Matching Items (16)
Filtering by

Clear all filters

156834-Thumbnail Image.png
Description
Exposure of blood plasma/serum (P/S) to thawed conditions, greater than -30°C, can produce biomolecular changes that misleadingly impact measurements of clinical markers within archived samples. Reported here is a low sample-volume, dilute-and-shoot, intact protein mass spectrometric assay of albumin proteoforms called “ΔS-Cys-Albumin” that quantifies cumulative exposure of archived P/S samples

Exposure of blood plasma/serum (P/S) to thawed conditions, greater than -30°C, can produce biomolecular changes that misleadingly impact measurements of clinical markers within archived samples. Reported here is a low sample-volume, dilute-and-shoot, intact protein mass spectrometric assay of albumin proteoforms called “ΔS-Cys-Albumin” that quantifies cumulative exposure of archived P/S samples to thawed conditions. The assay uses the fact that S-cysteinylation (oxidation) of albumin in P/S increases to a maximum value when exposed to temperatures greater than -30°C. The multi-reaction rate law that governs this albumin S-cysteinylation formation in P/S was determined and was shown to predict the rate of formation of S-cysteinylated albumin in P/S samples—a step that enables back-calculation of the time at which unknown P/S specimens have been exposed to room temperature. To emphasize the capability of this assay, a blind challenge demonstrated the ability of ΔS-Cys-Albumin to detect exposure of individual and grouped P/S samples to unfavorable storage conditions. The assay was also capable of detecting an anomaly in a case study of nominally pristine serum samples collected under NIH-sponsorship, demonstrating that empirical evidence is required to guarantee accurate knowledge of archived P/S biospecimen storage history.

The ex vivo glycation of human serum albumin was also investigated showing that P/S samples stored above their freezing point leads to significant increases in glycated albumin. These increases were found to occur within hours at room temperature, and within days at -20 °C. These increases continued over a period of 1-2 weeks at room temperature and over 200 days at -20 °C, ultimately resulting in a doubling of glycated albumin in both healthy and diabetic patients. It was also shown that samples stored at lower surface area-to-volume ratios or incubated under a nitrogen atmosphere experienced less rapid glucose adduction of albumin—suggesting a role for oxidative glycation in the ex vivo glycation of albumin.
ContributorsJeffs, Joshua W (Author) / Borges, Chad R (Thesis advisor) / Van Horn, Wade (Committee member) / Williams, Peter (Committee member) / Arizona State University (Publisher)
Created2018
135187-Thumbnail Image.png
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 disease. The cold-activated TRP Melastatin-8 (TRPM8) channel, also recognized as the human body's primary cold sensor, is among the few

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.
ContributorsWaris, Maryam Siddika (Author) / Van Horn, Wade (Thesis director) / Redding, Kevin (Committee member) / School of Molecular Sciences (Contributor) / Department of English (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
136317-Thumbnail Image.png
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 human diseases, including obesity and cancer. Despite sustained interest in TRPM8 since its discovery in 2001, many of the molecular

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.
ContributorsHelsell, Cole Vincent Maher (Author) / Van Horn, Wade (Thesis director) / Wang, Xu (Committee member) / Barrett, The Honors College (Contributor) / School of Mathematical and Statistical Sciences (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2015-05
136377-Thumbnail Image.png
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 by normalization using an internal standard of heavy permethylated glycans. Glycans are complex biological sugars that have a variety of

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.
ContributorsMiyasaki, Tyler Takeo (Author) / Borges, Chad (Thesis director) / Van Horn, Wade (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / Chemical Engineering Program (Contributor)
Created2015-05
134567-Thumbnail Image.png
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 nuclear magnetic resonance – amino acid analysis (NMR-AAA) workflow where errors accrue due to lack of an optimized protocol.

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.
ContributorsJordan, Jacob Smith (Author) / Yarger, Jeffery (Thesis director) / Van Horn, Wade (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
Description

Alzheimer’s disease (AD) is a common neurodegenerative disorder affecting approximately 10% of people aged 65 and up and 30-50% over 85. In pathological AD representations, a way to recognize early onset AD is the increased levels of pro-NGF in BFCNs that come from the downregulation of NGF with age. Pro-NGF

Alzheimer’s disease (AD) is a common neurodegenerative disorder affecting approximately 10% of people aged 65 and up and 30-50% over 85. In pathological AD representations, a way to recognize early onset AD is the increased levels of pro-NGF in BFCNs that come from the downregulation of NGF with age. Pro-NGF has a higher affinity for p75NTR, which binds and participates in the pro-NGF-p75NTR-sortilin complex sequentially cleaved by α- and γ-secretase. Pro-NGF triggers apoptosis through the cleavage of the intracellular membrane by γ-secretase. Since γ-secretase physically cleaves off the intramembrane portion that promotes TNF- and Fas-dependent apoptotic signaling pathways, it has a crucial role in AD and must be better understood. This research aims to understand better and visualize γ-secretase and its actions, specifically with its interactions with the substrate p75NTR in the RIP process. To analyze γ-secretase function, the proteins must be produced and analyzed through the protein expression protocol. During protein production, DNA, cell concentrations, and optical density measurements were difficult to produce due to the incompetency of e. coli cells (DH5α), contamination of the Sf9 insect cell culture, and decreased viability of aged insect cells. We identified the problems and improved the conditions for future project development.

