Characterization of Mitochondrial DNA Heteroplasmy at Five Hotspots within the HVI Region of Post-Mortem, Formalin Fixed Paraffinized Human Liver Cells
Year of Dissertation:
2012
Within the field of mitochondrial DNA (mtDNA) analysis, heteroplasmy is a widely recognized and yet poorly-understood event. Heteroplasmy is defined as the presence of more than one mitochondrial genome within a tissue sample from a single individual, such that the mtDNA sequence shows the presence of a mixed base or regions of homologous bases that vary in length. Due to the highly conserved nature of the mitochondrial genome, these heteroplasmic events occur at a variety of well-documented hotspots, a majority of which occur within the hypervariable control region that flanks the origin of replication. This control region is the same area that is tested in forensic mtDNA analysis, and is the most useful for establishing the link between evidentiary samples and maternally-related individuals due to the polymorphisms that accumulate in this region. However, when heteroplasmic events are uncovered in forensic mtDNA analysis, issues arise due to the lack of clear understanding of the origin of heteroplasmy. The occurrence of mtDNA heteroplasmy between different tissues within a single individual has been well-established, and heteroplasmic events have been shown to increase with age. However, what is presently unclear is whether or not mtDNA heteroplasmy exists within a single cell when heteroplasmy is present within a tissue. In this regard, three possibilities exist; 1) a single cell contains a solitary pool of mtDNA genomes (defined as homoplastic), and heteroplasmy exists as a mixture of different homoplastic cells within tissue, 2) a single cell contains a mixture of multiple mtDNA genomes, and heteroplasmy is present within a single cell, or 3) heteroplasmic tissue contains a mixture of homoplastic and heteroplasmic cells due to random cellular distribution throughout the tissue. To investigate this question, laser dissection microscopy will be used to isolate individual cells from a tissue sample with known mtDNA heteroplasmy. Typing of single nucleotide polymorphisms at specific hotspots within the HVI region will then be done to detect possible mtDNA heteroplasmy within a single cell.
Strategic targeting of curcumin to eliminate brain tumors
Year of Dissertation:
2011
Glioblastoma, the most common form of primary brain cancer, is highly aggressive and associated with very poor prognosis. Curcumin (or diferuloylmethane), a natural molecule, which is not toxic to normal tissue, has been shown to inhibit proliferation, induce apoptosis and inhibit angiogenesis and metastasis in a wide range of cancer cells. However, the effective delivery of curcumin to cancers presents a problem because curcumin is poorly soluble in water and metabolizes quickly. Our preliminary work has established that solubilized curcumin can cross the blood-brain barrier and is harmless to normal brain cells, that solubilized curcumin blocks brain tumor formation when introduced by injection into the blood or directly into the brain, and that it markedly decreases cell viability in several cell lines, including murine melanoma B16F10 and murine glioblastoma GL261. Targeted drug delivery is frequently used to deliver drugs selectively and at high concentrations to cancer tissue. Antibody-mediated targeting, in addition to delivering drugs selectively, serves to increase the water solubility of attached drugs. We postulated that antibody-mediated targeting would be an effective means of eliminating brain tumors. Nonetheless, a number of structural features had to be carefully considered. Curcumin has several functional groups, which potentially can be used to target the molecule to cancer cells; however, curcumin's functional groups have been shown to be critical for its anticancer activity. With this in mind, after weighing different options, we synthetically modified curcumin at its phenolic hydroxyl position to enable the formation of a cleavable antibody attachment. Intracellular hydrolysis of an ester bond returns curcumin to its original state after its delivery into target cells. Murine infiltrating melanoma (B16F10) and primary glioblastoma (GL261) brain tumor models were utilized. We created two adducts, curcumin-MUC18 for targeting to B16F10 cells and curcumin-CD68 for targeting to GL261 cells. Our studies show that both adducts are highly effective at eliminating B16F10 and GL261 cancer cells in vitro, and that these adducts destroy cancer cells at far lower concentrations than does free curcumin. Our molecular analyses show that, in GL261 cells, curcumin causes a dramatic increase in caspase 3/7 activity and suppression of tumor-promoting proteins NF-κB, Akt1, VEGF, cyclin D1, and BclXL. We show in GL261 cells that overexpressed NF-κB is protective against curcumin treatment. Lastly, we show that animals implanted with B16F10 or GL261 cells receiving targeted curcumin treatment live longer and have significantly reduced tumor size.
