diagnostic assay. Computational biologists have developed programs to automatically generate primers for a given nucleic acid sequence. The programs utilize parameters which have been experimentally determined to carry out a heuristic search among many possible primers. Often diagnostic tests need to differentiate between very similar pathogens, introducing the need for specificity in primer design. A program was created which was optimized to design specific primers, given a nucleic acid multiple alignment and a taxonomic structure. The program can also design degenerate primers to amplify divergent sequences. The program was tested by designing primers to five serotypes of the Adenovirus family. The program selected primers based on melting temperature, GC content, degeneracy, amplicon size and ability to speciate. In singleplex and multiplex PCR, the primers amplified their target sequences. Some pairs were able to amplify 50 molecules from a large pool of human genomic DNA. The program cuts the development time of diagnostics primers by automating the tedious and difficult task of sequence comparison. Also, it provides a method for evaluating and optimizing primers which will be used in the same PCR conditions, aiding the development of multiplexes.

Current Research in Prion Diseases: Understanding the Molecular Biology of PrP Proteins

Jennifer M. Jolivet, M.A. 2004

Prion diseases are rare, neurological disorders that affect both animals and humans with devastating consequences.  Prion diseases have been recognized since the 18th century, and many advances in our understanding of the diseases have been made since then.  In humans, three groups of prion diseases have been identified: inherited, infectious, and sporadic.  The discovery of the prion protein in the 1980’s revolutionized our understanding of the disease because it was suggested to be the infectious agent.  The involvement of proteins in the disease is described by the protein-only hypothesis, which states the infectious prion is a protein devoid of nucleic acids that adopts an abnormal conformation.  The gene responsible for the prion protein is PRNP, and what makes it unique is its ability to code for two proteins with the same amino acid sequence.  The only difference between the proteins is their conformational structure.  PrPC, the normal cellular protein, is composed primarily of α helices, while PrPSc, the pathologic protein, contains β sheets and α helices.  For establishment of the disease, PrPC is converted to PrPSc.  Although there is a general description of how prion diseases develop, there lacks substantial information with regards to the key biological and molecular aspects of the PrP proteins, including the normal function of PrPC , the exact infectious agent, the location of conversion, mechanism of conversion, and factors involved in conversion.  Current research concentrates on answering many of these questions because until they are answered, prion diseases will continue to be a major public health problem.      

Therapeutic Targets for Resistant Gastrointestinal Stromal Tumors (GIST)

Gastrointestinal stromal tumors (GIST) are mesenchymal neoplasms of the digestive tract.  They are characterized by the expression of a constitutivelyactive KIT receptor tyrosine kinase (RTK) that results from mutations in the c-KIT gene.  One drug, imatinib mesylate (more commonly known as Gleevec®), has shown tremendous promise by targeting the KIT receptor and selectively inhibiting phosphorylation at the ATP-binding site.  Recent developments have shown a growing resistance to this drug.  A main feature of resistant GIST is the presence of multiple nodules either intra-abdominally or within the liver.  An investigation was carried out on a small subset of patients, each with multiple tumor nodules.  These patients developed resistance to imatinib mesylate after an initial tumor shrinkage response.  A comparison was made of the histologic response of each individual nodule in the molecular phenotype, since independent genetic events within individual nodules can lead to loss of response to imatinib mesylate.  DNA was obtained from each histologically distinct nodule.  PCR was performed for the exons of c-KIT and PDGFRα associated with mutations.  Sequence data was obtained and analyzed for any secondary mutations.  The data presented in this study shows that there is a high probability that the c-KIT gene within an individual nodule is capable of undergoing secondary mutations leading to loss of response to imatinib mesylate.  This data is however limited to a small subset of patients and further analysis needs to be done to determine the exact genetic mechanism in the development of these secondary mutation events. The data concludes that with resistance as a threat, new agents inhibiting the KIT receptor tyrosine kinase need to be investigated.  Furthermore, new targets for GIST therapy need to be researched.

