Elasticity of Skeletal Muscle PEVK Exons Determined by Single-Molecule Techniques  abstract
Advisor:  Julio Fernandez

The Genetic Structure of Human Population  abstract
Advisor: James Russo 

Jonathan Wei-Ji Chang, M.A.

Avastin The Champion of Angiogenesis Inhibitors  abstract
Advisor:  Monica Motwani

Tiffany C. Chang, M.A.

Enhancing Pathogen Resistance in Crop Plants through Transgenic Technology abstract
Advisor: Thomas Eulgem

Julie L. Cheong, M.A.

The Role of Dopamine and DOPAC on Mitochondrial Inhibition: Implications for Parkinson’s Disease abstract
Advisors:  Martin Gluck and Alexander Tzagoloff

Aaron I. Davis, M.A.

Extension of Life-Span through Telomerase: Future Implications in Cancer Research abstract
Advisor:  Carol Lin

John DiClerico Jr., M.A.

Effects of FTDP-17 Mutated Tau Pseudophosphorylation at Alzheimer’s Disease Phosphorylation Sites on Self-Assembly and Microtubule Stability abstract
Advisor:  Alejandra Alonso

Daniel Anthony Ferrara, M.A.

Forensic DNA Fingerprints: Matching Substance with Source  abstract
Advisor:  Harmen Bussemaker

Meng-Tien Hsieh, M.A.

HBV- and HCV-Related Hepatocarcinogenesis: Mechanisms and Implication of Treatment abstract
Advisor:  Lili Yamasaki

Hirotaka Ishibashi, M.A.

The Past, Current, and Future Development of Antiobesity Drugs  abstract
Advisor:  Kuan Zhang

Miki Kim, M.A.

The X Chromosome and Cognitive Ability Differences Between the Sexes abstract
Advisor: J. Russo

Mihajlo Krsmanovic, M.A.

Regulation of the Cell Cycle: Potential Cancer Therapeutic Target  abstract
Advisor:  Ron Prywes

Grace S. Lee , M.A.

Disease-Modifying, Neuroprotective, and Stem Cell Strategies for the Development of Mechanism-Based Therapeutic Interventions in Alzheimer’s Disease abstract
Advisor: Tae-Wan Kim

Yelena Luzhanovskaya, M.A.

Strategies for Suppressing CD4+CD25+ Regulatory T Cells Function to Enhance the Antitumor Immune Response of Cancer Vaccines  abstract
Advisor: Carol Lin

The Potentials and Limitations of Monoclonal Antibody Therapy in the Treatment of Cancer  abstract
Advisor: Ron Prywes

The Underestimated Role of Placental Growth Factor in Tumor Angiogenesis abstract
Advisor: Jan Kitajewski

A Critical Review of Colorectal Cancer with Specific Emphasis on Current Screening Options
and Newly Developed  Targeted Therapies  abstract
Advisor: Daniel Kalderon

An in vitro Co-culture System for Studying the Macrophage Regulation of Adipocyte Metabolic  Expression   Profile abstract
Advisor: Anthony Ferrante

Derek S.C. Park, M.A.

The Application of Oral Genomics with Microarray Technology to Bioinformatics for Better Treatment of Oral Diseases   abstract
Advisor:  Shan Lal

A Review Of The Production And Engineering Of Human Monoclonal Antibodies As Therapeutic Agents  Against Disease abstract
Advisor: Amer Beg

Chemokine Receptors as HIV-1 Coreceptors: Recent Advances in the Understanding of Viral Entry and the Discovery of Therapeutic Agent  abstract
Advisor:  Jerry Di Salvo

Nelson Scharadin, M.A.

A Potential New Use for Aptamers: Real-time Biological and Chemical Detection  abstract
Advisor:  Lawrence Chasin

The Ubiquitin-Proteasome System: Roles in Tumor Suppressor Destabilization and Prospects for Targeted Therapies   abstract
Advisor: Carol Prives

Psoriasis: History, Etiology and Monoclonal Therapies abstract
Advisor: Robert Winchester

Hua Zhong, M.A.

The Role of Soluble Guanylyl Cyclase (sGC) in Tumorgenesis and as a Potential Target for Cancer Therapy  abstract
Advisor: I. Bernard Weinstein

A Situational Analysis of Multiple Sclerosis: Current and Future Disease Modifying Therapies  abstract
Advisor:  Joan W. Witkin

Elasticity of Skeletal Muscle PEVK Exons Determined by Single-Molecule Techniques

The giant muscle protein titin’s I band region contains a domain enriched for the amino acids proline, glutamate, valine, and lysine, and is denoted the PEVK domain. The PEVK domain of titin encodes a random coil shown to be an important factor in titin’s passive elasticity. Multiple exons encode the PEVK region of titin, and it is believed that muscle specific PEVK exon splicing adjusts the flexibility of the random coil. To better understand the determinants of titin’s passive elasticity, we have used single molecule atomic force microscopy to examine the role of three PEVK exons, 120, 161 and 184. All three exons vary in both PEVK as well as proline composition. Because the random coil nature of the PEVK domain does not produce a mechanical unfolding force signature, we have engineered a protein chimera containing the PEVK exons combined with the well-characterized I27 domain. Such a construct, [(exonPEVK)3I27]4, allows us to unambiguously define single molecule mechanical events during which the PEVK region has been stretched. By fitting the worm like chain model to our data we measured the persistence length of the PEVK domain. All three exons showed a wide distribution of persistence length values ranging from 0.26 - 2 nm. These results suggest that neither proline nor overall PEVK content determines the flexibility of PEVK. One possible way to interpret this data is that the large number of PEVK exons may serve as equally flexible random coil spacers adjusting the length of the PEVK region of titin and thereby affecting passive elasticity.

