Biochemistry Honors Theses and Research Papers

Large scale production of stable hemolymph juvenile hormone binding protein

Large scale production of stable hemolymph juvenile hormone binding protein Arifin, Trina To better understand the pathways of the insect juvenile hormone (JH) action, milligram amounts of pure hemolymph juvenile hormone binding protein (hJHBP) are required. Hence the goal of this study is to scale up the system obtaining for purified and stable hJHBP. A drosophila S2 cell line was previously established in the laboratory and stably transfected with a plasmid containing the coding region of hJHBP. The construct included a metallothionein promoter which induces the recombinant JHBP (rJHBP). Purification of the protein produced involved column chromatography using a HisBind resin (Novagen). The production, purification and cleavage of rJHBP had to be significantly modified from previous results to obtain a functional Manduca sexta hJHBP. 1 p. (abstract only). YOU CANNOT OPEN THE COMPLETE PAPER. It is not available to the public, in accordance with the author's wishes.

Probing the chemistry in the active site of P4H

Probing the chemistry in the active site of P4H Pua, Khian Hong Prolyl 4-hydroxylase (P4H), a mononuclear, non-heme FeII, 2-oxoglutarate and O2 dependent enzyme, plays an integral role in the biosynthesis of collagen. It catalyzes the post-translational hydroxylation of proline residues that leads to the stabilization of the collagen triple helix. Mononuclear, non-heme FeII, 2-oxoglutarate and O2 dependent enzymes are known to catalyze a wide variety of reactions including oxidative aromatic ring cleavage, cis-dihydroxylation of double bonds, and the hydroxylation of aliphatic C-H bonds. Non-heme FeII enzymes which carry out oxidative halogenations have recently been discovered. Halogenase SyrB2 and hydroxylase P4H are related enzymes, both 2-oxoglutarage and oxygen dependent. It is thought that both the halogenations and hydroxylation reactions proceed via a similar mechanism, each performing an extremely difficult reaction, abstracting a hydrogen atom from an aliphatic carbon, to allow the formation of a halogenated or hydroxylated product. Here we report that replacing the aspartate residue in the active site of P4H with alanine, so that the active site now mimics that of the halogenase SyrB2 was not successful in converting P4H to do halogenations chemistry. In addition, switching the relative positions of residues involve in the active site in wild-type P4H results in the loss of hydroxylase activity and no gain in halogenase activity when critical residues in P4H wer substituted to mimic that of a halogenase. 1 p. (abstract only). YOU CANNOT OPEN THE COMPLETE PAPER. It is not available to the public, in accordance with the author's wishes.

In vitro screenings predict polymer ability to facilitate RNAi through its use as a delivery vehicle of siRNA

In vitro screenings predict polymer ability to facilitate RNAi through its use as a delivery vehicle of siRNA Scarpace, Katie Gene therapy techniques have emerged in recent decades as a method of combating genetic disorders. One prospective branch of gene therapy is using RNA Interference (RNAi) to regulate gene expression. RNAi employs small interfering RNA (siRNA) to knockdown gene expression posttranscriptionally. The challenge for scientists is figuring out how to deliver the siRNA to target cells. Many groups are currently researching siRNA delivery techniques. It is hypothesized that in vitro screening of delivery techniques by measuring gene expression knockdown and viability will predict delivery success in vivo. For my senior thesis I will be studying a delivery mechanism of foreign siRNA and using in vitro screening techniques to analyze siRNA delivery by measuring gene knockdown and technique toxicity. It can be concluded that in vitro screening can predict in vivo delivery success. 1 p. (abstract only). YOU CANNOT OPEN THE COMPLETE PAPER. It is not available to the public, in accordance with the author's wishes.

Cleavage and Polyadenylation Specific Factor 6 (CPSF6): A possible protein mediator for CARM1 and pre-MRNA splicing