Use of Statistical Analysis Methods on Hippocampal Multivariate Data to Avoid Acceptance of False Positives

Microarray technology has permitted a paradigm shift from hypothesis-driven to discovery-driven research, permitting analysis of gene expression for thousands of genes simultaneously.  The shift has brought with it the challenge of determining which analytical methods are best suited to the task.  This research compared two different approaches to analyze microarray data from a murine contextual fear conditioning experiment to distinguish genes involved in the early and late phases of long-term memory consolidation: an ad hoc “fold change” analysis method using only the Change Calls and Signal Log Ratios of Affymetrix Mas 5.0 software versus the statistical t-test and Anova methods.  The comparison of the two analytical approaches draws the conclusion that the statistical methods are more powerful and avoid the potential of deriving misleading results due to noise in the data from biological as well as experimental sources.  The research also revealed several critical guidelines in the design of an expression profiling microarray experiment: a) the original design of the experiment must take into consideration the statistical method to be employed and insure that the biological question being asked will be addressed by the statistical analysis; b) a preliminary test should be done to establish expression patterns of known genes relevant to the biological question being asked by the experiment and thus establish the minimum replicates required to observe that pattern; c) care must be taken to insure consistency in sample preparation; d) microarray data must be checked for good correlation of the microarrays, good signal detection, and good reproducibility of genes; and e) data must be normalized.

Alzheimer's Disease: Drugs in Development

Alzheimer’s disease can be described as the accumulation of plaque-like deposits in brain tissue. The disease is characterized by cognition impairments and partial or total memory loss. As the population ages, the incidence of Alzheimer’s disease is increasing substantially. An estimated 4.5 million people are affected by this disease in the United States and the annual incidence of Alzheimer’s disease is expected to begin a sharp incline around the year 2030, when the baby boomer population will be over age 65. Four acetylcholinesterase (AchE) inhibitor drugs are presently on the market. These AchE inhibitors include Cognex (tacrine), Aricept (donepezil), Exelon (rivastigmine), and Reminyl (galantamine). These drugs are helpful in that they alleviate the symptoms of Alzheimer’s disease, but they do not help indefinitely. There is no effective treatment available to stop the progression of Alzheimer’s disease and scientists still do not fully understand what causes the disease. These issues have inspired many pharmaceutical and biotechnology companies to continue their research efforts and search for options to improve memory, treat, and delay or prevent the disease altogether. Several AchE drugs are in clinical trials that bind to the enzyme acetylcholinesterase and block its action. Other companies are studying the role of muscarinic and nicotinic receptors in Alzheimer’s disease. When these receptors are stimulated, they cause extra AchE to be released and provide short term improvement and an increase in nerve signaling in the hippocampus. Other brain sites are believed to play a major role in Alzheimer’s disease. One drug, memantine, acts on the NMDA receptor, which is a neuronal receptor that can be stimulated by neurotransmitters such as glutamate. Glutamate is thought to contribute to normal mental function. Ampakines, a new class of drug, modulate the AMPA receptor, a subtype of glutamate receptors and increase excitatory synaptic responses in the hippocampus. These drugs have shown the ability to enhance learning and memory in some small study groups. Scientists are also looking at the effects of products that are already on the market, such as vitamins, supplements, statins, and non-steroidal anti-inflammatory drugs, in order to prevent or slow the progression of Alzheimer’s disease. Ultimately, there is an enormous market opportunity, with only half of the population with Alzheimer’s disease diagnosed, and therefore companies are trying to capitalize on this market need with numerous products in development.