The Genetic Structure of Human Population

Kathy Jo Carstarphen, M.A. 2005

The determination of the sequence of the human genome is one of the most important human achievements of our time. Although somethink that it has limited potential, for medical and historical purposes, knowledge of the human genome reveals the past, present and futureof humanity.

The study of the genetic structure of human populations reveals the genetic diversity of humans and thus has anthropological, political and medical significance and implications. This paper will describe the differences among approaches for studying the genetic structure of human populations. The outcome and conclusions differ in these studies mainly as a result of the approaches taken to the analysis of the allelic frequencies. Some of the theories presented in this paper actually refute the importance of population structures.   In addition to addressing these different approaches, this paper will also discuss the effects our new understanding of genomics will have on medicine, especially pharmacogenetics, and the social and ethical implications of genetic diagnostics. The genetic structure of human populations is a foundation for pharmacogenetics, which is the study of individualized medicine. This could be the future of medicine, most importantly for cancer therapeutics. It is highly likely that this also is a workable solution for genetically identified diseases. The market for this treatment is extremely large, and for many reasons, including financial, this type of treatment system is a highly discussed topic in heath care.  The social and ethical implications of anything genetically diagnostic are unavoidable. Throughout history, information about races and “pre-dispositions” have at times been helpful but have also led to horrible decisions. The misuse of genetic predisposition information has life-altering consequences for the patients involved.  Another important aspect of this field is the development and use of biotechnology and genome-scale approaches. Haplotyping has become important for many reasons, including disease mapping and determining human history through migration patterns. The technologies that go into haplotype studies are varied, and the analysis of these technologies are just as plentiful.  This paper therefore also reviews the technologies and methods of haplotyping. This paper attempts to link the knowledge of the human genome, the biotechnological developments in the area of haplotyping, the importance of personalized medicine for the understanding of an individual’s genetic makeup, and how the genetic structure of human populations sets a basis or framework for understanding the differences of races and therefore the racial propensity for disease.

Avastin -The Champion of Angiogenesis Inhibitors

 Jonathan Wei-Ji Chang, M.A. 2005

Tumor growth and metastasis is dependent on angiogenesis as oxygen and nutrients are supplied by the vasculature.  The vascular endothelial growth factor (VEGF) is the key regulator of angiogenesis.  Avastin, a humanized monoclonal antibody directed at VEGF, has been shown to reduce tumor growth in vivo.  The FDA’s approval of Avastin as first-line therapy for metastatic colorectal cancer validates the theory that disrupting angiogenesis, particularly through the VEGF pathway, can help in the battle against cancer.  The field of angiogenesis inhibitors continues to bloom as additional oncology indications for Avastin are explored, alternative strategies targeting VEGF are tested, and anti-VEGF therapies for non-cancer diseases are approved.

Enhancing Pathogen Resistance in Crop Plants through Transgenic Technology

Tiffany C. Chang, M.A. 2005

 As the world population continues to rapidly grow, food supplies must meet the demands of each person’s nutritional requirement. One means to increase food supplies is by mitigating crop loss caused by plant pathogens. Traditional approaches include plant breeding and chemical applications, but these approaches can not effectively protect plants from pests. Transgenic technology, such as manipulation of gene expression and utilization of RNA silencing mechanisms, can more efficiently enhance plant resistance against pathogens. During the past few years, the increasing knowledge of plant defense mechanisms and the identification of key regulatory genes in plant’s defense systems allowed the development of sophisticated transgenic approaches to enhance plant resistance. The transgenes can come from the same plant species, from different plant species, and even from pathogens. In theory, these genes can be manipulated by overexpression, induced expression, tissue-specific expression, stable gene knockouts, and silencing by RNA interference. Transgenic technology has been applied to crops, and it is leading to promising results. However, public concerns regarding safety and ethical issues need to be addressed before genetically modified (GM) crops can be to successfully introduce to the food supply. Transgenic technology shows great promise, some limitations and exciting opportunities to develop many new tools for combating plant pests.

Role of Dopamine and DOPAC on Mitochondrial Inhibition: Implications for Parkinson’s Disease 

 Julie L. Cheong, M.A. 2005

Parkinson’s disease results primarily from the selective loss of dopaminergic neurons in the substania nigra.  The current available medications for Parkinson’s disease treat the symptoms but are not efficacious in halting or retarding the neurodegeneration of the dopaminergic neurons.  The main obstacle for developing neuroprotective therapies is due to the limited understanding of the key molecular events that provoke neurodegeneration.  In recent years, however there is continual evidence supporting a possible role for mitochondria dysfunction in its cause.The work presented here uses freeze-thawed mitochondria preparations as the experimental system for exploring what the chemical and biochemical mechanisms of  dopamine and its metabolite, 3,4-dihydroxyphenylacetic acid have on mitochondrion that are compromised due to oxidative stress or aging.  Investigating and characterizing the non-enzymatic pathways are the main focus of this experimental research.  We have acquired evidence in our laboratory to suggest possible mechanisms operating in compromised dopaminergic neurons. Anti-oxidants such as catalase, deferoxamine (an iron chelator), Trolox (a vitamin E derivative) and glutathione were examined to see if inhibition to the electron transport chain activity could be prevented. Glutathione was shown to be a potent protector against the inhibitory effect of dopamine on mitochondrial respiration.

