Alumni Dissertations

 

Alumni Dissertations

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  • SEROTONIN 1A RECEPTOR MEDIATED NEUROGENESIS IN THE DEVELOPING HIPPOCAMPUS

    Author:
    Buddima Ranasinghe
    Year of Dissertation:
    2009
    Program:
    Biochemistry
    Advisor:
    Probal Banerjee
    Abstract:

    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

    Author:
    Zhen Ren
    Year of Dissertation:
    2012
    Program:
    Biochemistry
    Advisor:
    Ranajeet Ghose
    Abstract:

    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.

  • MOLECULAR INTERACTIONS OF ADIPOCYTE FATTY ACID-BINDING PROTEIN WITH ACTIVATING AND NON-ACTIVATING LIGANDS: PROTEIN OLIGOMERIZATION AND LIGAND BINDING SITES

    Author:
    Samar Rizk
    Year of Dissertation:
    2013
    Program:
    Biochemistry
    Advisor:
    Ruth Stark
    Abstract:

    Intracellular lipid binding proteins involved in fatty acid transport and metabolism include adipocyte fatty acid-binding protein (AFABP), a 15 kDa polypeptide that plays a central role in the development of diabetes and atherosclerotic cardiovascular disease in experimental animals; the significant degree to which the protein is released into the bloodstream is thought to predict the development of Metabolic Syndrome. Upon binding of activating ligands such as linoleate and troglitazone (TDZ) or inactivating ligands such as oleate, AFABP has been proposed to adopt two alternative modes of self-association that activate or deactivate a nuclear localization signal. The goal of this study is to develop a molecular rationale for these contrasting ligand-associated signals. Both apo and liganded AFABP proteins were shown to maintain an overwhelmingly monomeric form in solution using size exclusion chromatography and static light scattering methods. Multidimensional solution-state nuclear magnetic resonance experiments were used to make sequential resonance assignments of the polypeptide backbone 1H and 15N nuclei. These assignments made possible ligand titration experiments that identified the key protein residues involved in the binding and the defined binding site. Comparative analysis of the binding sites in the three holo proteins demonstrated that oleate and linoleate bind similarly in a U-shaped configuration within the protein binding cavity despite their contrasting functional behavior, whereas the activating linoleate and TDZ ligands bind at dissimilar sites with the AFABP protein.

  • Metabolic Checkpoints in Cancer Cell Cycle

    Author:
    Mahesh Saqcena
    Year of Dissertation:
    2014
    Program:
    Biochemistry
    Advisor:
    David Foster
    Abstract:

    Growth factors (GFs) as well as nutrient sufficiency regulate cell division in metazoans. The vast majority of mutations that contribute to cancer are in genes that regulate progression through the G1 phase of the cell cycle. A key regulatory site in G1 is the growth factor-dependent Restriction Point (R), where cells get permissive signals to divide. In the absence of GF instructions, cells enter the quiescent G0 state. Despite fundamental differences between GF signaling and nutrient sensing, they both have been confusingly referred to as R and therefore by definition considered to be a singular event in G1. Autonomy from GF signaling is one of the hallmarks in cancer; however, cancer cells also have metabolic rewiring enabling them to engage in anabolic biosynthetic pathways. In the absence of GF instructions and nutrients, cells commonly undergo apoptotic cell death. Thus, it is of importance to elucidate the differences between GF and nutrient deregulation in cancer to develop novel strategies in targeting tumor cell proliferation and survival. Here, we report that the GF-mediated mid-G1 restriction point (R) is distinct and distinguishable from a series of late-G1 metabolic checkpoints mediated by essential amino acids, conditionally essential amino acid - glutamine, and mTOR - the mammalian target of rapamycin. Our data indicate that the arrest sites mediated by various blocking conditions are in the order of GF -> EAA -> Q -> mTOR. We temporally mapped the EAA and glutamine checkpoints at 12 hr from G0 and mTOR mediated arrest occurring at 16 hr from G0. Distinct profiles for cell cycle regulator expression and phosphorylation was observed when released from restriction point relative to the metabolic checkpoints. These data are consistent with a mid-G1 R where cells decide whether they should divide, followed by late-G1 metabolic checkpoints where cells determine whether they have sufficient nutrients to divide. Since mTOR inhibition using rapamycin or Torin1 arrested the cells latest in G1, mTOR may serve as the final arbiter for nutrient sufficiency prior to replicating the genome. Significantly we also observed that in addition to GF autonomy, several cancer cells also have dysregulated nutritional sensing, and arrest in S- and G2/M phase upon essential amino acid and glutamine deprivation. We identified K-Ras mutation as the underlying genetic cause for this phenomenon. We found that treating cancer cells harboring K-Ras mutation with aminooxyacetate (AOA) - drug that interferes with glutamine utilization - causes them to arrest in S- and G2/M-phase, where synthetic lethality could be created to phase-specific cytotoxic drugs. Thus, besides addressing the long standing assumption of GF and nutrients regulating G1 cell cycle progression, our work provides rationale and proof of principle for targeting metabolic deregulations in cancer cells.