Development of Methodology for Performing Sequence-Based Typing of Psoriatic Arthritis Patients in a Large Cohort of Irish Families, Using the HLA-B Locus

Psoriatic arthritis is a disease of the joints that is associated with psoriasis and is mediated through a CD8 T-cell pathway.  Since, CD8 T-cells recognize peptides presented by class I molecules that are encoded by the HLA locus of the Major Histocompatibility Complex, and there is evidence that certain alleles of the class I HLA-B and HLA-C loci are associated with psoriasis and psoriatic arthritis triggered by peptides presented we believe this to be one of the accessible but still incompletely understood factors responsible for this autoimmune disease.  As such, we sought to optimize the methodology to type a large population of individuals that are mostly genetically isolated, as well as, well-documented.  For this we will use a patient population of approximately 500 patients (composed of probands, family members of said probands, and control patients) in order to seek out any alleles that may confer susceptibility to psoriatic arthritis. The study design involves determining the inheritance of HLA-B and HLA-C alleles in simplex and multiplex families to determine whether there is linkage between particular HLA-B and HLA-C haplotypes and the development of disease.  This project involves amplifying patient DNA via PCR, and sequencing of their HLA-B and HLA-C loci to determine the particular allele.  We accomplished this by first acquiring blood samples from a clinical unit we are working with that is in Ireland, isolating DNA, amplifying through intronic primers and sequencing these samples using modern DNA sequencing techniques.  Afterward, the samples, were typed using an allele-calling software, and these results were compared to a master database for quality checks and for allele comparison.  The major portion of this thesis deals with development of the typing procedure.

Pre-Erythrocytic Malaria Vaccines: Identifying and Developing a Candidate Vaccine that will Induce Sterile Protective Immunity

  Sheila Ramgopal, M.A. 2004

Malaria is a parasitic disease of major health and socioeconomic significance in several regions of the world, causing nearly 3 million deaths annually.  With increased awareness and funding for improving malaria control efforts, developing a vaccine that induces effective protection against infection is crucial to curbing this devastating parasite.  With the only proven protective efficacy of vaccination seen with radiation-attenuated sporozoites, a pre-erythrocytic vaccine targeting the sporozoite and liver stages of Plasmodium is most likely to be effective.   Since vaccination through bites of irradiated mosquitoes is not feasible, a subunit vaccine containing candidate immunogens that stimulates protective immune responses is being studied.  With recent technology advances in vaccine delivery design, recombinant DNA techniques, and immune response detection assays combined with the new knowledge gleaned from the recently sequenced Plasmodium genome, identification of new immunogenic candidates and improving immunogenicity of old ones is being studied.  This review covers studies on these various candidate vaccines and trials evaluating their safety and efficacy of immune response and protection.  

 The Role of Fas-Mediated Apoptosis in the Development of Hashimoto’s Thyroiditis

 Jeannette Richmond, M.A. 2004

Hashimoto’s Thyroiditis is an autoimmune thyroid disease characterized by lymphocytic infiltration of the thyroid, as well as the destruction of this organ.  Fas-mediated apoptosis is believed to play an important role in the destruction of thyrocytes.  However, it is unclear if Fas-mediated apoptosis of thyrocytes is initiated by infiltrating lymphocytes, or neighboring thyrocytes. The expression patterns of Fas and FasL on thyrocytes and lymphocytes may indicate which cell type is responsible for thyrocyte destruction. 

In addition, there are other factors which affect the extent to which Fas-mediated apoptosis is able to take place.  These include the possible removal of a labile protein inhibitor, the glycosylation status of Fas and the cytokine microenvironment.  In addition the expression of various anti-apoptotic genes affects whether apoptosis can take place.  Surprisingly, some groups found that Fas-mediated apoptosis may be involved in the resolution of experimental form of HT.  Further work is required to unravel the role of this important pathway.

Evidence the Spliceosome is a Ribozyme

 Nima Shah, M.A. 2004

Though many have the misconception of RNA as being only a transitional phase in making proteins, RNA can be both informational and catalytic. The catalytic property of RNA has led to the creation of an RNA World.  The RNA world hypothesizes that billions of years ago RNA was the catalytic material and was responsible for carrying out fundamental biological functions.  Though catalytic RNA exists as a fossil of the pre-biotic era, ribozymes still function at the core of fundamental biological processes, for example, the ribosome.