Extension of Life-Span through Telomerase: Future Implications in Cancer Research

 Aaron Davis, M.A. 2005

 Cancer is one of the most devastating diseases known in the world. Contrary to the name, cancer refers to a group of over one hundred diseases, any of which will end up ultimately effecting 50% of males and 35% of females in the USA during their lifetime. Details supporting telomere biology, and the end-replication problem leading to genomic instability and re-stimulation of telomerase, are critical to the understanding of cancer. It is scientifically established that telomerase up-regulation is a major event responsible for continuous tumor cell growth. The telomere-telomerase hypothesis of cancer and aging is based on the findings that telomere length is maintained by a balance between processes that lengthen telomeres (telomerase) and those that shorten telomeres (the end-replication problem). Research has been done to prove telomere shortening may be the “molecular clock” counting the number of times a cell has divided to trigger when cellular senescence should occur. Telomerase is the reverse transcriptase ribonucleoprotein complex required for maintenance of telomeric DNA “capping” of eukaryotic chromosomes. Without telomerase, telomeres eventually shorten to a critical length and chromosomes become unstable and activate cell cycle arrest and/or cell suicide pathways. Human telomerase is highly repressed in most normal somatic cells, sometimes expressed in certain stem or progenitor cells for homeostatic purposes, and constitutively activated in germline and tumor cells. In contrast to normal cells, tumor cells do not display a loss of telomere length with cell division. Research has suggested that telomere stability is required for these cells to escape replicative senescence and proliferate indefinitely. Since immortalization can occur through telomerase expression, reactivation of telomerase is believed to be the rate-limiting step required for continued proliferation of advanced cancers. There are many publications that support the association between tumorigenesis and activation of telomerase, and many researchers have targeted telomeres or telomerase for the development of potential therapeutic agents within the last several years. The knowledge developed on telomerase and its extensive presence and activity in tumorigenesis will have broad implications as a therapeutic mechanism for future anti-cancer therapies.

Effects of FTDP-17 Mutated Tau Pseudophosphorylation at Alzheimer’s Disease Phosphorylation Sites on Self-Assembly and Microtubule Stability

Hyperphosphorylated tau, which is the major protein of neurofibrillary tangles in Alzheimer’s Disease, FTDP-17 and many other neurodegenerative diseases, is most likely the result of both an imbalance in the regulation of kinases and phosphatases as well as mutations in tau that allow it to be a more favorable substrate for phosphorylation. Therefore, in order to understand the importance of site specific phosphorylation and its effects on tau’s ability to bind microtubules variants of a full-length tau construct were generated for Serine 199, 235, 262, 396 and Threonine 212. Through site directed mutagenesis these sites were changed to either inhibit phosphorylation by alanine substitution or mimic phosphorylation by glutamic acid substitution. Biochemically, since phosphorylation was not induced and only basal levels where present mutations at these sites did not significantly change phosphorylation levels at other sites. When assayed to determine if these constructs where indeed cytotoxic values were found to be statistically insignificant. However, certain trends existed for the FTDP-17 point mutation R406W and its glutamic mutants. Although these mutations proved to not be cytotoxic, caspase assays showed an elevated level of caspases 3&7 in PC-12 cells expressing the point mutation R406W and R406W constructs with glutamic mutations suggesting a potential link between expression of mutant tau’s and apoptosis.  The most promising and convincing results came from immunocytochemical staining of total tau and tubulin for microtubule network stability. Upon expression in PC-12 cells tau for the most part colocalized with tubulin and the microtubule network was intact, with the exceptions of sites 235, 262 and most notably 212. Here tau was found as an unbound protein which aggregated in the cell body in R406W 235 E, 262 E and 212 E. However, R406W 212 E had more then just aggregation; tau appeared to be forming filamentous structures that were negative for tubulin filament suggesting that phosphorylation at this site induces tau polymerization and filament formation.

 Forensic DNA Fingerprints: Matching Substance with Source 

Daniel Anthony Ferrara, M.A. 2005

Forensic DNA fingerprints make use of the natural genetic polymorphisms present among humans to identify biological substances.  Beginning with blood types, different biological markers have been used throughout history for this purpose.  Short Tandem Repeats (STRs) are the current polymorphism of choice for forensic DNA analysis.  STRs consist of repeated short “core” sequences that are present at specific locations in the human genome.  DNA typing methods have also evolved with time.  Forensic scientists now use automated capillary electrophoresis to separate different STR alleles by subjecting test DNA to an electrical field in a sieving matrix.  STR loci are identified by fluorescent signals which have been attached to each locus via the polymerase chain reaction.  Each DNA fragment’s migration through the capillary can be used to determine its size and therefore the number of “core” repeats present.  The resulting set of STR loci and their repeats constitute a genetic fingerprint that is unique to every individual.  One must remember that a DNA fingerprint is evidence that must be weighed by a jury with all other evidence for a particular case.  Forensic DNA labs maintain strict quality assurance programs to ensure that their data will hold up in court.  The Federal Bureau of Investigation has standardized 13 STR loci which are used to guarantee that this fingerprint can be shared throughout the law enforcement community.  It seems likely that forensic DNA analysis will eventually shift to typing Single Nucleotide Polymorphisms (SNPs) using more sophisticated mass spectrometry techniques.  Though we possess these powerful tools of identification, we must also protect the rights of individual citizens.  The government must assure us that genetic information will be used for criminal justice purposes only.  If used properly by law enforcement agencies, nucleic acids remain a priceless tool for matching a biological substance with its source.  This paper will explore the assorted polymorphisms, typing methods, history and ethics of genomic biotechnology as it is used to serve justice.

