Biochemistry Commons^™

The Characterization Of A Recombinant Virophage Integrase, Martin Christopher Chacon

Open Access Theses & Dissertations

Virophages are satellite-like dsDNA viruses that parasitize giant viruses of the family Mimiviridae. Mavirus is the second virophage discovered that associates with its host virus Cafeteria roenbergensis Virus (CroV). When co-infecting their common host cell Cafeteria roenbergensis, a marine zooplankton that is widely spread throughout the oceans, mavirus will inhibit CroV's replication. In addition, mavirus was shown to share high similarities to the Maverick/Polinton eukaryotic DNA transposons. A coding sequence in mavirus genome (MV02) reveals high homology to retroviral integrases such as those found in HIVs. The putative integrase MV02 is predicted to integrate mavirus DNA into the host genome. …

Structure And Biological Activity Of A D3g Mutation In The Human Mitochondrial Chaperonin Hsp60, Jihui Li

Open Access Theses & Dissertations

Proteins are essential components in all cellular activities, including signal transduction, gene expression regulation, immune response, structural support and many others. Following their synThesis on the ribosome, proteins need to fold into their precise three-dimensional structure to obtain functionality. If they fold incorrectly they are prone to aggregation and cause a wide range of neurodegenerative diseases. Some proteins are capable of folding without assistance but others need help from protein complexes known as chaperonins. Chaperonins are a family of proteins that assemble into barrel-shaped cages the create a protected chamber for misfolded proteins to refold into their biologically active state. …

Expression And Functional Analysis Of Lipids And Glycolipids From The Mammal-Dwelling Stages Of Trypanosoma Cruzi, Felipe Gazos Lopes

Open Access Theses & Dissertations

Trypanosoma cruzi is the causative agent of the life-threatening Chagas disease, in which increased platelet aggregation related to myocarditis is observed. Platelet-activating factor (PAF) is a potent intercellular lipid mediator and second messenger that exerts its activity through a PAF-specific receptor (PAFR). Previous data from our group suggested that T. cruzi synthesizes a phospholipid with PAF-like activity. The structure of T. cruzi PAF-like molecule, however, remains elusive. Here, we have purified and structurally characterized the putative T. cruzi PAF-like molecule by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). Our ESI-MS/MS data demonstrated that the T. cruzi PAF-like molecule is actually a lysophosphatidylcholine …

Role Of Small Molecules In Rescuing Protein Folding Under Oxidative Stress, Mahmoud Fawzi Megahed Helal Khalil

Open Access Theses & Dissertations

Increased levels of nitrosative stress intracellularly within the endoplasmic reticulum is a key factor involvled in the pathogenesis of both Parkinson's (PD) and Alzheimer's (AD) diseases. Previous in-vitro studies in our lab showed that increased levels of nitrosative stress lead to aggregation of misfolded proteins and formation of Lewy Bodies, the main biomarker of PD and AD diseases. Although this was mainly through nitrosylation of Protein Disulfide Isomerase (PDI), the chief endoplasmic reticulum (ER) resident oxidoreductase chaperone responsible for maturation of disulfide-bond-containing proteins, we demonstrate in this project that increased levels of nitrosative stress has an additional direct effect on …

Evaluation Of Small Molecules On The Excitatory Amino Acid Transporter 3 (Eaat3) For The Treatment Of Ocd, Emmanuel Zubia

Open Access Theses & Dissertations

We seek to discover new drug candidates for the excitatory amino acid transporter 3 (EAAT3) for the treatment of Obsessive Compulsive Disorder (OCD). We hypothesize that decreasing the glutamatergic transmission via EAAT3 intervention will create an amelioration of the symptoms of OCD. For this we have prepared in-silico binding calculations to identify a better compound that can stimulate EAAT3 and decrease glutamatergic transmission. This research will help us further understand and elucidate the role of glutamate in the neural mechanisms of this illness, which to date remain inconclusive. Those areas of study are significant because, until now, there is no …

Characterization Of The Nodamura Virus Rna Dependent Rna Polymerase And Formation Of Rna Replication Complexes In Mammalian Cells, Vincent Ulysses Gant

