SEROTONIN 1A RECEPTOR MEDIATED NEUROGENESIS IN THE DEVELOPING HIPPOCAMPUS
Author:
Buddima Ranasinghe
Year of Dissertation:
2009
The importance of the brain serotonin 1A receptor (5-HT1A-R) during postnatal brain development has been established, but the mechanism of its action in brain neurons remains unclear. It is currently known that the 5-HT1A-R plays a crucial role in the brain by regulating mood and behavior and 5-HT1A-R stimulation in adult mice has been suggested to induce neurogenesis in the adult neurogenic niches such as the subventricular zone (SVZ) and the dentate gyrus (DG) of the hippocampus. In mice, absence of the 5-HT1A-R during development results in heightened anxiety and depressive behavior. However, the 5-HT1A-R mediated signaling activity that is responsible for its role during development is unknown. Among the hippocampal signaling pathways stimulated by the agonist-bound 5-HT1A-R, the mitogen activated protein kinase (MAPK) pathway is an important regulator of both division and survival of neuronal cells in the brain. Additionally, the Protein Kinase C (PKC) isozyme PKCɛ is an important signaling molecule that is highly expressed during early postnatal brain development particularly postnatal day 2-6 (P2-P6). Here we show that neurogenesis in the developing hippocampus at P6 in mice is dependent on both MAPK and PKCɛ. Our initial experiments use pharmacological inhibitors to confirm that PKCɛ mediates 5-HT1A-R-linked activation of MAPK. We then demonstrate that neurogenesis is increased upon stimulation of this 5-HT1A-R→PKCɛ→MAPK pathway both in a hippocampal-derived neuronal cell line stably expressing the 5-HT1A-R (HN2-5) and in the DG of P6 mice. Further, inhibition of either MAPK or PKCɛ considerably disrupts the burst in bromo-deoxy-uridine (BrdU) labeling and Ki-67 staining, showing neuroblast number, in the DG. As for a downstream signal that relays the proliferative signal from MAPK, we have identified the Retinoblastoma protein (Rb) as a potential target of MAPK, and shown that its phosphorylation dynamics may be tightly regulated in response to 5-HT1A-R stimulation. Therefore, our findings reveal a novel pathway involving PKCɛ, MAPK, and Rb through which the 5-HT1A-R potentially regulates neurogenesis during early postnatal hippocampal development.
DYNAMICS ON MULTIPLE TIMESCALES IN THE CYSTOVIRAL RNA-DIRECTED RNA POLYMERASE
Year of Dissertation:
2012
The RNA-dependent RNA polymerase P2 from cystovirus ϕ6 directs the replication and transcription of the double-stranded RNA genomes. In spite of the availability of multiple crystal structures at various points along its catalytic pathway, the dynamics role involving in the catalytic cycle and fidelity control remain unclear. Isoleucine residues are distributed over the three-dimensional fold of P2. By using the δ1 positions of 25 Ile residues as probes, we measured the dynamic modes and their alterations along P2 catalytic cycle with CPMG-based multiple quantum relaxation dispersion experiments. The results indicate that P2 is dynamic on the fast (ps-ns) and slow (μs-ms) timescale. The characteristics of several motional modes are altered in the presence of substrate analogs and single-stranded RNA templates. The timescale of the lower frequency motional modes that involve several conserved functional motifs coincides with the catalytic timescale (1-2 ms), which was determined from kinetic analyses of representative RdRPs. We further investigated the influence of the extreme 3'-end sequence of the single-stranded RNA templates and the nature of the substrate nucleotide triphosphates on the slow motional modes using multiple-quantum relaxation dispersion. We found that P2, in the presence of templates bearing the proper genomic 3'-ends or the preferred initiation nucleotide (GTP), displays unique dynamic signatures that are different from those in the presence of nonphysiological templates or substrates. This further suggests that dynamics may play a role in the fidelity of recognition of the correct substrates and template sequences to initiate RNA polymerization.