HBV- and HCV-Related Hepatocarcinogenesis: Mechanisms and Implication of Treatment  

 Meng-Tien Hsieh, M.A. 2005

 Hepatocellular carcinoma (HCC) ranks as the fifth most common cancer in the world, and the fourth leading cause of death, with 1 million new cases per year. Among the risk factors of HCC, chronic infection with hepatitis B and C viruses (HBV and HCV) appear to be the strongest two. The genome structures of hepatitis B and C viruses have been plotted, and yet the detail functions of specific viral proteins, such as HBx protein of HBV and core protein of HCV, are not well established. It is now well accepted that carcinogenesis is a multi-step process including inactivation of tumor suppressor genes and deregulation of cellular oncogenes, as well as other genetic alterations. Hepatocellular carcinoma is of no exception. However, the altered genes and pathways in HCC are less characterized comparing to cancers like Hereditary Nonpolyposis Colorectal Carcinoma (HNPCC). Since over 50% of HCC incidences are HBV- and/or HCV-related, the molecular mechanisms underlying HBV- and HCV-related hepatocarcinogenesis are reviewed in this article, as well as choices of treatment indicated by these players.

The Past, Current, and Future Development of Antiobesity Drugs

Hirotaka Ishibashi, M.A. 2005

Obesity is a chronic condition defined by an excess amount of body fat. The accumulation of too much fat in the body increases the risk of developing type II diabetes mellitus, heart disease, stroke, hypertension, high cholesterol, certain forms of cancers, respiratory complications, and other disorders. The number of obese American adults has doubled and the number of obese children and teenagers has almost tripled over the last two decades. The obesity (ob) gene, which encodes the molecular components of the physiological system that regulates body weight, was identified in 1994. It has been further showed that leptin, a16 kDa protein, which is encoded by the ob gene and is synthesized in adipose tissues, makes mice grossly obese and diabetic. This discovery opened the doors to greater understanding of obesity research and brought new hope to millions of obese people. Leptin was hailed as a potential wonder drug; however, leptin research discontinued due to insufficient efficacy. Consequently, many pharmaceutical and biotech firms are designing second-generation versions of leptin.

Why are there not many antiobesity drugs today? It is simply that obesity is a complex disease. Therefore, the identification of major pathways and molecular mechanisms regulating body weight plays a crucial role in the development of new antiobesity drugs. Understanding the feedback model for modulating the central controller regulating body weight, there might be four important strategies for primary drug action that might lead to significant weight loss: 1) reducing food intake, 2) blocking nutrient absorption, 3) modulating fat or protein metabolism or storage, and 4) increasing thermogenesis.

The X Chromosome and Cognitive Ability Differences Between the Sexes

Miki Kim, M.A. 2005

Since the late 1800’s, surveys of census data have shown that males are more susceptible to mental retardation than females, leading to the hypothesis that genes related to cognitive ability may be found on the X chromosome.  Additionally, research in psychology using techniques such as fMRI has shown that on average, men and women “think differently,” at least with regard to how they approach visuospatial and verbal tasks.  These correlations have led to the possibility that differences between the sexes may be attributable to the X chromosome.  Recent research has been able to broadly support this hypothesis, although not definitively prove it.  The recent sequencing of the X chromosome has shown that many genes related to cognitive ability may be X linked.  Studies of X-linked mental retardation have identified certain X-linked genes that are fundamental to neurobiological processes.  Additionally, the phenotypic variability that is observed in cognitive abilities can be explained by mechanisms of the X chromosome such as X chromosome inactivation and genomic imprinting, as recapitulated (reproduced? modeled?) by RNAi, as well as monozygotic twin studies.

Regulation of the Cell Cycle: Potential Cancer Therapeutic Target 

 Mihajlo Krsmanovic, M.A. 2005

The ability of cells to maintain genomic integrity is vital for cell survival and proliferation. Lack of fidelity in DNA replication and maintenance can result in deleterious mutations leading to cell death or, in multicellular organisms, cancer. Many known and well-described signal transduction pathways play critical roles in regulating cell cycle progression. In addition cells have employed mechanisms that insure DNA stability in the face of genotoxic stress. In particular, the mammalian cell cycle checkpoint functions, and their role in maintaining DNA stability during the cell cycle following exposure to genotoxic agents, and the gene products that act in checkpoint function signal transduction cascades. Key transitions in the cell cycle are regulated by the activities of various protein kinase complexes composed of cyclin and cyclin-dependent kinase (Cdk) molecules. Surveillance control mechanisms that check to ensure proper completion of early events and cellular integrity before initiation of subsequent events in cell cycle progression can generate a transient delay that provides the cell more time to repair damage before progressing to the next phase of the cycle.  Several human heritable cancer-prone syndromes known to alter DNA stability have been found to have defects in checkpoint surveillance pathways. Furthermore, the disruption of normal cell cycle regulation, which is the hallmark of cancer, presents numerous opportunities for targeting checkpoint controls to develop new therapeutic strategies for this disease. Such strategies include induction of checkpoint arrest leading to cytostasis and ultimately apoptosis, arrest of proliferating cells in stages of the cell cycle which may sensitize them to treatment with other therapeutic agents such as radiation, and targeting of therapies toward specific regulatory components of the cell cycle.