Cleavage and Polyadenylation Specific Factor 6 (CPSF6): A possible protein mediator for CARM1 and pre-MRNA splicing Ang, SiangYun Coactivated associated methyltransferase 1 (CARM1), an enzyme that regulates cellular processes via arginine methylation, has been suggested to play a role in RNA processing and alternative splicing. The focus of this project is to identify if cleavage and polyadenylation specific factor 6 (CPSF6), a protein essential for pre-mRNA3'-end processing, is a substrate of CARM1. We hypothesize that CPSF6 may bridge CARM1 to mRNA splicing machinery and CPSF6 itself is modified. The results suggest that CPSF6 is an in vitro substrate of CARM1 and the regions on CPSF6 methylated by CARM1 were narrowed down. Further research involves examining the in vivo interaction of CARM1 and CPSF6 and further mapping of CARM1 methylation sites on CPSF6. We will also determine whether CPSF6 is important in CARM1-mediated splicing process and whether CPSF6 methylation by CARM1 regulates dynamic association of CPSF6 with the spliceosome. Since currently CARM1 regulation of alternative splicing is not well defined, this project will provide insight into this mechanism 1 p. (abstract only). YOU CANNOT OPEN THE COMPLETE PAPER. It is not available to the public, in accordance with the author's wishes.

Inflammatory response and extracellular matrix turnover following pulsed dyed laser treatment of vocal fold and vocal fold fibroblasts

Inflammatory response and extracellular matrix turnover following pulsed dyed laser treatment of vocal fold and vocal fold fibroblasts Ya, Lin The 585-nm pulsed dye laser (PDL) is commonly utilized for the treatment of vascular and epithelial vocal fold lesions; however there are anecdotal reports that it may improve scar outcomes, perhaps by altering extracellular matrix (ECM) turnover; in certain patients. The purpose of this study was to evaluate the effects of PDL treatment on a selected group of inflammatory and procollagen/collagenase genes in normal rat vocal folds and vocal fold fibroblast cells. Vocal folds and fibroblasts were treated with 5-50J/cm2 PDL fluence and harvested at several time points. The mRNA expression profiles of TGF-Beta1, COX-2, IL-1Beta, IL-6, MMP-13, Procollagen I and Procollagen III were examined using real time RT-PCR. Our results indicated changes in inflammatory response alongside alterations in procollagen/collagenase expression, suggesting that PDL treatment does alter gene expression of fibroblasts within the focal fold ECM. 1 p. (abstract only). YOU CANNOT OPEN THE COMPLETE PAPER. It is not available to the public, in accordance with the author's wishes.

Pre-steady state product formation rates for mutant and wild type toluene 4-monooxygenases

Pre-steady state product formation rates for mutant and wild type toluene 4-monooxygenases Hauser, Andrew David This study measures the pre-steady state product formation rate for toluene 4-monooxygenase (T4MO). T4MO catalyzes the NADH and O2 dependent hydroxylation of toluene to form p-cresol. By using alternative substrates with altered benzene ring substitution patterns, the hypothesis that the chemical mechanism of hydroxylation by T4MO is an electrophilic aromatic substitution will be tested. T4moH has been expressed and purified with active site mutants to investigate the role of specific amino acid side chains during catalysis. Rapid chemical-mix quench was used to obtain the product which was analyzed with gas chromatography. The percent turnover for nitrobenzene and chlorobenzene was well below 100% in both the wild-type (wt) and T201A mutant. The percent turnover of both toluene and methoxybenzene in the wt and T201A mutant was almost 100%. It was observed that each alcohol group was added to the para position of each substrate and that the rate of product formation was higher in mutant than the wild-type. 1 p. (abstract only). YOU CANNOT OPEN THE COMPLETE PAPER. It is not available to the public, in accordance with the author's wishes.

Second Sphere Effects of O2 Activation in Stearoyl-Acyl Carrier Protein Delta9 Desaturase

Second Sphere Effects of O2 Activation in Stearoyl-Acyl Carrier Protein Delta9 Desaturase Ng, Yi Han Stearoyl-acyl carrier protein Delta9 desaturase (Delta9D) from Ricinus communis converts stearic acid into monounsaturated oleic acid and functions to maintain the lipid composition of the cell membrane. While the desaturase active site glutamate and histidine residues are crucial to catalysis, there are a number of conserved "second sphere" residues. One such residue is Threonine 199 found ~5Angstroms from the diiron center and it has been hypothesized to stabilize the peroxo intermediate formed during catalysis. In this work, the role of this residue has been probed through studies of a series of mutants. These mutations show a lower kcat and an increase in the rate of decay of the peroxo intermediate, providing evidence of stabilization of the intermediate. 18 p.