Open Access Theses & Dissertations

Positive-strand RNA viruses amplify their genomes in membrane-bound structures associated with intracellular membranes and organelles called replication complexes (RCs). Here, we begin to elucidate mechanisms of Nodamura virus (NoV; family Nodaviridae) RC assembly. The literature reports that NoV-infected muscle tissue exhibits mitochondrial aggregation and rearrangement of mitochondrial structure, leading to disorganization of the muscle fibrils. However, the molecular basis for this pathogenesis and the role of mitochondria in NoV infection remained unclear until now. We tested the hypoThesis that NoV establishes RCs in association with mitochondria in cultured mammalian cells at physiological temperature. We used immunofluorescence confocal microscopy and biochemical …

Biochemical Characterization Of Four Distinct Proteins, Gustavo A. Avila

Open Access Theses & Dissertations

Proteins are ubiquitous in all living organisms, executing the majority of cellular functions in distinct ways. Understanding a protein's role necessitates investigating its structure and function, which are closely related. My research couples these two aspects by delving into the biochemical and structural characterization of proteins in four distinct systems, all playing central roles in numerous significant disease progressions. These four original research endeavors were all targeted for structural studies with a unifying relationship to establish our new structural biochemistry lab. These four systems are: (1) Gam1, an early adenovirus protein globally inhibiting host SUMOylation; (2) Anthrax toxin complexed with …

A Comparative Approach To Assessing The Functional And Structural Characteristics Of Human Fkbp52 In The Regulation Of Steroid Hormone Receptor Signaling Pathway, Diondra Crystal Harris

Open Access Theses & Dissertations

The 52 kDa FK506 binding protein (h52) is a key positive regulator of Androgen Receptor (AR) in cellular and animal models and is an attractive target for the treatment of prostate cancer. Human FKBP52 is a known regulatory protein and co-chaperone that has been shown to play an important role in the regulation of the AR signaling pathway, and in the development of the male sexual phenotype. Cellular studies in mammalian and yeast cells reveal that FKBP52 is a positive regulator of AR, glucocorticoid receptor (GR), and progesterone receptor (PR), potentiating receptor-mediated gene expression up to 60-fold in some systems. …

Mechanisms To Mitigate Neurodegeneration By Maintaining Mitochondrial Health, Parijat Kabiraj

Open Access Theses & Dissertations

Cerebral accumulation of amyloidogenic protein aggregates is most frequently observed in the pathogenesis of neurodegenerative diseases. Recent studies showed prion like spreading of beta amyloid (A beta) in Alzheimer's disease (AD) and alpha synuclein protein in Parkinson's disease (PD) brain. Failure or compromise to the chaperone activity of protein disulfide isomerase (PDI) is also been reported as a major factor of aggregate formation. Nitrosative stress mediated S-nitrosylation (SNO) of protein disulfide isomerase (PDI), a housekeeping oxidoreductase, has been implicated in the pathogenesis of sporadic PD and AD. Mitochondrial dysfunction, leading to elevated levels of reactive oxygen species (ROS), is associated …

Expression And Characterization Of The Major Capsid Protein (Mcp) Of A Giant Marine Virus: Cafeteria Roenbergensis Virus, Sayan Chakraborty

Open Access Theses & Dissertations

The oceans play significant roles in maintaining the climate on Earth and providing nutrients to the whole biosphere. Oceanic microbes produce approximately half of the Earth's oxygen. It has been shown that the populations of microbial communities in oceans are largely regulated by viral infection and thus affect the marine ecosystem. Cafeteria roenbergensis Virus (CroV) is a giant marine virus with a linear, double stranded, and AT-rich DNA genome. The genome of CroV has a size of 744kb and encodes for 544 predicted genes. CroV derived its name from its host, a unicellular marine zooplankton Cafeteria roenbergensis (Cro) that is …

Giardia Lamblia: Regulation Of Cyst Production By Glycosphingolipids, Tavis Lyle Mendez

Open Access Theses & Dissertations

The diplomonad protist, Giardia lamblia, colonizes and replicates in the small intestine of mammals. In humans, Giardia infection (known as giardiasis) can be symptomatic or asymptomatic with the former being associated with fatty stool, abdominal pain, malnutrition, and weight loss. Both cell-mediated and humoral immune responses against Giardia infection are possible, and adaptive responses have been reported to be important for controlling the infection. Non-immune components such as secretory immunoglobulin, intestinal lipids, and bile acids also play significant roles in determining the severity of the infection by Giardia.