Disease-Modifying, Neuroprotective, and Stem Cell Strategies for the Development of Mechanism-Based Therapeutic Interventions in Alzheimer’s Disease

Grace S. Lee, M.A. 2005

Currently, there are no diagnostic biomarkers, no preventative or disease modifying treatment, and no cure for Alzheimer’s disease (AD), a condition considered to be the most devastating unmet need amongst neurological disorders.  Progressively after disease onset, the clinical cognitive and functional status of the patient deteriorates and inevitably ends in death.  Unfortunately, the insidious nature of neurodegeneration has thus far been unstoppable and there is an urgent demand for safe and effective therapies.  Much interest has been vested towards elucidating the pathogeniccascade, identifying target for pharmacologic intervention, and adapting emerging biotechnology to develop mechanism-based therapeutics.  Particularly, stem cell therapy holds tremendous potential as an alternate therapy for neurodegenerative conditions.  This review summarizes the existing hypotheses on AD pathogenesis and presents the scientific rationale behind current disease-modifying or neuroprotective strategies in the development of chemically synthesized therapeutics.  The practical utility, potential benefits, perceived challenges, and general limitations of stem cell strategy are critically evaluated.  The successful mastery of this biotechnology and integration with existing disease-modifying and neuroprotective strategies are recommended.  Once achieved, this three-pronged approach may evolve to be the elusive fulcrum of a paradigm shift away from amelioration of symptoms using pharmacologic monotherapy and towards a more comprehensive treatment regimen for Alzheimer’s disease utilizing stem cell and adjuvant therapy.

Strategies for Suppressing CD4+CD25+ Regulatory T Cells Function
to Enhance the Antitumor Immune Response of Cancer Vaccines

Yelena Luzhanovskaya, M.A. 2005

Immunotherapy has already become a standard approach of cancer treatment. While much of the success in this area lies within passive immunotherapy, optimism for cancer vaccination strategies is increasing. Numerous clinical studies evaluating different cancer vaccine approaches have established the safety and bioactivity of some vaccines, with a low rate of clinical responses. The recent studies have revealed that CD25+CD4+ regulatory T (Treg) cells play essential roles in inhibiting naturally occurring and therapeutically induced protective immune responses against tumors. Thus, the depletion of suppressive action of Treg cells is in the focus of present cancer vaccine research. The suppressive mechanism of action of Treg cells is multifaceted and there is no single effective method to suppress Treg cell in order to break tumor tolerance. Several strategies targeting the negative influence of Treg cells during cancer vaccination in combination with standard cancer therapies and adjuvants are just entering clinical trials. Therefore, developing the most potent strategies for breaking immune tolerance, while maintaining the efficacy of traditional approaches to cancer management, will ensure successful integration of vaccines into the standard cancer care.

The Potentials and Limitations of Monoclonal Antibody Therapy in the Treatment of Cancer 

 Charlotte McKinley, M.A. 2005

Since the discovery of monoclonal antibodies for the use in treating disease 30 years ago, the potential of their use has evolved.  From the original “naked” mouse monoclonal antibodies, complex drug or toxin-conjugated monoclonal antibodies have developed.  These antibodies provide longer lasting results than their ancestors.  The FDA has approved eight monoclonal antibodies for use in the United States, and there are currently many more in industry pipelines.  The monoclonal antibodies that are in clinical and pre-clinical trials at this time are building on what has worked in the past with this type of therapeutic agent.  More drugs, toxins and radioactive particles are being tested for their ability to destroy a cancerous cell.  Also, more tumor antigen sites are being investigated so that more monoclonal antibodies can be made against a variety of cancers.  

The Underestimated Role of Placental Growth Factor in Tumor Angiogenesis

 Izabella M. Messina, M.A. 2005

 The FDA approval in 2004 of Avastin (Genentech), a humanized anti-VEGF monoclonal antibody, was the ultimate realization of Dr. Judah Folkman’s groundbreaking theory that tumorigenesis could be inhibited by blocking the development of new blood vessels.  Yet present therapies are still not fully successful in blocking tumorigenesis by the efficient inhibition of angiogenesis.  Here, I explore the possibility of utilizing both the VEGF/VEGFR2 and PLGF/VEGFR1 axes as a therapeutic target to successfully block tumor angiogenesis and consequently induce tumor necrosis and regression. Vascular Endothelial Growth Factor (VEGF) and Placental Growth Factor (PLGF) work in tandem during development.  PLGF’s role, however, is particularly pronounced during pathological processes and it may constitute the key regulator of the angiogenic switch.  It was shown that there are tumors resistant to the anti-VEGF antibodies, which suggests the possibility that the PLGF/VEGFR1 axis may be in control of their growth.  Additionally, there is emerging evidence that circulating endothelial progenitor cells (CEPs) and hematopoietic stem cells (HSCs), derived from bone marrow, may also play a critical role in tumor neo-vascularization.  Thus, I discuss the possibility of developing an antibody against PLGF by utilizing conventional hybridoma technology, which could be used in a bidirectional therapy together with an anti-VEGF antibody.  Such therapy should be properly tailored to spatial and temporal activation of both growth factors: targeting VEGF at the outset to launch a starvation mode in tumors, and later inhibiting both VEGF and PLGF to block the recruitment of bone marrow derived cells.  Despite the positive therapeutic aspects of such a design, this course of treatment may also have unfavorable side effects, chief among them bone marrow toxicity.