The Effects of Doa4 and Other Multivesicular-Body Proteins on Brome Mosaic Virus Replication in Yeast

The Effects of Doa4 and Other Multivesicular-Body Proteins on Brome Mosaic Virus Replication in Yeast Zaban, Nick Brome Mosaic Virus (BMV) replication was measured in yeast single and double deletion mutants of Doa4 and other proteins in the multivesicular-body (MVB) pathway in order to determine the role of Doa4 and other MVB proteins in BMV replication. BMV is a positives-strand RNA virus that is studied as a model for many other RNA viruses. The MVB pathway packages proteins into endosomes and designates them for degradation. One MVB protein, Doa4, has been shown to affect BMV replication. Deletion mutants were transformed with plasmids encoding for BMV. Northern blotting and Western blotting were used to detect the accumulation of BMV RNAs and proteins in order to determine the degree of BMV replication in each mutant. One mutant containing the deletion of Doa4 and another MVB protein, Bro1, demonstrated BMV replication 60% of wild-type. This indicates that the deletion of Bro1 partially restores BMV replication when Doa4 is deleted. 20 p.

Characterization of Tau Phosphorylation During the Differentiation of Human Embryonic Stem Cells

Characterization of Tau Phosphorylation During the Differentiation of Human Embryonic Stem Cells Kaltcheva, Maria Alzheimer's disease is the seventh leading cause of death in the United States with an estimated cost of 100 billion dollars. The severity of this neurodegenerative disease is strongly correlated with the number of neurofibrilary tangles (NFT) composed of hyperphosphorylated tau, a microtubule associated protein. The phosphorylation of tau is known to be tightly regulated by glycogen synthase kinase 3Beta (GSK-3Beta) and cyclin-dependent kinase 5 (Cdk-5). These two kinases also play a vital role in neuronal cell development. We characterized the phosphorylation of tau and expression of tau, GSK-3Beta, and Cdk-5 during early embryo development using human embryonic stem cells (hESC) as our model system. We determined that GSK-3Beta plays an important role in the regulation of proliferation, tau phosphorylation, and neuroectodermal differentiation of hESC. However, the inhibition of Cdk-5 did not significantly change hESC proliferation and the expression of tau, GSK-3Beta, and Cdk-5 throughout hESC differentiation. 30 p.

Defining the PIPKIgamma707/SNX5 interaction and its possible association with E-cadherin

Defining the PIPKIgamma707/SNX5 interaction and its possible association with E-cadherin Christianson, Lisa Nicole E-cadherin, a cell adhesion molecule and tumor suppressor plays an important role in suppressing metastasis of cancers of epithelial origin by acting as the cornerstone of adherens junctions, which facilitate adhesion between epithelial cells. The type I gamma phosphatidylinositol phosphate kinases (PIPKIgamma) directly bind E-cadherin and produce phosphatidylinositol 4,5-bisphosphate (PI4,5P2), a lipid messenger whose localized generation is necessary for E-cadherin transport and AJ formation. The sorting nexins are a family of proteins that bind multiple phosphoinositides and function in endocytic and endosomal trafficking pathways via vesicles which internalize extracellular components. We have identified a novel splice variant, PIPKIgamma707 that associates with both E-cadherin and sorting nexin 5 (SNX5) independently in vivo. Data presented here demonstrate that amino acids 645- 651 of PIPKI?707's C-terminus are largely responsible for mediating the interaction between Igamma707 and sorting nexin 5. Results also indicate the possible existence of a PIPKIgamma707/SNX5/ECD complex within epithelial cells, suggesting that PIPKIgamma707 and SNX5 may play a role in the proper trafficking of E-cadherin. 17 p.

Chronic and acute seizure activity affects NKCC1 and KCC2 expression and alters GABA-induced inhibition in the adult and developing hippocampus

Chronic and acute seizure activity affects NKCC1 and KCC2 expression and alters GABA-induced inhibition in the adult and developing hippocampus Gielissen, Katherine A. Temporal lobe epilepsy is a relatively common nervous system disorder characterized by abnormal activity in the hippocampus, often caused by a decrease in GABA inhibition. GABAA receptors are Cl- ion channels whose effects are medicated by inward Cl- rectifying (excitatory) NKCC1 and outward Cl- rectifying (inhibitory) KCC2 cotransporters. The expression of these cotransporters is modified by both aberrant neural activity and "critical periods" in development. We hypothesized the hippocampi class V seizures would cause a permanent upregulation of NKCC1 and/or downregulation of KCC2. We also hypothesized rats with seizures during postnatal days 20-30 would cause long term dysfunction in hippocampal expression of these cotransporters. Western blotting results suggest that increased propensity for seizures is due to permanent changes in NKCC1 levels, which subsequently reduces GABAA-mediated inhibition. Furthermore, developing rats that experience a seizure at P25 display decreased KCC2 expression levels both 24 hours and 90 days after the event, with little change in NKCC1 expression?with similar physiological effects as in adults. 23 p.