Giardia exists in two morphologic forms--trophozoites and cysts--and maintains a simple life …

Development Of A Chaperonin Activity Assay, Timothy P. Brown, Danielle Y. Martinez, Ricardo A. Bernal

COURI Symposium Abstracts, Summer 2012

Chaperonin-mediated protein folding by the Escherichia coli GroEL/GroES complex is pivotal in preventing premature aggregation and improper folding of proteins. In fact E. coli that have had the groE operon knocked out are not able to survive. The explicit details of facilitated folding in the interior of the chaperonin complex remain relatively unknown, although it is understood that ATP hydrolysis is essential to the chaperonin protein-folding cycle. The goal of this project is to develop an assay to detect the protein folding activity of chaperonins. ATP hydrolysis reactions allow chaperonins to undergo a series of conformational changes that alter the …

Purification And Characterization Of Mycobacterium Tuberculosis Esx-1 Secreted Protein Esat-6, Sebastian Montalvo, Gouzhong Jiang, Jianjun Sun

COURI Symposium Abstracts, Summer 2012

Mycobacterium tuberculosis ESX-1 secreted protein ESAT-6 has been shown to contribute to the pathogenesis and virulence of M. tuberculosis. Despite many studies, little is known about how this protein interacts with the host’s membrane and evades the host’s defense mechanism. In this project, we successfully purified and characterized of ESAT-6 wild type (E6-WT) and ESAT-6 mutant with either a single (E6-S) or double (E6-D) amino acid mutation that are supposed to diminish the membrane-interacting activity of ESAT-6. The methods used in our project include: cloning in plasmid vectors, cloning in PCR products with T-Vectors, chemical transformation of E. coli, affinity …

Binding Efficacy Of Different Polyphenolic Phytochemicals With Β-Lactoglobulin And Human Serum Albumin: Implication For Therapeutics Against Neurodegenerative Diseases, Rene Duran^, Andres Ortiz^, Mahesh Narayan*, Vladik Kreinovich*

COURI Symposium Abstracts, Spring 2012

No abstract provided.

Derivation Of Hill's Equation From Scale Invariance, Andres Ortiz^, Vladik Kreinovich*

COURI Symposium Abstracts, Spring 2012

No abstract provided.

Interaction Of Trypanosoma Cruzi Sterol 14-Alpha-Demethylase Cyp51 With Ru-Azole Derivatives, Arturo Ortega^, Rosa A. Maldonado*, Roberto A. Sanchez-Delgado, Hugues Ouellet*

COURI Symposium Abstracts, Spring 2012

No abstract provided.

Cholesterol Side-Chain Degradation Enzymes Of Mycobacterium Tuberculosis, Brian Villalba^, Itzel Chavarria, Bernardo Terres, Johnathan Abou-Fadel, Rhone A. Mendoza, Hugues Ouellet*

COURI Symposium Abstracts, Spring 2012

No abstract provided.

Engineering The Human Immunodeficiency Virus To Infect Drosophila Melanogaster Cells, Laura F. Porras ^, Elizabeth R. Aguilera, Eduardo Urias, Daniel Reyes, Adarsh Saheba, Manuel Llano *

COURI Symposium Abstracts, Spring 2012

HIV continues to infect at pandemic levels despite the significant progress in research, treatment and prevention. Advances in anti-HIV retroviral drugs have led to significant reduction in AIDS-related deaths, delayed the progression of the HIV infection to AIDS and diminished the rates of HIV transmission. However, the current antiretroviral treatment frequently encounters drug-resistant viruses due to the high mutation rate of HIV proteins. Therefore, identification of cellular cofactors of HIV infection is critical to identify new targets for anti-HIV drug development. However, the genetic complexity of mammalian cells imposes additional obstacles to the identification of HIV-1 cellular cofactors. To mitigate …

Validation On Sodium Channel Dependence By Electrical Stimulation-Induced Dopamine Release, Alice E. Hernandez ^, Mabel N. Terminel, Edward Castaneda *

COURI Symposium Abstracts, Spring 2012

No abstract provided.