An In vitro Co-culture System for Studying Macrophage Regulation of Adipocyte Metabolic Expression Profiles

Rachel D. Ngo, M.A. 2005

Adipose tissue releases pro-inflammatory molecules that have been implicated in the obesity-induced complications.  Recently macrophages have been identified as an important source for pro-inflammatory molecules released from adipose tissue.  We report here an in vitro co-culture system for studying the interaction between adipose tissue macrophages and adipocytes.  We established this system using differentiated 3T3-L1 adipocytes and stromal vascular cells (SVCs) isolated from lean and obese mice.  Co-culture of these two cell populations induces expression of pro-inflammatory genes by SVCs and an alteration in the expression of important metabolic genes by the 3T3-L1 adipocytes.  The alterations in metabolic gene expression are consistent with in vivo finding and are hypothesized to perturb lipid metabolism and storage in 3T3-L1 adipocytes.  Alterations in adipocyte metabolism have been strongly implicated in obesity induced insulin resistance.  Depletion of the macrophage population with clodronate liposomes leads to a downregulation of some of the pro-inflammatory molecules, notably Tnfa and Mip1a, indicating a major contribution of the macrophages to the release of these factors.  A significant improvement in the metabolic profile of the 3T3-L1 adipocytes is likewise observed.  The intercellular signals that are responsible for the modulation of pro-inflammatory gene expression in SVCs and metabolic gene expression in adipocytes are not known, though our data suggest that Pparg or Ccr2 may be targets in adipocytes.  Nevertheless, macrophage-induced alterations in 3T3-L1 adipocyte gene expression are consistent with expression profiles obtained from adipose tissue isolated from obese, insulin resistant mice.  The system described here will provide a powerful tool to investigate the functional interaction between adipocytes, macrophages and other cell population in the SVC.  

A Critical Review of Colorectal Cancer with Specific Emphasis on Current Screening Options and Newly Developed Targeted Therapies

Sally A. Nicholas, M.A. 2005

Colorectal cancer is responsible for over 50,000 deaths every year in the United States. Once this condition is diagnosed at an early stage, it can be readily treated via surgical resection. Unfortunately though, in over 30% of colorectal cancer cases the tumor is not localized upon diagnosis. At present the American Cancer Society has specific guidelines regarding colorectal cancer screening in individuals over age 50. However, public compliance with these guidelines is low. Therefore, in many patients metastatic spread of the tumor is already present at the time of diagnosis. Patients with advanced colorectal cancer can expect to live no more than 2 years. Two new targeted drugs, bevacizumab and cetuximab received FDA approval in early 2004 for the treatment of advanced colorectal cancer. While these drugs have been found to be of some benefit to patients, giving them a few extra months of life, a significant amount of research still needs to be conducted into these drugs and into the development of newer, more efficacious drugs.

The Application of Oral Genomics with Microarray Technology to Bioinformatics for Better Treatment of Oral Diseases

 Derek S.C. Park, M.A. 2005

We are living in an extraordinary time in human history punctuated by the convergence of major scientific and technological progress.  The knowledge base from human and non-human genomics, bioinformatics, and microarray technology is serving to revolutionize the methods of treatment for oral diseases.  The information from the Human Genome Project (HGP) and the integration of information from related areas of study and technology will dramatically change health care for the craniofacial complex.  In particular, the convergence of knowledge from oral genomics and microarray analysis will play a pivotal role in creating a bridge of opportunity for integrating scientific and clinical specialties to promote the advances in treatment, risk assessment, diagnosis, therapeutics, and oral health-care outcomes.  This application of oral genomics can aid in the molecular understanding of the genes and proteins, their interactions, pathways, and networks that are responsible for the development and progression of oral diseases and disorders.  As a result of the HGP, new advances have prompted high-throughput technologies, such as DNA microarrays, which have become accepted tools in the biomedical research community.  With such technology, simultaneous analysis of tens of thousands of genes for the purpose of gene discovery, diseases diagnosis, drug development, and therapeutics tailored to specific oral diseases are possible.  Aiding in this new concept of treatment comes other disciplines from the “informatics revolution”, such as bioinformatics.  Bioinformatics has established itself as an emerging field in biomedical research and has been gaining much attention in its application to dental medicine.  This area of study has created a steady stream of large and complex genomic data, which in turn, has transformed the way a clinical or basic science researcher approaches genomic research.  With the incorporation of microarray technology-based research and bioinformatics, we can look forward to more accurate diagnosis and drug therapy based on an individual patient’s genetic profile.  Integration of information from the human genome, comparative and microbial genomics, proteomics, bioinformatics, and related technologies will provide the basis for proactive prevention and intervention and novel and more efficient treatment approaches to several oral diseases. 

    A Review of the Production and Engineering of Monoclonal Antibodies as Therapeutic Agent Against  Disease

Sophia Weise-Riccardi, M.A. 2005

This review will focus on the production and engineering of monoclonal antibodies, which has found great interest over the last 30 years, especially since Koehler and Milstein’s discovery of antibody-producing cells in culture, and therefore unlimited supplies of homogeneous antibodies due to advances in fusion technology, has been well established.  I will introduce the subject by providing information about history, structure and mechanism of monoclonal antibodies.  Because immunogenicity against the produced murine antibodies and toxicity first limited their competence as ‘magic bullets’ that could potentially be used to treat many diseases, expectations seemed high for further understanding of the mechanism of action and new developments in genetic engineering techniques, which will be further explored in this review. They generally involve production of chimeric and humanized antibodies by cloning and replacement of murine with human antibody regions.  They are particularly acceptable because of reduction of immune reactions against murine proteins and exchangeability of the relevant genes of antibodies.  Other strategies for the production of fully human and human-like antibodies, such as the phage display library, will be discussed here, which mainly involve fusion of cloned spleen DNA and a coat protein of a bacteriophage and consequent expression and further engineering of the fusion gene to result in useful therapeutics.  Also, high affinity mAb’s that appear to be more effective for certain treatments due to their smaller size are said to be produced using only certain fragments of antibody genes and tailoring them according to special needs using genetic engineering. In addition, the creation of transgenic animals and plants, whose relevant original antibody genes have been inactivated through gene targeting, are discussed, which allow further production of fully human antibodies.  Other expression systems for production of recombinant antibodies will be questioned in this review, such as bacterial, yeast and mammalian cell cultures, each of which has its own advantages and disadvantages.  The conclusion will be concentrated on examples of biological molecules approved in clinical trials, and on future directions of the development of antibodies as therapeutic and diagnostic agents.    