Inhibition of the Escherichia coli replication cycle by a mutant RecA protein

Inhibition of the Escherichia coli replication cycle by a mutant RecA protein Li, Hao Abstract: A conserved [K/R]x[K/R] motif is found at the subunit-subunit interface in bacterial RecA protein filaments. In the Escherichia coli RecA protein, this motif is made up of residues K248 and K250. Both of these residues have a role in ATP hydrolysis (in trans across the interface) and in the transmission of conformational information across the interface. A K250R mutation creates a RecA protein that promotes both ATP hydrolysis and DNA-strand exchange at a six-fold lower rate than the wild type protein. E. coli strains overexpressing this mutant RecA protein also grow much slower than wild type strains. Suppressor mutations appear quickly, and most suppressor mutations inactivate RecA (leading to high levels of UV sensitivity). One suppressor mutation, A11V, did not inactivate the RecA protein, but instead produced a RecA variant that allowed normal rates of growth under normal growth conditions, while partially restoring UV resistance. We found that this suppressor mutation has no effect on the rate of ATP hydrolysis of the mutant RecA K250R protein, but has slightly better strand exchange activity than RecA K250R. We also found that RecA K250R/A11V has a higher rate of disassembly than RecA K250R, showing that this suppression mutation allowed the double mutant RecA nucleoprotein filament to be more dynamic. Our hypothesis is that the [KR]x[KR] motif, especially RecA residue K250 is directly involved in RecA functions. Through mutational studies, we can get a better understanding of the role of this residue in RecA-catalyzed reactions. 23 p.

The effects of erythropoietin on transferrin receptor concentration in rat duodenum tissue

The effects of erythropoietin on transferrin receptor concentration in rat duodenum tissue Pittner, Christa Preliminary studies showed factors contained in human milk retain their mitogenic activity following pasteurization. Iron is critical in cell proliferation in early life. Erythropoietin (Epo), a factor found in human milk, stimulates iron utilization, but its role in iron absorption is unclear. We hypothesized that expression of duodenal iron transporter, TfR, would be higher in damfed (DF) or rats with iron deficiency anemia (IDA) fed enteral Epo from postnatal day 4-12, compared to control. Duodenum and liver were stained for iron and duodenal TfR immunohistochemistry was performed. Body or liver iron content was measured. TfR density was higher in IDA, compared to DF (p=0.05). Liver iron content was greater in IDA and IDA+Epo than Dam or Dam+Epo, p<0.0005, but liver Prussian blue staining was lower in IDA and IDA+Epo, compared to Dam or Dam+Epo (p<0.0001). These data support increased TfR density with iron deficiency, but no appreciable effect with Epo. 22 p.

Polyamide-chlorambucil conjugates

Polyamide-chlorambucil conjugates Mohandas, Appesh Polyamides are chemically engineered molecules made up of sequences of pyrolle and imidazole rings. In particular sequences, these molecules have been shown to have a very high affinity and specificity for nucleic acids, and can therefore be arranged to bind a specific sequence of DNA. By combining such a compound with a known anti-cancer agent, Chlorambucil, and using microarray technology to query the specificity and affinity with which this conjugate binds DNA, and in particular which sequences are bound 27 p.

Mutations in CNGC2 in Arabidopsis thaliana lead to a reduction in fertility due to a sporophytic defect

Mutations in CNGC2 in Arabidopsis thaliana lead to a reduction in fertility due to a sporophytic defect Strohm, Allison While cyclic nucleotide gated channels (CNGCs) are known to play an important role in sensory transduction in animals, little is known about their functions and the mechanisms by which they act in plants. cngc2 plants exhibit reduced size and fertility. These phenotypes become more severe when the plants are treated with physiologically relevant levels of calcium. To determine the cause of reduced fertility, we investigated pollination and fertilization in cngc2 plants. These plants have short anthers, poor pollen tube growth, and a fertilization defect. By genetic analysis, we conclude that this is a sporophytic, not gametophytic, defect. 29 p.