POTENTIAL ROLE OF ATP8A1 AS PLASMA MEMBRANE AMINOPHOSPHOLIPID TRANSLOCASE IN PROLIFERATING NEURONAL CELLS
Year of Dissertation:
2009
The inner leaflet-localized phospholipid PS undergoes a translocation to the outer leaflet of the plasma membrane in apoptotic cells to trigger recognition and phagocytic removal of these dying cells by PS receptor-bearing scavenger cells, such as microglia and macrophages. The enzyme activity responsible for the inner-membrane localization of PS is the plasma membrane aminophospholipid translocase (PLAPLT), also known as flippase, which translocates PS from the outer to the inner leaflet of the plasma membrane. Attempts to identify the PLAPLT molecule of mammalian cells have revealed a candidate molecule, Atp8a1, which is a P-type Mg-ATPase. After much controversy, it is currently believed that Atp8a1 translocates PS across internal membranes but not the plasma membrane. Based on our earlier studies showing overexpression of Atp8a1 in proliferating hybrid neuroblastoma cells causes an increase PLAPLT activity, we postulated that Atp8a1 functions as PLAPLT only in fast dividing cells, such as neurotumor cells or neuroblasts. This study used the fluorescent PS analogue, NBD-PS, to show that ectopic expression of Atp8a1 in the N18 neurotumor cells causes no significant change in the Km value of PLAPLT, but an increase in the Vmax for this enzyme, which suggests that overexpression of Atp8a1 causes an increase in the PLAPLT molecules. This indicates that Atp8a1 is possibly identical to the PLAPLT molecule of the N18 cells. As a confirmation of this hypothesis we expressed phosphorylation-site mutants of Atp8a1 in the N18 cells to elicit a decrease in the Vmax value of PLAPLT, without significantly altering the Km value. The inhibition of the PLAPLT activity in N18 cells was also evidenced by a striking increase in surface staining of these cells with the PS-binding protein annexin V. According to our postulate Atp8a1 deletion should also cause PS exposure in neuroblasts harbored within the dentate gyrus (DG), which is a proliferative niche within the memory center termed hippocampus. In corroboration, we observed pronounced annexin V staining in both dissociated DG cells as well as cultured hippocampal slices of Atp8a1 (-/-) mice but not wild type mice. Such PS externalization should trigger phagocytosis of DG cells, which in turn could lead to a loss of hippocampal function. In support of this postulate we have observed that the Atp8a1 (-/-) mice suffer from possibly hippocampal-related learning defects. Therefore, Atp8a1 may play a crucial role in the maintenance of the functional integrity of the hippocampus. Additionally, our study reveals a potential strategy for the selective removal of the brain tumor cells through targeted suppression of Atp8a1 activity in brain cancer cells, which would lead to PS externalization and elimination of the cells by phagocytosis.
Conformational Dynamics of Guanine Residues Within the Human Telomeric G-quadruplex
Year of Dissertation:
2012
The human telomeric single-stranded guanine-rich DNA (HT4: d(TTAGGG)4) located at the end of chromosomes forms an intramolecular G-quadruplex in the presence of K+ or Na+ in vitro. The formation and stabilization of this structure by quadruplex interactiveagents (QIAs) can inhibit the activity of the enzyme complex telomerase, which is overactivated in cancer cells, and is thus a target for potential cancer therapeutics. However, the solution quadruplex conformation is complex and varies with the presence of stabilizing Na+ or K+-ions. In addition, details about the contribution of individual guanine residues of the single stranded HT4 sequence required for G-quadruplex formation and stabilization remain unclear.