 Chemokine Receptors as HIV-1 Coreceptors: Recent Advances in the Understanding of Viral Entry and the Discovery of Therapeutic Agents 

 Anthony T. Sanfiz, M.A. 2005

Since the discovery of human immunodefiency virus (HIV) as the causative agent of acquired immune deficiency syndrome (AIDS), extensive research has been conducted into the biochemistry of the pathogenesis of this disease. Over the years, it has been shown that infection of human cells by HIV type 1 (HIV-1) is initiated by the binding and fusion of the virus to the host cell. The binding and fusion pathways use glycoproteins present on the viral surface to interact with the target cell. The glycoprotein, Env, first interacts with CD4. After a conformational change, the glycoprotein then interacts with a secondary receptor, the chemokine receptor. This secondary interaction leads to a further conformational change in the glycoprotein, which then initiates the fusion process. The chemokine receptors are a family of seven transmembrane G-protein coupled receptors that are involved in leukocyte trafficking. These receptors and their natural ligands, the chemokines, are important during the inflammatory response, helping in the recruitment of specific leukocyte subpopulations to sites of tissue damage. Two members of this chemokine receptor family are known to have important roles in the HIV-1 entry/fusion process. One protein earlier identified as “fusin” turned out to be the receptor for the CXC chemokine SDF-1α (CXCL12) and subsequently termed CXCR4. The other chemokine receptor CCR5, was also identified as a fusion coreceptor, interacting naturally with RANTES (CCL5), MIP-1 α (CCL3) and MIP-1b (CCL4). The interesting twist to this chemokine coreceptor story is that evidence shows that different isolates of HIV-1 viruses use different chemokine coreceptors. Many primary isolates and macrophage-tropic viruses use CCR5 as the coreceptor, while CXCR4 is the coreceptor for HIV-1 isolates that are T-cell-tropic. This tropism presents many challenges to researchers that hope to block HIV-1 entry. This review follows the discovery of the chemokine “coreceptor” and looks into recent research that may provide added clues into the HIV-1 fusion mechanism. The paper will also describe some current therapies developed to block the HIV-1 entry process.

 A Potential New Use for Aptamers: Real-time Biological and Chemical Detection

Aptamers, single-stranded nucleic acids developed in vitro to bind not only their complementary DNA or RNA strands, but almost anything that can be selected for including small molecules, proteins, and living cells, are attracting interest in the areas of therapeutics and diagnostics.  However, with high sensitivity and binding affinities, and autocatalytic activity, allosterically controlled aptazymes have potential use as ideal candidates for components in biosensing.  As enzymatic molecules, aptazymes can be synthesized for their catalytic applications.  By selecting for binding and catalytic activity, aptazymes can be used to target certain ligands, and with the introduction of fluorogenic molecules, transduce a recognizable signal.  Due to aptamers’ high binding affinities, sensitivities, and specificities, RNAzymes and more likely DNAzymes may provide excellent tools as rapid biological and chemical sensors.  Currently it is now anticipated that the use of allosterically controlled immobilisable DNAzymes will be exploited in the biosensor field, a mode of detection suited to aptamer-based sensors that facilitates a reagentless one step analysis. 

The Ubiquitin-Proteasome System: Roles in Tumor Suppressor Destabilization and Prospects for Targeted Therapies

Tanaz Sharifnia, M.A. 2005

 Ubiquitin is a 76-amino-acid globular protein whose canonical role is to mediate the degradation of unwanted cellular proteins. In a tightly regulated process, the tagging of protein substrates with the ubiquitin molecule destines them to the disposal machinery of the cell, an abundant protease known as the 26S proteasome. Ubiquitination, as this tagging process is called, is extraordinary not only for the diversity of proteins it targets—ranging from misfolded or mistargeted proteins to cell cycle-specific or developmental stage-specific proteins—but the specificity with which it accomplishes this task. Indeed, as this system is so pervasive in its role as a regulator of cellular protein levels, it is perhaps not surprising that abnormalities in its function account for quite a few malignancies, including cancer, neurodegenerative diseases, immune and inflammatory disorders, and muscle wasting. In the case of cancer, gain-of-function mutations in ubiquitin system enzymes (particularly E3 ubiquitin ligase enzymes) have been implicated in the accelerated degradation of the p53 and p27 tumor suppressors; such destabilization subsequently renders cells susceptible to various malignant transformations. Yet the specificity of the ubiquitin system simultanously makes it an apt therapeutic target for these diseases. Efforts at inhibiting specific E3 ligases or ancillary proteins affecting the ubiquitination of p53, p27, and other proteins have yielded promising results. In these cases, the use of peptide aptamers and small molecules to block specific ubiquitination mediators have had the effect of inducing apoptosis or G₁ arrest in carcinoma cell lines. Additionally, the development of a novel technique that manipulates a cell’s own ubiquitin machinery to selectively degrade virtually any cellular protein suggests a new paradigm for drug target evaluation and protein knockout studies. In this way, the ability to exploit this complex and extensive system may prove to have momentous implications for the areas of targeted therapies and proteomics.

