Matching Items (32)

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Expression of Pleiotrophin as Separate Domains to Examine Glycosaminoglycan Binding Sites

Description

Glycosaminoglycan (GAG) binding by the cytokine pleiotrophin (PTN) was examined by expressing both thrombospondin 1 type-1 repeat domains of PTN separately, as PTN-N and PTN-C. PTN-N contains residues 31-89, and

Glycosaminoglycan (GAG) binding by the cytokine pleiotrophin (PTN) was examined by expressing both thrombospondin 1 type-1 repeat domains of PTN separately, as PTN-N and PTN-C. PTN-N contains residues 31-89, and PTN-C contains residues 90-146. Nuclear magnetic resonance (NMR) experiments were conducted on both PTN-N and PTN-C to elucidate GAG binding regions. Titration with heparin dp6 showed a twofold increase in affinity when expressing PTN-N and PTN-C separately rather than as intact PTN. Paramagnetic relaxation rate enhancement experiments and surface paramagnetic relaxation enhancement (PRE) perturbation experiments were used to determine which residues were involved in GAG binding. One binding site was observed in PTN-N, around residue T82, and two binding sites were observed in PTN-C, one around residue K93 and the other around residue G142. These observed binding sites agree with the binding sites already proposed by the Wang lab group and other studies. Future work on the subject could be done on confirming that other varieties and length GAGs bind at the same sites, as well as examining the effect longer GAG fragments have on the affinity of intact PTN versus separate domains.

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Date Created
  • 2015-12

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Refining the structure of hPIRT, a modulator of TRP channels, via measurement of residual dipolar couplings in nuclear magnetic resonance spectroscopy

Description

Transient receptor potential channels (TRP channels) are a family of ion channels that mediate a wide variety of sensations, including pain, temperature, and mechanosensation. Human phosphoinositide-interacting regulator of TRP (hPIRT)

Transient receptor potential channels (TRP channels) are a family of ion channels that mediate a wide variety of sensations, including pain, temperature, and mechanosensation. Human phosphoinositide-interacting regulator of TRP (hPIRT) is a 15.5 kDa, relatively uncharacterized membrane protein that has been shown to modulate the activity of certain TRP channels and some other ion channels. hPIRT is also able to interact with phosphatidylinositol-4,5-bisphosphate (PI(4,5)P¬2), a phospholipid that modulates the activity of many important signaling proteins, including TRP channels. Some information is already known about the structure of hPIRT: it contains a relatively unstructured N-terminus, two transmembrane helices, and a juxtamembrane region at the C-terminus that plays a role in binding PI(4,5)P2 and TRPV1. However, more detailed structural data about this molecule would be very informative in understanding how these interactions occur. In order to accomplish this, this thesis investigates the measurement of residual dipolar couplings (RDCs) in nuclear magnetic resonance spectroscopy (NMR) to refine the structure of hPIRT. RDCs are a measurable effect in NMR experiments caused by partial alignment of molecules solubilized in a weakly anisotropic medium. The resulting data set can be used to calculate bond angles within the protein relative to the axis of the external magnetic field, which will assist efforts to further constrain the structure of hPIRT.

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Date Created
  • 2017-05

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Modifying and Optimizing 1H NMR for Amino Acid Analysis

Description

The parameters of microwave-assisted acid hydrolysis (MAAH) and 1H NMR highly affect the quantitative analysis of protein hydrolysates. Microwave-induction source, NMR spectral resolution, and data analysis are key parameters in

The parameters of microwave-assisted acid hydrolysis (MAAH) and 1H NMR highly affect the quantitative analysis of protein hydrolysates. Microwave-induction source, NMR spectral resolution, and data analysis are key parameters in the nuclear magnetic resonance – amino acid analysis (NMR-AAA) workflow where errors accrue due to lack of an optimized protocol. Hen egg white lysozyme was hydrolyzed using an 800W domestic microwave oven for varying time points between 10-25 minutes, showing minimal protein hydrolysis after extended time periods. Studies on paramagnetic doping with varying amounts of gadolinium chloride for increased NMR resolution resulted in little T1 reduction in a majority of amino acids and resulted in significant line broadening in concentrations above 1µM. The use of the BAYESIL analysis tool with HOD suppressed 1H-NMR spectra resulted in misplaced template peaks and errors greater than 1% for 10 of 13 profiled amino acids with the highest error being 7.6% (Thr). Comparatively, Chenomx NMR Suite (v7.1) analysis resulted in errors of less than 1% for 9 of 13 profiled amino acids with a highest error value of 3.6% (Lys). Using the optimized protocol, hen egg white lysozyme C was identified at rank 1 with a score of 64 in a Gallus gallus species wide AACompIdent search. This technique reduces error associated with sample handling relative to previously used amino acid analysis (AAA) protocols and requires no derivatization or additional processing of the sample prior to analysis.