GENE EXPRESSION IN HUMAN KERATINOCYTES CONTAINING INTEGRATED COPIES OF SV40 EARLY REGION
Year of Dissertation:
2013
ABSTRACT
MODELING SMAD DOMAINS AND THEIR INTERACTION WITH SMURF-1, C-SKI AND DNA PROMOTER MOTIF TO DESIGN INHIBITORY COMPOUNDS
Year of Dissertation:
2010
Advisor:
Boojala Vijay Reddy
Transforming Growth Factor-&beta (TGF-&beta) superfamily members are known for regulating wide array of cellular processes such as growth, differentiation, proliferation, and apoptosis. In the downstream of TGF-&beta signaling there are important growth and differentiation factors known as Smad proteins, which carry out the TGF-&beta responsive signaling and elicit various responses once inside the nucleus. The goal of this dissertation is texplore the available structural data of some of the molecules involved in TGF-&beta signaling process and to apply state of the art molecular modeling, docking and virtual screening tools and techniques to gain insight into the TGF-&beta signaling pathway. This study mainly concentrates on the interaction of Smad proteins with the DNA promoter motif, and other proteins c-Ski and Smurf-1 with which they interact in the signaling process. Initially MH1 domain of mammalian Smad proteins were modeled based on known crystal structure of Smad3 MH1-DNA complex (PDB ID: 1OZJ) followed by modeling of interaction pose of MH1 domain of BMP regulated Smads (Smad1/5/8) with their corresponding DNA sequence motif 5'-GCCG-3'. In this work the key residues of MH1 domain of Smad1/5/8 interacting with `GCCG' motif were identified. To investigate further the solvent accessibile contact area of key residues and binding energy calculations of modeled Smad1/5/8 MH1 with the GCCG DNA motif and GTCT DNA motif were computed. Higher free energy of binding for Smad1/5/8-MH1 complexed with nonspecific `GTCT' DNA motif compared to the GCCG motif confirmed high specificity of Smad1/5/8 with `GCCG' motif indicating that these Smads may not bind with `GTCT' DNA. Further, homology modeling approach was followed to build Smad binding domain of c-Ski, a proto-oncoprotein, which acts as co-repressor in Smad mediated TGF-&beta signaling. Various protein-protein docking methods were applied to study the interactions between the model c-Ski domain and Smad3-MH2 domain. Knowledge of biochemical data, contacts observed between key residues and solvent accessibility calculations of residues of both proteins in our top models were applied to finalize four best favored complexes of Smad3-Ski that can be used to design small molecule inhibitors antagonizing the c-Ski binding which may lead to anti-cancer drug design by appropriately regulating Smad3-Ski interaction.
Structure/Function Correlations in Pseudomonas aeruginosa DNA Ligase LigD
Year of Dissertation:
2011
The ATP-dependent DNA ligase D (LigD) performs a major role in the non-homologous end-joining (NHEJ) pathway. Pseudomonas aeruginosa LigD contains a N-terminal phosphoesterase domain (PE) domain followed by a ligase domain and a C-terminal polymerase domain. The PE domain (187 residues), belonging to a class of unique 3'- end-processing enzymes, possesses manganese dependent phosphodiesterase and phosphomonoesterase activities as it sequentially removes the 3'-ribonucleoside from the primer strand of the primer-template DNA duplex and hydrolyzes the 3'-PO4 produced finally to a 3'-OH group. Extensive mutagenesis and biochemical studies have identified critical residues and important features required for 3'- ribonuclease and 3'- phosphatase activities. Lack of sequence homology to other known nucleases lead to the belief that this enzyme possesses some unique motifs. However, in the absence of atomic level structural information clear structure/function correlations were lacking. This thesis describes the procedures used to obtain a high-resolution structure of PE domain obtained using solution NMR methods and to ascertain its interaction with DNA substrates.
Role of p53, CstF-50, and BARD1 in the Regulation of mRNA Processing following DNA Damage
Year of Dissertation:
2010
Following UV irradiation, cellular mRNA levels are transiently decreased due to the inhibition of transcription and mRNA polyadenylation. The UV-induced inhibition of 3' processing reflects the interaction of the polyadenylation factor CstF-50 with the tumor suppressors BRCA1/BARD1 as well as the BRCA1/BARD1-mediated degradation of RNA polymerase II (RNAP II). As CstF-50 can interact with BRCA1/BARD1 and RNAP II inhibiting or activating polyadenylation, respectively, and cells with reduced levels of CstF show an enhanced sensitivity to UV and reduced ability to ubiquitinate RNAP II and repair DNA, we propose a coordinating role for this factor in the DNA damage response.
Exploration of Aporphines as MDMA and AChE Inhibitors
Year of Dissertation:
2010
PART A
CHARACTERIZATION OF PAXILLIN, PHOSPHOLIPASE D AND THEIR FUNCTIONAL INTERACTION
Year of Dissertation:
2012
The actin cytoskeleton plays a fundamental role in various processes including differentiation, migration, endocytosis and exocytosis. An adapter protein, paxillin, as well as an enzyme, Phospholipase D (PLD), have been associated with processes based on actin cytoskeleton regulation. Such regulation is critical for the development of Dictyostelium discoideum. To gain better insight into the roles of paxillin and PLD and to investigate their