Psoriasis: History, Etiology and Monoclonal Therapies

Grace C. Wong, M.A. 2005

Psoriasis is a genetically determined autoimmune disease involving T cells. It is putatively linked to seven chromosomes - 6p, 17q, 4q, 1q, 1p, 3q, and 19p. With the exception of 6p, none of the other suspected alleles have yielded a gene that plays a role in the pathogenesis of psoriasis to date. The region of interest in chromosome 6p is the Major Histocompatibility Complex (MHC) that governs the interactions in the immune system and how antigen presenting cells (APCs) present antigenic peptides. Additionally, other factors such as chemokines and cytokines play a pivotal role in maintaining on-going immunopathogenesis by secreting chemoattractants to recruit CD8+ T cells to the site of active inflammation and activating them for clonal expansion. The CD8+ T cells release pro-inflammatory cytokines and this sustains the inflammation. Of particular interest are the highly damaging effects that TNF-a produces. Current monoclonal therapies that target T cells, their interactions and TNF-a have seen a breakthrough in the treatment and compliance of psoriatic patients. Thus, we can expect to see a trend shifting towards monoclonal antibodies in the management of psoriasis as more work is being elucidated on how best to inhibit or antagonize the immune system to tip the scales in the psoriatic patient’s favor.

The Role of Soluble Guanylyl Cyclase (sGC) in Tumorgenesis and as a Potential Target for Cancer Therapy 

Hua Zhong, M.A. 2005

Nitrogen oxide (NO) and cyclic GMP (cGMP) have emerged as important signal transduction mediators of several biologic functions including the effects of hormones, inter- and intracellular signals, toxins and various drugs. NO is a physiologic activator of sGC, thus increasing the intracellular level of cGMP, which activates the enzyme protein kinase G (PKG). In addition to its known biologic functions, activation of PKG by cGMP can induce growth inhibition and apoptosis, and inhibit cell migration, in human colon cancer cells. Therefore, the NO/cGMP/PKG pathway may play an important role in cancer therapy. Most of the previous studies in cancer cells have activated this pathway by using inhibitors of cGMP phosphodiesterases (PDEs), especially PDE5, to increase cellular levels of cGMP. A novel approach would be to use compounds that activate sGC, alone or in combination with other agents, to inhibit growth and induce apoptosis in cancer cells. So in the first part of my thesis, I will focus on the NO/cGMP/PKG pathway, describe the key elements inside this pathway and its downstream biologic effects, as well as the relationship between NO/cGMP/PKG pathway and the tumorgenesis. In addition, in the research part of my thesis, I characterized the levels of expression of sGC in a series of human colon cancer cell lines, and picked up a pair of cell lines which have very low sGC expression level (HCT116) and very high sGC expression level (HT29). During the research, it was found that in HT29 cell line, sGC activator and other NO-donors could induce the cell growth inhibition and also induce the increase of the phosphorylation of the proteins which related to the NO/cGMP/PKG pathway. The compounds to be used include YC-1 and BAY41-2272, two known activators of PKG, nitro-aspirin and other potential NO-donors. ODQ was also used, which as a known inhibitor of sGC, could be used to confirm that the compounds I use exert their effect, by activating sGC. The gene expression profiles in normal tissue and human cancer have also been searched in order to 1) demonstrate that the lower expression level of sGC indeed exists in different cancer cell lines which in turn, is not only happening in colon cancer cells; 2) identify which types of human cancer cells express higher levels of sGC among different cancer cell lines, and may, therefore, be candidates for therapy with sGC activators.

A Situational Analysis of Multiple Sclerosis: Current and Future Disease Modifying Therapies

Tricia E. Zubal, M.A. 2005

Multiple Sclerosis (MS) is a chronic recurrent inflammatory disorder of the central nervous system (CNS). The disease results in injury to the neuronal myelin sheaths, the oligodendrocytes and, to a lesser extent, the axons and nerve cells themselves. There are at least three potential kinds of therapy for patients with MS. The first is treatment aimed at reducing the biological activity of MS in order to prevent or delay future neurological injury; the second is symptomatic treatment for specific clinical complaints, such as bladder dysfunction and fatigue; and the third is treatment to repair the damage already done to the CNS, especially the myelin sheaths. Currently approved disease modifying therapies include the immunomodulators interferon-beta (IFN-β) (Betaseron, Avonex, Rebif) and glatiramer acetate (Copaxone), as well as mitoxantrone (Novantrone), an immunosuppressant. Although the mechanisms of action of these products have not been completely elucidated, they all have been moderately effective in their ability to reduce clinical relapse rates and modify the natural disease course in patients with the relapsing-remitting disease subtype. However, advances in the understanding of the molecular mechanisms that contribute to the pathogenesis of MS have greatly increased the number of knowledge-based drugs that are currently being developed. The idea is that better understanding of both the disease state and the normal functioning of the immune system and the CNS, as well as their interaction, will ultimately lead to superior drug candidates. The design of emerging and future therapies is directed towards the development of more specific and less toxic treatments. Fueled by the progress of biotechnology, research is dominated by orally active small molecule immunomodulators and intravenous humanized monoclonal antibodies. Strategies include preventing the breakdown of the blood-brain barrier and/or inhibiting the trafficking and infiltration of lymphocytes; initiating an immune deviation in CD4+ T cell cytokines from a Th1 to a Th2 phenotype; and targeting the oligodendrocytes in attempt to influence remyelination. It is likely that within the next decade much of this research will result in many new treatments approved for MS.