The current debate is whether the spliceosome, similar to its sister the ribosome, is also a ribozyme. Group II introns and group I introns can splice in an autocatalytic manner.  Nuclear splicing has an uncanny resemblance to group II splicing.  The evidence that the spliceosome is a ribozyme is threefold:

1)    Group II introns and the spliceosome undergo the same two transesterification reactions and essential residues are conserved in both systems.

2)    Similar to many ribozymes, the spliceosome has Mg2+ dependency

3)    There are strong interactions between snRNAs and the pre-mRNA at the catalytic core of the spliceosome.

 RNA Interference: A Revolutionary Approach to Defining Gene Function 

Timothy J. Smith, M.A. 2004

The completion of the sequencing of the human genome provided scientists with the nucleotide-by-nucleotide sequence of each of the approximately 30,000 genes that comprise our DNA.  While forward genetics has provided the scientific community with a toolbox of methods to define gene function, more efficient and cost-effective methods are needed to describe the functions of the vast number of new genes uncovered by the Human Genome Project.  During the past several years, RNA interference has emerged as a useful tool for reverse genetic analysis and has the potential to evolve into a revolutionary drug development tool.  Unique to eukaryotes, RNAi is an evolutionarily conserved, post-transcriptional gene silencing mechanism mediated by double-stranded RNA. 

The double-stranded RNA is processed into 21-23 nucleotide short-interfering RNAs, which associate with and degrade a specific mRNA target.  Gene transcription is allowed to proceed as normal, but protein translation is prevented by selective degradation of the encoded mRNA.  Because the entire sequence of the human genome is known and siRNA’s can be designed in a nucleotide specific fashion, RNAi theoretically has the potential to silence any gene that is related to human disease.  While obstacles such as effective delivery to the cells of interest and siRNA stability exist, an enormous potential for RNAi to develop novel therapeutics exists.

Gene Therapy for Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the most common cancers, and its mortality is very high.  The major etiology of HCC is chronic hepatitis B virus (HBV) infection followed by chronic hepatitis C virus (HCV) infection, although nonviral causes also play a role in minority of cases.  HCC is associated with cirrhosis in most cases. Researchers have been trying to discover efficient treatment methods for HCC without side effects and recurrence.  I examine here the genetic characteristics of HCC and possible gene therapies to treat it.

Advances in the Treatment of Acromegaly

Chiemi Suzuki, M.A. 2004

Acromegaly is a rare endocrine disorder resulting from the hypersecretion of growth hormone from a benign pituitary adenoma.  Researchers have developed numerous treatments for acromegaly – transsphenoidal surgery, radiation, dopamine agonists, and somatostatin analogs – however these treatments have been met with limited success, providing a cure for only some patients.  Further, there is no consensus on the best course of treatment for acromegaly.  Through genetic engineering and rational drug design researchers have developed a novel therapy, growth hormone receptor antagonists.  Pegvisomant, the first drug in this class, has demonstrated efficacy in over 97% of patients, far out-pacing the effectiveness of traditional therapies.  Due to its increased predictability of response, high cure rate, normalization of biochemical markers of disease, good safety and adverse event profile, and ability to correct metabolic co-morbidities, pegvisomant should be considered the best treatment for acromegaly. 

Human Papillomavirus: The Silent Predator

Human Papillomavirus is predominately a sexually transmitted disease that accounts for approximately one-third of all new sexually transmitted disease infections.  Over the last decade, evidence has shown that infection with specific types of Human Papillomavirus can lead to the development of cervical cancer and high-grade precursors.  For this reason, several molecular methods have been developed to identify these viruses in clinical specimens.  Unfortunately, most cases of Human Papillomavirus remain undetected because women do not seek the appropriate medical attention.  This is due, in part, to the lack of information available to the general public.  While many women have begun to make the connection between Human Papillomavirus, abnormal cytology, and cervical cancer, many have not.  Therefore, it is essential that the risks associated with Human Papillomavirus infections are made known.