Examination Of The Neurochemical Mechanisms That Mediate Nicotine Withdrawal In Adolescent And Adult Rats, Luis Alberto Natividad

Open Access Theses & Dissertations

Introduction: The mechanisms that mediate nicotine withdrawal are presently unclear and age group differences in the neurochemical effects of withdrawal have been largely unexplored. Previous studies in our laboratory demonstrated that adult rats display a decrease in extracellular levels of dopamine in the nucleus accumbens (NAcc) during nicotine withdrawal and this decrease is reduced in adolescent rats (Natividad et al., 2010). The goal of this dissertation was to examine whether these age group differences in dopamine during withdrawal are mediated via excitatory and inhibitory mechanisms that modulate dopamine in the cell body region of the ventral tegmental area (VTA). Methods: …

Arachidonic Acid Signaling In Invasive And Non-Invasive Breast Cancer Cells, Debarshi Roy

Open Access Theses & Dissertations

Breast cancer is the second largest cause of cancer-related deaths in women all over the world. Epidemiological studies suggest that the consumption of high-fat diets can promote the incidence of breast cancers in both developed and developing countries. In particular, the lipid-rich diet contains arachidonic acid (AA, a C20:4 polyunsaturated fatty acid), which has been shown to be associated with tumor formation in breast tissues. Nevertheless, the actual mechanism by which AA induces the metastatic transformation and malignancy is not well understood. The goal of my dissertation, therefore, is to identify the molecules and unravel the pathways that participate in …

Map Kinases Couple Hindbrain-Derived Catecholamine Signals To Hypothalamic Adrenocortical Control Mechanisms During Glycemia-Related Challenges, Arshad Khan, Kimberly Kaminski, Graciela Sanchez-Watts, Todd Ponzio, J. Kuzmiski, Jaideep Bains, Alan Watts

Selected Works Temporary Series

No abstract provided.

Map Kinases Couple Hindbrain-Derived Catecholamine Signals To Hypothalamic Adrenocortical Control Mechanisms During Glycemia-Related Challenges, Khan, Kimberly Kaminski, Graciela Sanchez-Watts, Todd Ponzio, J. Kuzmiski, Jaideep Bains, Alan Watts

Departmental Papers (Biology)

No abstract provided.

Cloning, Expression, And Characterization Of The Aminoethylphosphonate Pathway In Trypanosoma Cruzi, Samuel I. Rodriguez^, Esteban M. Cordero, Igor C. Almeida*

COURI Symposium Abstracts, Summer 2011

No abstract provided.

Structural Study Of Mcry2: A Circadian Rhythm Regulator, Brenda L. Rodarte^, Gustavo A. Avila, Adrian S. Enriquez, Nicolas D. Silva, Tiffany H. Blankenship, Erquan Eric Zhang*, Chuan Xiao*

COURI Symposium Abstracts, Spring 2011

A circadian clock is a 24 hour biological rhythm that regulates physiological, psychological and behavioral processes in living organisms. Understanding the regulation mechanism of such circadian rhythms can have great impact in certain diseases such as sleeping disorders and cancers. There are many genes regulating circadian rhythms in different species. Cryptochromes (CRY) are photosensory receptors mediating light regulation of growth and development in certain species. Mouse cryptochrome protein mCRY2 has been found to encode a blue light photoreceptor and is important for mice circadian rhythm. The main goal of the project is to determine the atomic structure of mCRY2, via …