A TALE OF TWO NUTRITIONAL TARGETS: STUDIES OF MOLECULAR STRUCTURE AND PEDAGOGY
Year of Dissertation:
2012
Nutrition is one of the allied branches of biochemistry. Numerous nutritional targets in lipid metabolism and energy homeostasis are widely studied in biochemical research. For instance, liver fatty acid-binding protein is an important player in lipid metabolism. In this dissertation, two nutritional targets: physiological liver fatty acid-binding protein (LFABP) and natural tomato were studied from basic research and pedagogical perspectives. Three different projects were undertaken involving LFABP and tomato. (1) In the first project, solution state NMR was used to investigate ligand binding to LFABP. Oleate and linoleate exhibited moderately different binding locations in spite of apparently similar binding stoichiometries to LFABP. Additionally, oleate liganded holo LFABP showed chemical shift perturbations in presence of the anticoagulant drug warfarin, indicative of possible competition between oleate and warfarin as ligands of LFABP. However, reported claims of glucose and phytanic acid as ligands of LFABP could not be validated by solution state NMR. (2) In the second project, the basic research protocol concerning LFABP inspired a redesign of the City College undergraduate biochemistry laboratory course with an aim to incorporate a "research-inspired" module involving a new in-silico exercise. (3) In the third project, basic physical and spectroscopic research techniques used to study biopolymers in tomato were used to develop an adaptable suite of chemistry experiments for undergraduate and high school students. Both curricula were successfully tested on undergraduate and high school students in a cost effective fashion, demonstrating the feasibility of their implementation.
THE CATALYTIC MECHANISMS OF MYCOBACTERIUM TUBERCULOSIS CATALASE-PEROXIDASE (KATG) AND THE ORIGIN OF ANTIBIOTIC RESISTANCE IN THE KATG[S315G] MUTANT.
Year of Dissertation:
2009
Advisor:
Richard Magliozzo
Catalase-peroxidase (KatG) in M. tuberculosis is a bifunctional heme protein that exhibits both high catalase activity (2H2O2 → 2H2O + O2) and a broad-spectrum peroxidase activity (2AH + H2O2 → 2A* + 2H2O) and is responsible for activation of isoniazid (INH), a pro-drug used to treat TB infections. Resistance to INH is a global health problem most often associated with mutations in the katG gene. Nevertheless, there is a big gap in the M. tuberculosis literature with respect to the molecular origins of isoniazid resistance due to mutations in KatG. Here, we examined the origin of INH resistance caused by the KatG[S315G] mutant enzyme. Overexpressed KatG[S315G] was characterized by optical, EPR and resonance Raman spectroscopy and by studies of the INH activation mechanism in vitro. INH resistance is suggested to arise from a redirection of catalytic heme intermediates into non-productive reactions that interfere with oxidation of INH.
BIOPHYSICAL CHARACTERIZATION OF HCV Q65H NS5B AND EIF4B : PROTEIN - NUCLEIC ACID INTERACTIONS
Year of Dissertation:
2010
Hepatitis C Virus (HCV) infection is one of the most common chronic blood borne infections. The mechanism underlying persistence of HCV infections are not well understood. Mutation of Gln65His (Q65H), which develops in the NS5B dependent RNA polymerase (RdRp) during chronic infection, has 1.8 fold enhanced invitro RdRp activity, may have major implications in the efficiency of HCV RNA replication and viral persistence. The reason for this increase in activity is not well understood (Virology 2003 Dec 5; 317(1):65-72). We used endogenous tryptophan fluorescence to characterize the interactions of Q65H protein with GTP, GDP, magnesium and a 19mer RNA template. The Q65H protein exhibits 3 fold lower Kd (11.6 ± 1.2 µM) than wildtype (36.8 ± 1.0 µM) for GTP, whereas the Kd values of the mutant for magnesium and 19 mer RNA template were similar to wildtype protein. Thermodynamic studies on the binding reaction of the Q65H variant were also performed to provide details of the energetic and entropic characteristics.
