Molecular Biology Commons^™

Evaluating The Role Of Evolutionarily Conserved Regions Of Ledgf/P75 In Hiv-1 Infection, Jose Adrian Garcia

Open Access Theses & Dissertations

LEDGF/p75 is an important cellular co-factor for lentiviral integration. LEDGF/p75-deficient cells are markedly resistant to HIV-1 infection and re-expression of the wild type protein rescues infectivity. Although the molecular mechanism of LEDGF/p75 in HIV-1 integration is not yet known, this co-factor activity requires the interaction of LEDGF/p75 with both the host chromatin and viral integrase. In order to evaluate the involvement of other LEDGF/p75 regions in HIV-1 infection we constructed a panel of deletion mutants targeting clusters of charged residues that are evolutionarily conserved and predicted to be post-translationally modified. These mutants were evaluated for their ability to rescue HIV-1 …

Dielectrophoretic Choking Phenomenon In A Converging-Diverging Microchannel, Ye Ai, Shizhi Qian, Sheng Liu, Sang W. Joo

Mechanical & Aerospace Engineering Faculty Publications

Experiments show that particles smaller than the throat size of converging-diverging microchannels can sometimes be trapped near the throat. This critical phenomenon is associated with the negative dc dielectrophoresis arising from nonuniform electric fields in the microchannels. A finite-element model, accounting for the particle-fluid-electric field interactions, is employed to investigate the conditions for this dielectrophoretic (DEP) choking in a converging-diverging microchannel for the first time. It is shown quantitatively that the DEP choking occurs for high nonuniformity of electric fields, high ratio of particle size to throat size, and high ratio of particle's zeta potential to that of microchannel. © …

Electric Pulses To Prepare Feeder Cells For Sustaining And Culturing Of Undifferentiated Embryonic Stem Cells, Lauren M. Browning, Tao Huang, Xiao-Hong Nancy Xu

Chemistry & Biochemistry Faculty Publications

Current challenges in embryonic-stem-cell (ESC) research include inability of sustaining and culturing of undifferentiated ESCs over time. Growth-arrested feeder cells are essential to the culture and sustaining of undifferentiated ESCs, and they are currently prepared using gammaradiation and chemical inactivation. Both techniques have severe limitations. In this study, we developed a new, simple and effective technique (pulsed-electric-fields, PEFs) to produce viable growth-arrested cells (RTS34st) and used them as high-quality feeder cells to culture and sustain undifferentiated zebrafish ESCs over time. The cells were exposed to 25 sequential 10- nanosecond-electric-pulses (10nsEPs) of 25, 40 and 150 kV/cm with 1s pulse interval, …

On The Mechanism Of Protein Fold-Switching By A Molecular Sensor, Margaret M. Stratton, S N. Loh

Biochemistry & Molecular Biology Department Faculty Publication Series

Alternate frame folding (AFF) is a mechanism by which conformational change can be engineered into a protein. The protein structure switches from the wild-type fold (N) to a circularly-permuted fold (N'), or vice versa, in response to a signaling event such as ligand binding. Despite the fact that the two native states have similar structures, their interconversion involves folding and unfolding of large parts of the molecule. This rearrangement is reported by fluorescent groups whose relative proximities change as a result of the order-disorder transition. The nature of the conformational change is expected to be similar from protein to protein; …

Development Of Ultra-Sensitive Fluorescence Photoamplification Assays For The Detection Of Molecular Recognition Events, Tiffany Priscilla Gustafson

Electronic Theses and Dissertations

During the course of this research a novel method which couples the molecular recognition-triggered photoamplification chain in diaryl ketone adducts of dithiane with a "turn-off" or "turn-on" fluorescence-based assay for the detection of biological targets and ligands, regardless of their nature, through a molecular recognition event has been developed. This research has included several key steps, the most significant being: (1) the design of fluorophore adducts or dyads which recover fluorescence upon photocleavage for a "turn-on" assay and the identification of fluorophores which are quenched upon the photochemical release of a quencher for a "turn off" assay; (2) Optimization of …

Uncultivated Environmental Tm7 Model To Study Human Disease-Associated Tm7 Bacteria, David Barton

Master's Theses

The TM7 bacterial phylum has no cultivated species and includes members that span a broad range of environmental and human habitats, some of which are associated with human periodontitis. In this project, activated wastewater TM7 bacteria were analyzed and their relatedness compared to human-associated TM7 bacteria for the long-term goal of using an environmental TM7 to better understand TM7 pathogenesis in humans. DNA was extracted from activated wastewater and PCR amplified using TM7 16S rRNA gene- specific primers. The ~1,170 base pair PCR products were then cloned and sequenced. DNA sequencing and phylogenetic analysis identified environmental TM7 clones with high …

Electroporation-Mediated Delivery Of A Naked Dna Plasmid Expressing Vegf To The Porcine Heart Enhances Protein Expression, W. G. Marshall Jr., B. A. Boone, J. D. Burgos, S. I. Gografe, M. K. Baldwin, M. L. Danielson, M. J. Larson, D. R. Caretto, Y. Cruz, B. Ferraro, L. C. Heller, K. E. Ugen, M. J. Jaroszeski, R. Heller