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Date Created
  • 2017-05

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Can Replacement of 5-HT2AR Expression in the Prefrontal Cortex of Egr3 -/- Mice Rescue the Schizophrenia-like Phenotypes?

Description

About 1% of the United States adult population currently suffers from schizophrenia. The symptoms of schizophrenia can be broken down into three main categories including: positive symptoms such as psychoses,

About 1% of the United States adult population currently suffers from schizophrenia. The symptoms of schizophrenia can be broken down into three main categories including: positive symptoms such as psychoses, negative symptoms such as anhedonia, and cognitive symptoms such as memory difficulties. The early growth response 3 (Egr3) is part of a family of genes known as the immediate early genes (IEGs), which are zinc-finger transcription factors. IEGs are not protein synthesis dependent, which means that they can be activated quickly, within 30-45 minutes, in response to certain environmental stimuli such as sleep deprivation. Egr3, an activity dependent gene, may be up-regulated by both genetic and environmental cues. Egr3 is thought to play an integral role in a biochemical pathway that may explain the onset of schizophrenia. However, the exact causes of schizophrenia remain unknown. Egr3 is not only activated in response to environmental factors, but has also been linked to many genes that are associated with schizophrenia in humans (Huentelman et al., 2015). Post-mortem brain tissue studies of patients with schizophrenia have decreased levels of EGR3 in their prefrontal cortex (PFC) and mice lacking Egr3 (Egr3 -/-) exhibit schizophrenia-like phenotypes such as locomotor hyperactivity. Egr 3 -/- mice also exhibit a diminished head twitch response to 2,5-Dimethoxy-4-iodoamphetamine (DOI), a 5-HT2A agonist (Yamada, et al., 2007; Gallitano-Mendel, et al., 2008). A link was established between schizophrenia patients and the serotonin 2A receptor (5-HT2AR) upon recognizing that 5-HT2AR agonists like lysergic acid diethylamide (LSD) create hallucinations similar to those in schizophrenic patients and 5-HT2AR antagonists such as the second-generation antipsychotic clozapine can reverse those hallucinations (Sommer, 2012). Paradoxically, however, post-mortem studies of schizophrenia patients have actually shown a decrease in PFC 5-HT2ARs as well as a 70% decrease found in the PFC of Egr3 -/- mice (Rasmussen, et al., 2010; Williams, et al., 2012). Therefore, we hypothesize that EGR3 directly regulates expression of 5-HT2ARs. To test this we will use virus-mediated overexpression of 5-HT2ARs in the PFCs of mice to see if we can rescue the schizophrenia-like phenotypes of the Egr3 -/- mice. After bilateral PFC stereotaxic injection of herpes simplex virus (HSV) with enhanced green fluorescent protein (EGFP) or HSV-Htr2a-EGFP in both wild type (WT) and Egr3 -/- mice, the mice were behaviorally tested using locomotor activity and DOI-induced head twitch response. We found that Egr3-/- mice, compared to WT mice, demonstrated locomotor hyperactivity and a decreased DOI-induced head twitch response, confirming prior findings, but no significant main effect of virus. A significant effect of the HSV-Htr2a-EGFP was seen when comparing DOI-induced head twitch response in WT mice to Egr3 -/- mice. WT mice showed a higher number of head twitches in comparison to the knockout mice. These findings suggest further research must be conducted in order to investigate whether a functional 5-HT2AR is being translated and correctly transported to the membrane. These findings may also point to an unknown factor mediating the regulation between Egr3 and 5-HT2ARs.