Enzymatic Characterization Of An Endolysin Mediated By Chaperonin In Bacteriophage Phi El, Diana A. Tafoya^, Nadia Herrera, Sudheer Molugu, Zachariah Hildenbrand, Maxim V. Filchikov, Vadim V. Mesyanzhinov, Konstantin A. Miroshnikov, Ricardo A. Bernal*

COURI Symposium Abstracts, Spring 2011

Bacteriophage phi-EL is a virus that attacks the human pathogen Pseudomonas aeruginosa. One of the gene products from phi-EL is a putative endolysin. Endolysin is an enzyme produced during gene expression in the lytic cycle of the bacteriophage. Its function is to digest the peptidoglycan layer of the host cell wall, thereby releasing the newly formed virions. In order to confirm the identity of this putative endolysin, the gene product was transformed into competent E. coli cells, expressed to high levels and purified to high homogeneity using nickel affinity and size exclusion chromatography. The peptidoglycan-hydrolyzing activity of the protein …

Synthesis, Self-Assembly And Biological Properties Of Self-Folding Cu(Ii) Coordination Polymers, Alejandro Arzola-Rubio

Open Access Theses & Dissertations

The synthesis of materials via molecular self-assembly is a powerful bottom-up approach for fabricating matter in the nano- and micro-meter scales. This methodology involves the spontaneous and reversible organization of small molecules that interact with one another to create larger structures driven by non-covalent interactions such as hydrogen bonding, hydrophobic forces and metal-ligand coordination interactions. In this thesis, the design, synthesis and characterization of a new set of molecules that spontaneously self-organize in water to form three-dimensional nanoscale toroidal structures was investigated. These supramolecular structures were designed with an intrinsic affinity to bind and condense DNA strands with the intent …

Phospholipid Analysis In Trypanosoma Cruzi, Melissa Rashonda Harris

Open Access Theses & Dissertations

Chagas disease, caused by the flagellate protozoan Trypanosoma cruzi is one of the most endemic and deadly infectious diseases. T.cruzi is distributed throughout most of South and Central America, where it infects 12 to 19 million people, with an annual incidence of 561,000. Another 35 million are exposed to infection, 2 to 3 million people have clinical symptoms of chronic Chagas disease, and about 45,000 of these may die every year due to cardiac failure. In addition to the growing number of incidences in other countries the disease is an emerging infectious disease in the U.S due to the migration …

Free Radical Stress-Induced Parkinsonian Lewy-Like Aggregation Prevented Through Polyphenolic Phytochemical Analog Intervention: Implications For Subcellular Trafficking And Neurodegenerative Disorders, Rituraj Pal

Open Access Theses & Dissertations

Protein disulfide isomerase (PDI), the chief endoplasmic reticulum (ER)-resident oxidoreductase chaperone, is known to catalyze the maturation of disulfide-bond-containing proteins primarily through oxidation-reduction and isomerization functions. The rate-determining step in the oxidative regeneration path of disulfide-bond-containing proteins generally couples chemical thiol-disulfide-exchange reactions to a physical conformational folding reaction. I have determined the impact of PDI and its subdomains on the rate-determining step in ribonuclease A folding and on the physical structure-forming step of select ER-processed proteins including RNase A. This was facilitated through application of a novel chemical tool to exclusively populate native-disulfide-containing intermediates in unstructured forms. The described biochemical …

Structural And Functional Investigation Of Φ-El-Chaperonin Mediated Protein Folding, Sudheer Kumar Molugu

Open Access Theses & Dissertations

Chaperonins are ubiquitous, sequence related protein complexes that aid in the folding of nascent and misfolded polypeptides in an ATP driven pathway. Recently a GroEL-like, 860 kilo Dalton chaperonin protein complex was identified and isolated from the bacteriophage EL, a virus that infects the Gram-negative bacterium Pseudomonas aeruginosa. The bacteriophage EL contains 201 predicted open reading frames and is the only known phage that encodes for its own chaperonin known as Φ-EL-chaperonin.

To understand the importance of Φ-EL-chaperonin in phage EL life cycle, the recombinant Φ-EL-chaperonin protein was expressed in Escherichia coli (E. coli) purified to homogeneity and the structure …