  • A TALE OF TWO NUTRITIONAL TARGETS: STUDIES OF MOLECULAR STRUCTURE AND PEDAGOGY

    Author:
    SAYANTANI SARKAR
    Year of Dissertation:
    2012
    Program:
    Biochemistry
    Advisor:
    RUTH STARK
    Abstract:

    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.

    Author:
    Javier Suarez
    Year of Dissertation:
    2009
    Program:
    Biochemistry
    Advisor:
    Richard Magliozzo
    Abstract:

    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. Previous studies have shown the formation of amino acid based radicals in KatG upon reaction with alkyl peroxide. However, the location and the possible function of these radicals are far from being resolved. In this study we tried to gain insights into the loci of radical formation through the analysis of cross-linking during turnover of KatG in the presence and absence of reducing substrate. SDS and Native-PAGE of KatG treated with peracetic acid or hydrogen peroxide under a variety of conditions demonstrate oligomers of molecular weight greater than that of the native dimer. The results are consistent with the hypothesis that cross-linking of KatG can occur in the absence of peroxidase substrates and that under physiological conditions, the activation of INH as well as the stability of KatG may be altered by this process. One of the most interesting structural features of Mtb KatG is the post-translational modification of residues Met 255, Tyr229 and Trp107, the side chains of which form an adduct on the distal side of the heme. Mutation of any of these three residues completely abolishes the catalase activity of KatG. A mechanism accounting for the robust catalase activity and the function of this adduct in catalase-peroxidases (KatG) presents a new challenge in heme protein enzymology. Here, optical stopped-flow spectrophotometry, rapid freeze-quench electron paramagnetic resonance (RFQ-EPR) spectroscopy both at X-band and at D-band, and mutagenesis were used to identify catalase reaction intermediates in Mtb KatG. Using rapid-freeze-quench EPR at X-band under catalase activity conditions (excess H2O2), a narrow doublet radical signal with an 11 G principal hyperfine splitting was detected within the first milliseconds of turnover. The radical persists in wild-type KatG only during the time course of turnover of excess H2O2 (1000-fold or more). Mutation of Met255, Tyr229, or Trp107, abolishes this radical and the catalase activity. Therefore, a catalytic role for an MYW adduct radical in the catalase mechanism of KatG is proposed.

  • Role of mammalian ubiquitin ligases UBR1 and UBR2 in cytosolic protein quality control

    Author:
    MST. RASHEDA SULTANA
    Year of Dissertation:
    2012
    Program:
    Biochemistry
    Advisor:
    Avrom Caplan
    Abstract:

    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. My studies with other Hsp90 clients showed that UBR1 promotes degradation of steroid hormone receptors GR and AR but not the ER- £\. Co-expression of rUBR1 with hGR led to reduce the levels of hGR in the presence and absence of GA. There is a direct correlation between increasing UBR1 concentration and decreasing GR levels. Further studies addressed the specificity of that function with analysis of hAR and hER-fÑ. In this case, there was a significant reduction of the hAR levels when UBR1 was overexpressed, even in the absence of GA. By contrast, similar experiments with transfected hER-£\ suggest that UBR1 does not play a similar role in the degradation of this receptor. My combined findings suggest that UBR1 acts specifically in the clearance of GR and AR but not in ER-fÑ. All of these findings suggest that UBR1 is involved in the cytosolic protein quality control in mammalian system and it also plays a role in determining the sensitivity of the cells to the Hsp90 inhibitors.

  • BIOPHYSICAL CHARACTERIZATION OF HCV Q65H NS5B AND EIF4B : PROTEIN - NUCLEIC ACID INTERACTIONS

    Author:
    Shemaila Sultana
    Year of Dissertation:
    2010
    Program:
    Biochemistry
    Advisor:
    Dixie Goss
    Abstract:

    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. The second part of this study involved interaction of eIF4B with zinc. eIF4B promotes RNA dependent ATP hydrolysis and ATP dependent RNA helicase activity of eIF4A and eIF4F. It has recently been reported that eIF4B also organizes assembly of the RNA, eIFiso4G, eIF4A, and poly-(A) binding protein and that zinc enhances the RNA binding and interaction with PABP. We previously reported that zinc binds tightly with eIF4B with a Kd of 19.7 ± 1.6 nM (J Biol Chem. 2008 Dec 26; 283(52):36140-53). The Kd of binding of eIF4B with 20mer poly (A) RNA is 77 ± 7 nM and in presence of zinc is reduced down to 45 ± 3 nM. Circular dichroism showed that addition of zinc resulted in a more than 50% decrease in alpha content of the eIF4B protein. To better understand the role of eIF4B in the selection of RNA, we also studied the interaction of eIF4B with structurally different RNAs and the effect eIF4A on this interaction using fluorescence anisotropy. This overall study on eIF4B helps towards the better understanding of basic translation initiation machinery and some of its regulators.