ContributorsRapacz, Elizabeth (Author) / Chiu, Po-Lin (Thesis director) / Van Horn, Wade (Committee member) / Munk, Barbara (Committee member) / Barrett, The Honors College (Contributor) / School of Human Evolution & Social Change (Contributor) / School of Molecular Sciences (Contributor)
Created2023-05
132081-Thumbnail Image.png
Description
Transient receptor potential (TRP) channels are a superfamily of ion channels found in plasma membranes of both single-celled and multicellular organisms. TRP channels all share the common aspect of having six transmembrane helices and a TRP domain. These structures tetramerize to form a receptor-activated non-selective ion channel. The specific protein

Transient receptor potential (TRP) channels are a superfamily of ion channels found in plasma membranes of both single-celled and multicellular organisms. TRP channels all share the common aspect of having six transmembrane helices and a TRP domain. These structures tetramerize to form a receptor-activated non-selective ion channel. The specific protein being investigated in this thesis is the human transient receptor potential melastatin 8 (hTRPM8), a channel activated by the chemical ligand menthol and temperatures below 25 °C. TRPM8 is responsible for cold sensing and is related to pain relief associated with cooling compounds. TRPM8 has also been found to play a role in the regulation of various types of tumors. The structure of TRPM8 has been obtained through cryo-electron microscopy, but the functional contribution of individual portions of the protein to the overall protein function is unknown.
To gain more information about the function of the transmembrane region of hTRPM8, it was expressed in Escherichia coli (E. coli) and purified in detergent membrane mimics for experimentation. The construct contains the S4-S5 linker, pore domain (S5 and S6 transmembrane helices), pore helix, and TRP box. hTRPM8-PD+ was purified in the detergents n-Dodecyl-B-D-Maltoside (DDM), 16:0 Lyso PG, 1-Palmitoyl-2-hydroxy-sn-glycero-3-phosphoglycerol (LPPG), and 14:0 Lyso PG, 1-Myristoyl-2-hydroxy-sn-glycero-3-phosphoglycerol (LMPG) to determine which detergent resulted in a hTRPM8-PD+ sample of the most stability, purity, and highest concentrations. Following bacterial expression and protein purification, hTRPM8-PD+ was studied and characterized with circular dichroism (CD) spectroscopy to learn more about the secondary structures and thermodynamic properties of the construct. Further studies can be done with more circular dichroism (CD) spectroscopy, planar lipid bilayer (BLM) electrophysiology, and nuclear magnetic resonance spectroscopy (NMR) to gain more understanding of how the pore domain plus contributes to the activity of the whole protein construct.
ContributorsMorelan, Danielle Taylor (Co-author) / Morelan, Danielle (Co-author) / Van Horn, Wade (Thesis director) / Chen, Julian (Committee member) / Luu, Dustin (Committee member) / Dean, W.P. Carey School of Business (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-12
166148-Thumbnail Image.png
Description
Ribulose-1,5-bisphosphate carboxylase/oxygenase enzyme (Rubisco) is responsible for the majority of carbon fixation and is also the least efficient enzyme on Earth. Rubisco assists 1,5-ribulose bisphosphate (RuBP) in binding CO2, however CO2 and oxygen have similar binding affinities to Rubisco, resulting in a low enzymatic efficiency. Rubisco activase (Rca) is an

Ribulose-1,5-bisphosphate carboxylase/oxygenase enzyme (Rubisco) is responsible for the majority of carbon fixation and is also the least efficient enzyme on Earth. Rubisco assists 1,5-ribulose bisphosphate (RuBP) in binding CO2, however CO2 and oxygen have similar binding affinities to Rubisco, resulting in a low enzymatic efficiency. Rubisco activase (Rca) is an enzyme that removes inhibiting molecules from Rubisco’s active sites, promoting the Rubisco activity. The binding of Rubisco and Rca stimulates a high-rate of carbon fixation and lowers the overall CO2 concentration in the atmosphere. To study the interaction between the two complexes, Rubisco was extracted from baby spinach (Spinacia oleracea) and purified using anion-exchange chromatography and size-exclusion chromatography. Rca was designed to use a recombinant gene and overexpressed in Escherichia coli (E. coli). The purified proteins were verified using SDS-PAGE. The two proteins were assembled in vitro and the interaction of the protein complex was stabilized using glutaraldehyde cross-linking. The samples were then deposited on a carbon-coated electron microscopy (EM) grid, stained with uranyl formate, and observed under a transmission electron microscope (TEM). The ultimate goal is to image the specimen and reconstruct the structure of the protein complex at high resolution.
ContributorsHart, Hayden (Author) / Chiu, Po-Lin (Thesis director) / Redding, Kevin (Committee member) / Van Horn, Wade (Committee member) / Barrett, The Honors College (Contributor) / School of Molecular Sciences (Contributor) / Department of Military Science (Contributor)
Created2022-05
Description