Bioelectrics Publications

Gene therapy is an attractive method for the treatment of cardiovascular disease. However, using current strategies, induction of gene expression at therapeutic levels is often inefficient. In this study, we show a novel electroporation (EP) method to enhance the delivery of a plasmid expressing an angiogenic growth factor (vascular endothelial growth factor, VEGF), which is a molecule previously documented to stimulate revascularization in coronary artery disease. DNA expression plasmids were delivered in vivo to the porcine heart with or without coadministered EP to determine the potential effect of electrically mediated delivery. The results showed that plasmid delivery through EP significantly …

Increased Perfusion And Angiogenesis In A Hindlimb Ischemia Model With Plasmid Fgf-2 Delivered By Noninvasive Electroporation, B. Ferraro, Y. L. Cruz, M. Baldwin, D. Coppola, R. Heller

Bioelectrics Publications

Gene therapy approaches delivering fibroblast growth factor-2 (FGF-2) have shown promise as a potential treatment for increasing blood flow to ischemic limbs. Currently, effective noninvasive techniques to deliver plasmids encoding genes of therapeutic interest, such as FGF-2, are limited. We sought to determine if intradermal injection of plasmid DNA encoding FGF-2 (pFGF) followed by noninvasive cutaneous electroporation (pFGFE+) could increase blood flow and angiogenesis in a rat model of hindlimb ischemia. pFGFE+ or control treatments were administered on postoperative day 0. Compared to injection of pFGF alone (pFGFE-), delivery of pFGFE+ significantly increased FGF-2 expression for 10 days. Further, the …

Epsvr And Epmeta: Prediction Of Antigenic Epitopes Using Support Vector Regression And Multiple Server Results, Shide Liang, Dandan Zheng, Daron M. Standley, Bo Yao, Martin Zacharias, Chi Zhang

School of Biological Sciences: Faculty Publications

Background: Accurate prediction of antigenic epitopes is important for immunologic research and medical applications, but it is still an open problem in bioinformatics. The case for discontinuous epitopes is even worse - currently there are only a few discontinuous epitope prediction servers available, though discontinuous peptides constitute the majority of all B-cell antigenic epitopes. The small number of structures for antigen-antibody complexes limits the development of reliable discontinuous epitope prediction methods and an unbiased benchmark to evaluate developed methods.

Results: In this work, we present two novel server applications for discontinuous epitope prediction: EPSVR and EPMeta, where EPMeta …

The Interictal State In Epilepsy And Behavior, Daniel Tice Barkmeier

Wayne State University Dissertations

Epilepsy is one of the most common neurological diseases, affecting up to 1% of the world population. Epilepsy remains poorly understood and there are currently no medications to cure it. Patients with epilepsy have both seizures as well as another type of abnormal activity between seizures, known as interictal spikes. Interictal spikes have thus far been poorly researched, yet growing evidence supports an important role for them in epilepsy. In this project, we first show the high variability between reviewers in marking interictal spikes on intracranial EEG, and then develop and test an automated detection method to solve this problem. …

Evolution Of Lactate Dehydrogenase Genes In Primates, With Special Consideration Of Nucleotide Organization In Mammalian Promoters, Zack Papper

Wayne State University Dissertations

Concomitant with an increase in brain volume and mass, the allocation of energetic resources to the brain increased during stem anthropoid evolution, leading to humans. One mechanism by which this allocation may have occurred is through greater use of lactate as a neuronal fuel. Both the production of lactate, and conversion to pyruvate for use in aerobic metabolism, are catalyzed, in part, by the tetrameric enzyme lactate dehydrogenase (LDH). The two primary LDH genes, LDHA and LDHB, confer different rates of substrate turnover to the LDH enzyme, and these rates lend to the argument that LDHA supports anaerobic while LDHB …

Characterization Of Arsd: An Arsenic Chaperone For The Arsab As(Iii)-Translocating Atpase, Jianbo Yang

Wayne State University Dissertations

Arsenic is a metalloid toxicant that is widely distributed throughout the earth's crust and causes a variety of health and environment problems. As an adaptation to arsenic-contaminated environments, organisms have developed resistance systems. In bacteria and archaea various ars operons encode ArsAB ATPases that pump the trivalent metalloids As(III) or Sb(III) out of cells. In these operons, an arsD gene is almost always adjacent to the arsA gene, suggesting a related function. ArsA is the catalytic subunit of the pump that hydrolyzes ATP in the presence of arsenite or antimonite. ArsB is a membrane protein which containing arsenite-conducting pathway. ArsA …

Regulatory And Functional Aspects Of Foxo3a Transcription Factor And Their Implications In Prostate Cancer, Melissa Elise Dobson