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Date Created
  • 2016-05

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Exploring Structure and Function of Human Cold Sensing Protein TRPM8 with ROSETTA Comparative Models

Description

Transient Receptor Potential (TRP) channels are a diverse class of ion channels notable as polymodal sensors. TRPM8 is a TRP channel implicated in cold sensation, nociception, and a variety of

Transient Receptor Potential (TRP) channels are a diverse class of ion channels notable as polymodal sensors. TRPM8 is a TRP channel implicated in cold sensation, nociception, and a variety of human diseases, including obesity and cancer. Despite sustained interest in TRPM8 since its discovery in 2001, many of the molecular mechanisms that underlie function are not yet clear. Knowledge of these properties could have implications for medicine and physiological understanding of sensation and signaling. Structures of TRP channels have proven challenging to solve, but recent Cryoelectron microscopy (Cryo-EM) structures of TRPV1 provide a basis for homology-based modeling of TRP channel structures and interactions. I present an ensemble of 11,000 Rosetta computational homology models of TRPM8 based on the recent Cryo-EM apo structure of TRPV1 (PDB code:3J5P). Site-directed mutagenesis has provided clues about which residues are most essential for modulatory ligands to bind, so the models presented provide a platform to investigate the structural basis of TRPM8 ligand modulation complementary to existing functional and structural information. Menthol and icilin appear to interact with interfacial residues in the sensor domain (S1-S4). One consensus feature of these sites is the presence of local contacts to the S4 helix, suggesting this helix may be mechanistically involved with the opening of the pore. Phosphatidylinositol 4,5-bisphosphate (PIP2)has long been known to interact with the C-terminus of TRPM8, and some of the homology models contain plausible binding pockets where PIP2 can come into contact with charged residues known to be essential for PIP2 modulation. Future in silico binding experiments could provide testable hypothesis for in vitro structural studies, and experimental data (e.g. distance constraints from electron paramagnetic resonance spectroscopy [EPR]) could further refine the models.

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Created

Date Created
  • 2015-05

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ELECTRON TRANSFER PROCESS BETWEEN COFACTORS OF HELIOBACTERIA'S REACTION CENTER

Description

ABSTRACT:
The experiment was conducted to analyze the role of menaquinone (MQ) in heliobacteria’s reaction center (HbRC). Their photosynthetic apparatus is a homodimeric of type I reaction center (1). HbRC

ABSTRACT:
The experiment was conducted to analyze the role of menaquinone (MQ) in heliobacteria’s reaction center (HbRC). Their photosynthetic apparatus is a homodimeric of type I reaction center (1). HbRC contains these cofactors: P800 (special pair cholorphyll), A0 (8-hydroxy-chlorophyll [Chl] a), and FX (iron-sulfur cluster). The MQ factor is bypassed during the electron transfer process in HbRC. Electrons from the excited state of P800 (P800*) are transported to A0 and then directly to Fx. The hypothesis is that when electrons are photoaccumulated at Fx, and without the presence of any electron acceptors to the cluster, they would be transferred to MQ, and reduce it to MQH2 (quinol). Experiments conducted in the past with HbRC within the cell membranes yielded data that supported this hypothesis (Figures 4 and 5). We conducted a new experiment based on that foundation with HbRC, isolated from cell membrane. Two protein assays were prepared with cyt c553 and ascorbate in order to observe this phenomenon. The two samples were left in the glove box for several days for equilibration and then exposed to light in different intensity and periods. Their absorption was monitored at 800 nm for P800 or 554 nm for cyt c553 to observe their oxidation and reduction processes. The measurements were performed with the JTS-10 spectrophotometer. The data obtained from these experiments support the theory that P800+ reduced by the charge recombination of P800+Fx-. However, it did not confirm the reduction of P800+ done by cyt c553¬ which eventually lead to a net accumulation of oxidized cyt c553; instead it revealed another factor that could reduce P800+ faster and more efficient than cyt c553 (0.5 seconds vs several seconds), which could be MQ. More experiments need to be done in order to confirm this result. Hence, the data collected from this experiment have yet to support the theory of MQ being reduced to MQH2 outside the bacterial membranes.

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Date Created
  • 2015-05

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Determining the Validity of Using Heavy-Isotope-Permethylated Glycans as Internal Standards for Glycan Node Analysis

Description

In this thesis, glycan nodes, the basic subunits of complex biological sugars, were studied to determine the reproducibility of gas chromatography-mass spectrometry (GC/MS) based methylation analysis of whole blood plasma

In this thesis, glycan nodes, the basic subunits of complex biological sugars, were studied to determine the reproducibility of gas chromatography-mass spectrometry (GC/MS) based methylation analysis of whole blood plasma by normalization using an internal standard of heavy permethylated glycans. Glycans are complex biological sugars that have a variety of applications in the human body and will display aberrant compositions when produced by cancerous cells. Thus an assay to determine their composition can be used as a diagnostic tool. It was shown that the assay may have potential use, but needs further refinement to become an improvement over current methods by analyzing the results of ratio-determination and replicate experiments.