In intracranial aneurysms, multiple factors and biochemical pathways are believed to be involved in the event of a rupture. The epidermal growth factor receptor (EGFR) activation pathway is of particular interest as a way to understand and target the mechanism of rupture due to its established role in cellular proliferation

In intracranial aneurysms, multiple factors and biochemical pathways are believed to be involved in the event of a rupture. The epidermal growth factor receptor (EGFR) activation pathway is of particular interest as a way to understand and target the mechanism of rupture due to its established role in cellular proliferation and inflammation. Furthermore, unfolded protein responses in vascular cells’ endoplasmic reticulum (ER), known as ER stress, have emerged as a potential downstream mechanism by which inflammatory EGFR activation may lead to aneurysm rupture. The purpose of this project was to investigate the role of EGFR inhibition on the aneurysm rupture rate in a preclinical model, investigate the role of ER stress induction on the aneurysm rupture rate, and confirm which cellular phenomenon lies upstream in this mechanistic cascade. Based on analyses of aneurysm rupture rate and gene expression in the Circle of Willis, ER stress and inflammatory unfolded protein responses were found to be downstream of initial EGFR activation, which may be an effective therapeutic target for preventing aneurysm rupture in a clinical setting.

ContributorsPolen, Kyle (Author) / Van Horn, Wade (Thesis director) / Martin, Thomas (Committee member) / Hashimoto, Tomoki (Committee member) / Barrett, The Honors College (Contributor) / School of Molecular Sciences (Contributor) / School of Human Evolution & Social Change (Contributor)
Created2022-12
157708-Thumbnail Image.png
Description
Phenotypic and molecular profiling demonstrates a high degree of heterogeneity in the breast tumors. TP53 tumor suppressor is mutated in 30% of all breast tumors and the mutation frequency in basal-like subtype is as high as 80% and co-exists with several other somatic mutations in different genes. It was hypothesized

Phenotypic and molecular profiling demonstrates a high degree of heterogeneity in the breast tumors. TP53 tumor suppressor is mutated in 30% of all breast tumors and the mutation frequency in basal-like subtype is as high as 80% and co-exists with several other somatic mutations in different genes. It was hypothesized that tumor heterogeneity is a result of a combination of neo-morphic functions of specific TP53 driver mutations and distinct co-mutations or the co-drivers for each type of TP53 mutation. The 10 most common p53 missense mutant proteins found in breast cancer patients were ectopically expressed in normal-like mammary epithelial cells and phenotypes associated with various hallmarks of cancer examined. Supporting the hypothesis, a wide spectrum of phenotypic changes in cell survival, resistance to apoptosis and anoikis, cell migration, invasion and polarity was observed in the mutants compared to wildtype p53 expressing cells. The missense mutants R248W, R273C and Y220C were most aggressive. Integrated analysis of ChIP and RNA seq showed distinct promoter binding profiles of the p53 mutant proteins different than wildtype p53, implying altered transcriptional activity of mutant p53 proteins and the phenotypic heterogeneity of tumors. Enrichment and model-based pathway analyses revealed dysregulated adherens junction and focal adhesion pathways associated with the aggressive p53 mutants. As several somatic mutations co-appear with mutant TP53, we performed a functional assay to fish out the relevant collaborating driver mutations, the co-drivers. When PTEN was deleted by CRISPR-Cas9 in non-invasive p53-Y234C mutant cell, an increase in cell invasion was observed justifying the concept of co-drivers. A genome wide CRISPR library-based screen on p53-Y234C and R273C cells identified separate candidate co-driver mutations that promoted cell invasion. The top candidates included several mutated genes in breast cancer patients harboring TP53 mutations and were associated with cytoskeletal and apoptosis resistance pathways. Overall, the combined approach of molecular profiling and functional genomics screen highlighted distinct sets of co-driver mutations that can lead to heterogeneous phenotypes and promote aggressiveness in cells with different TP53 mutation background, which can guide development of novel targeted therapies.
ContributorsPal, Anasuya (Author) / LaBaer, Joshua (Thesis advisor) / Roberson, Robert (Committee member) / Van Horn, Wade (Committee member) / Maley, Carlo (Committee member) / Arizona State University (Publisher)
Created2019