Wayne State University Dissertations

The P13K/Akt pathway is a critical mediator of growth factor signaling involving many cellular functions. The deregulation of this pathway has been shown to be involved in the development of various cancers. One of the main targets of this pathway is FoxO3a, a transcription factor whose target genes are involved in important cellular processes such as apoptosis, cell cycle control, and glucose metabolism. FoxO3a is regulated by various post translational modifications including acetylation, ubiquitination and phosphorylation. The transcription factor is directly phosphorylated by Akt on 3 residues: Threonine 32, Serine 253 and Serine 315. Phosphorylation by Akt generates binding sites …

Towards An Understanding Of The Etiology Of Abdominal Aortic Aneurysms: Identification Of Genes Implicated In Aaa Risk And Development, John Hunt Lillvis

Wayne State University Dissertations

Abdominal aortic aneurysm (AAA) is a common disease for which mechanisms of formation are still not well understood. Despite a strong genetic component to AAA risk, specific risk alleles are still largely unidentified. AAA is also a localized disease with a majority occurring in the infrarenal abdominal aorta and is six times more common than aneurysms of the thoracic aorta. To determine whether risk alleles are present in functional positional candidate genes. we: 1. performed a genetic association study using DNA from AAA cases and controls in ten candidate genes and 2. performed exon sequencing on three genes with evidence …

Tracking Profiles Of Genomic Instability In Spontaneous Transformation And Tumorigenesis, Lesley Lawrenson

Wayne State University Dissertations

The dominant paradigm for cancer research focuses on the identification of specific genes for cancer causation and for the discovery of therapeutic targets. Alternatively, the current data emphasize the significance of karyotype heterogeneity in cancer progression over specific gene-based causes of cancer. Variability of a magnitude significant to shift cell populations from homogeneous diploid cells to a mosaic of structural and numerical chromosome alterations reflects the characteristic low-fidelity genome transfer of cancer cell populations. This transition marks the departure from micro-evolutionary gene-level change to macro-evolutionary change that facilitates the generation of many unique karyotypes within a cell population. Considering cancer …

Animal Models Of Alzheimer's Disease, Gemma Casadesus, Gary Arendash, Frank Laferla, Mike Mcdonald

Molecular Biosciences Faculty Publications

No abstract provided.

Comparing Models Of Evolution For Ordered And Disordered Proteins, Celeste J. Brown, Audra K. Johnson, Gary W. Daughdrill

Molecular Biosciences Faculty Publications

Most models of protein evolution are based upon proteins that form relatively rigid 3D structures. A significant fraction of proteins, the so-called disordered proteins, do not form rigid 3D structures and sample a broad conformational ensemble. Disordered proteins do not typically maintain long-range interactions, so the constraints on their evolution should be different than ordered proteins. To test this hypothesis, we developed and compared models of evolution for disordered and ordered proteins. Substitution matrices were constructed using the sequences of putative homologs for sets of experimentally characterized disordered and ordered proteins. Separate matrices, at three levels of sequence similarity ( …

Group Ii Intron Dynamics In Heterologous Hosts, Venkata Raghavendra Aditya Chalamcharla

Legacy Theses & Dissertations (2009 - 2024)

Group II introns are ribozymes with an innate ability to self-splice. They are found predominantly in bacterial and bacterial-derived organellar genomes, but not in the nuclear genomes of eukaryotes. In bacteria, group II introns often behave as mobile retroelements, invading host DNA and exploiting its machinery to complete the retromobility process. The object of my studies is the group II intron found in the Lactococcus lactis relaxase gene. To determine the nature of the group II intron-host relationship, we performed a genetic screen and identified several host factors that affect group II intron retromobility in Escherichia coli, which provides a …

Saturation Transfer Difference Nmr Studies : Pyrazinamide And Pyrazinamide Analogs Coordinating With Mycobacterium Tuberculosis Fatty Acid Synthase I (Fas I), Halimah Sayahi

Legacy Theses & Dissertations (2009 - 2024)

One third of the world's population is infected with M. tuberculosis, the causative agent of tuberculosis (TB) that claims the life of approximately 2 million people every year. The rapid spread of multidrug-resistance tuberculosis as well the emergence of extensively drug resistance strains that are resistant to virtually any known antibiotic, has transformed this once curable disease into a major public health challenge. To regain control of this disease and the associated mortality and morbidity new antibiotics are urgently needed.

Preparation And Application Of Transgenic And Knockout Mouse Models For Studies On Cytochrome P450 Functions, Yuan Wei

Legacy Theses & Dissertations (2009 - 2024)

The overall objective of this dissertation is to study the in vivo function of microsomal cytochrome P450 monooxygenases (P450s), which metabolize numerous drugs, chemical carcinogens, environmental pollutants, as well as endogenous signaling molecules such as steroid hormones and eicosanoids. The major research tool of this study involves the development of transgenic and knockout mouse models. The specific aims are 1) to study the in vivo function of NADPH-cytochrome P450 reductase (CPR) and CPR-dependent enzymes using a mouse model with a reversible hypomorphic Cpr gene; 2) to study the in vivo function of CYP2A13 with a CYP2A13- transgenic model; and 3) …