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Date Created
  • 2015-05

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Elucidating Structural and Functional Information on the Human Cold-Sensing Protein TRPM8 via Isolating the Pore Domain and Cross-Chimeric Studies

Description

Transient Receptor Potential (TRP) ion channels are a diverse family of nonselective, polymodal sensors in uni- and multicellular eukaryotes that are implicated in an assortment of biological contexts and human

Transient Receptor Potential (TRP) ion channels are a diverse family of nonselective, polymodal sensors in uni- and multicellular eukaryotes that are implicated in an assortment of biological contexts and human disease. The cold-activated TRP Melastatin-8 (TRPM8) channel, also recognized as the human body's primary cold sensor, is among the few TRP channels responsible for thermosensing. Despite sustained interest in the channel, the mechanisms underlying TRPM8 activation, modulation, and gating have proved challenging to study and remain poorly understood. In this thesis, I offer data collected on various expression, extraction, and purification conditions tested in E. Coli expression systems with the aim to optimize the generation of a structurally stable and functional human TRPM8 pore domain (S5 and S6) construct for application in structural biology studies. These studies, including the biophysical technique nuclear magnetic spectroscopy (NMR), among others, will be essential for elucidating the role of the TRPM8 pore domain in in regulating ligand binding, channel gating, ion selectively, and thermal sensitivity. Moreover, in the second half of this thesis, I discuss the ligation-independent megaprimer PCR of whole-plasmids (MEGAWHOP PCR) cloning technique, and how it was used to generate chimeras between TRPM8 and its nearest analog TRPM2. I review steps taken to optimize the efficiency of MEGAWHOP PCR and the implications and unique applications of this novel methodology for advancing recombinant DNA technology. I lastly present preliminary electrophysiological data on the chimeras, employed to isolate and study the functional contributions of each individual transmembrane helix (S1-S6) to TRPM8 menthol activation. These studies show the utility of the TRPM8\u2014TRPM2 chimeras for dissecting function of TRP channels. The average current traces analyzed thus far indicate that the S2 and S3 helices appear to play an important role in TRPM8 menthol modulation because the TRPM8[M2S2] and TRPM8[M2S3] chimeras significantly reduce channel conductance in the presence of menthol. The TRPM8[M2S4] chimera, oppositely, increases channel conductance, implying that the S4 helix in native TRPM8 may suppress menthol modulation. Overall, these findings show that there is promise in the techniques chosen to identify specific regions of TRPM8 crucial to menthol activation, though the methods chosen to study the TRPM8 pore independent from the whole channel may need to be reevaluated. Further experiments will be necessary to refine TRPM8 pore solubilization and purification before structural studies can proceed, and the electrophysiology traces observed for the chimeras will need to be further verified and evaluated for consistency and physiological significance.

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Date Created
  • 2016-05

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The structural diversity of artificial genetic polymers

Description

Synthetic genetics is a subdiscipline of synthetic biology that aims to develop artificial genetic polymers (also referred to as xeno-nucleic acids or XNAs) that can replicate in vitro and eventually

Synthetic genetics is a subdiscipline of synthetic biology that aims to develop artificial genetic polymers (also referred to as xeno-nucleic acids or XNAs) that can replicate in vitro and eventually in model cellular organisms. This field of science combines organic chemistry with polymerase engineering to create alternative forms of DNA that can store genetic information and evolve in response to external stimuli. Practitioners of synthetic genetics postulate that XNA could be used to safeguard synthetic biology organisms by storing genetic information in orthogonal chromosomes. XNA polymers are also under active investigation as a source of nuclease resistant affinity reagents (aptamers) and catalysts (xenozymes) with practical applications in disease diagnosis and treatment. In this review, we provide a structural perspective on known antiparallel duplex structures in which at least one strand of the Watson–Crick duplex is composed entirely of XNA. Currently, only a handful of XNA structures have been archived in the Protein Data Bank as compared to the more than 100 000 structures that are now available. Given the growing interest in xenobiology projects, we chose to compare the structural features of XNA polymers and discuss their potential to access new regions of nucleic acid fold space.

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Date Created
  • 2015-12-15