  • Green Drugs: Anticancer Properties of Clerodendrum viscosum and Curcumin Conjugates

    Author:
    Chong Sun
    Year of Dissertation:
    2013
    Program:
    Biochemistry
    Advisor:
    Krishnaswami Raja
    Abstract:

    This thesis aims to find potential anti-cancer drug from natural sources like plants. (i) The objectives are to find and characterize the anticancer components from the plant Clerodendrum viscosum. (ii) the synthesis of curcumin derivatives with increased solubility and amplified bioactivity. Clerodendrum viscosum (Cv) is a traditional medicine plant in India for a long period of time employed to promote wound healing, and recently it has been found have some positive effects on cervical cancer. However, there were no direct v molecular and biochemical evidences for that. In this thesis, we made aqueous extracts (Cv-ap) from the Cv by ammonium sulfate precipitation. It was found that this extract had bioactivity against Hela cells - a human cervical cancer cell line. Caspase-3 immuno-staining proved that the Cv-ap can induce Hela cells apoptosis. It is also interesting that the Cv-ap can inhibit the movement of the cells at lower concentration without inducing cells apoptosis, which means it may stop the invasion of the cancer cells. MTT assays showed that at lower concentration the Cv-ap may not only inhibit the movement of the cells but also inhibit proliferation of the Hela cells. For the characterization of the Cv-ap, it was found that it had glycoproteins confirmed by SDS-PAGE staining with the Pro-Q emerald 300, and by binding to the concavalin A column. For further purification, the bio-assay guided method was used to purify the bioactivity components. After Cv-ap was passed through the column, the fraction with the most activity fraction was selected and followed by heating, and finally by precipitation with 66.7% ethanol. In the end we got the vi bioactivity component EPHP3 fraction which has a 30 fold increasing activity when compared to the Cv-ap. My second project aimed to overcome the difficulties associated with the low water/plasma solubility of the potent anti-oxidant, anti-inflammatory, anti-carcinogenic, anti- Alzheimer's active curcumin. We have successfully synthesized a curcumin derivate BSA-curcumin via a one-step successfully synthesized. BSA-curcumin had the high solubility in water and good cytotoxicity to the Hela cells. In conclusion, my research has characterized and produced novel plant derived components that have anti-cancer activity and may eventually be used in clinical therapies.

  • Identification and Mechanistic Study of Immunostimulatory Lipids in Juzen-Taiho-To

    Author:
    Anna Takaoka
    Year of Dissertation:
    2012
    Program:
    Biochemistry
    Advisor:
    Akira Kawamura
    Abstract:

    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. In this thesis, chapter 1 describes the establishment of the technique we used, biomarker–guided screening, to identify therapeutically relevant compounds from herbal medicines. The technique was established in our lab and refined using two different herbal medicines, namely Keishi–bukuryo–gan and Toki–shakuyaku–san. Chapter 2 describes the identification of common chemical components in MΦ–stimulating fractions from two different batches of JTT. This is an important achievement because batch–to–batch variability is a major concern in the natural product research. Chapter 3 describes the characterization of individual compounds identified in MΦ–stimulating fractions, which led to the discovery of one system that exhibit significant synergistic MΦ–stimulating activity, i.e. β–Glucocerebroside (GlcCer) and C16:0–ceramide (Cer). This finding is important because it is a rare example of a structurally defined, small molecule mixture that exhibited synergistic MΦ–stimulating activity. In addition, the activity assay showed that MΦ–stimulating activity of GlcCer/Cer mixture was time–sensitive and diminished within 3 hr of sample reconstitution in DMSO. Further mechanistic study of GlcCer/Cer mixture using transmission electron microscope (TEM) and dynamic light scattering (DLS) revealed that GlcCer/Cer mixture forms nanoparticles in both DMSO and an aqueous environment, and the size of the nanoparticles correlated with the MΦ–stimulating activity. Our study provided a new basis of novel combinational therapy, and served as an important step to systemically characterize the safe and effective immunostimulatory herbal medicine, JTT.