Role of mammalian ubiquitin ligases UBR1 and UBR2 in cytosolic protein quality control
Author:
MST. RASHEDA SULTANA
Year of Dissertation:
2012
UBR1 and UBR2 ubiquitin ligases function in the N-end rule degradation pathway in lower and higher eukaryotic cells. In yeast, the Ubr1 homologue also functions by N-end rule independent means to promote degradation of misfolded proteins generated via stress or with Hsp90 inhibitor GA. Based on these studies I examined the role of mammalian UBR1 in the degradation of protein kinase clients upon Hsp90 inhibition. I provide evidence that mammalian UBR1 promotes protein kinase quality control and sensitizes the cells to Hsp90 inhibition. The UBR1 deleted MEF cells showed reduced degradation of several protein kinases in the presence of GA. My findings also showed that Akt, p-Akt and Cdk4 the Hsp90 client protein kinases are still degraded in mouse UBR1 -/- cells treated with GA, but their levels recovered within 12-18 hours, in contrast to the wild type cells. The same findings were observed for human BT474 breast cancer cells with knocked down UBR1 by shRNA. These findings correlate with increased induction of Hsp90 expression in the Ubr1-/- cells compared with wild type cells. In addition, deletion of UBR1 and UBR2 showed resistance in terms of cell viability compared to wild type cells in the presence of GA and PU-H71. I also observed a reduction of UBR1 protein levels in GA-treated MEF and BT474 cells, suggesting that UBR1 is an Hsp90 client. I propose the existance of a novel feedback loop, where UBR1 negatively controls Hsp90 expression, while Hsp90 controls UBR1 stability. Further studies with CHIP reveal that CHIP and UBR1 have some functional overlap with respect to their E3 activities while UBR1 also affects the function of the Hsp90 chaperone machinery.
Green Drugs: Anticancer Properties of Clerodendrum viscosum and Curcumin Conjugates
Year of Dissertation:
2013
Advisor:
Krishnaswami Raja
This thesis aims to find potential anti-cancer drug from
Identification and Mechanistic Study of Immunostimulatory Lipids in Juzen-Taiho-To
Year of Dissertation:
2012
Juzen–taiho–to (JTT) is an herbal medicine known to exhibit safe and effective immunostimulatory activity. It is clinically used to improve the immune functions of cancer patients undergoing chemotherapy and radiation therapy in Japan. The chemical constituents responsible for its therapeutic effects are poorly characterized due to the chemical complexity of this formulation and possible synergism. Identification of the active constituents would be the first step to systemically characterize the safety and efficacy of JTT and to understand the mechanism. The main objective of this thesis was the identification of macrophage (MΦ)–stimulating compound(s) in JTT by biomarker–guided screening, and the characterization of individual components to identify possible new biological effects of those compounds.
BIOCHEMICAL AND STRUCTURAL CHARACTERIZATION OF DI-ASPARTYL INTRAMEMBRANE PROTEASES
Author:
Celia Mirtha Torres Arancivia
Year of Dissertation:
2011
A new era in the study of proteases was initiated with the discovery of intramembrane-cleaving proteases (I-CLiPs). In an unusual mechanism, I-CLiPs are able to catalyze peptide bond cleavage of substrates within the membrane bilayer, an environment poorly permeable to water. Here we will focus on gamma-secretase and presenilin(PS)-like proteases, membrane proteases that use two aspartic acids as catalytic residues and constitute the most relevant family of I-CLiPs. We first describe the purification of gamma-secretase from a mammalian expression system. We used this preparation for negative-stain single-particle electron microscopy and determined the structure of a native-like catalytically active gamma-secretase complex at a resolution of 25 Å. Antibody labeling of the extracellular domain of Nicastrin was employed to ascertain the topology of the reconstruction. In addition, active site labeling with a gold-coupled inhibitor demonstrated that gamma-secretase contains a single active site facing a large conical internal cavity.
A coarse-grained view of protein-protein recognition
Author:
Rodney Versace Babilonia
Year of Dissertation:
2012
One of the most important characteristics of proteins is their ability to specifically interact with other proteins and with other types of molecules to build supramolecular assemblies in order to perform different kinds of functions. Protein-protein interaction has been a subject of study in several sciences including biochemistry, structural biology, and computational biophysics/biology. One limitation that delays our understanding of molecular recognition is the lack of high-resolution three-dimensional structures of the protein-protein complexes. Because of this, the methods for computational prediction have gained popularity and importance but in many cases, the predicted complex is not accurate. Predicting the native three-dimensional conformation of protein-protein complexes still remains a big challenge.