Matching Items (146)
Description
Impairments to mitochondrial function and metabolism can make neurons vulnerable to stress and degeneration. Several studies have shown that aberrations in the electron transport chain (ETC) and the Krebs cycle are involved in the pathogenesis of Parkinson’s disease (PD). Therefore, targeting these pathways is becoming increasingly important in the discovery of new treatment for neurodegenerative diseases like PD. (−)-epigallocatechin-3-gallate (EGCG), the most common polyphenol found in Green tea, has been shown to exert neuroprotective effects and lower the risk of developing PD. However, the mechanism by which it accomplishes this remains to be elucidated. The purpose of this study was to shed light on these mechanisms by exploring the effects of EGCG against MPP+-induced mitochondrial dysfunction with PC12 cells being used as a PD pathological cell model. The cell viability differences between cells treated with varying combinations of MPP+ and EGCG were measured using a CCK-8 assay. The morphology changes induced by the different treatments were then identified with fluorescence microscopy. Next, a Seahorse assay was carried out to investigate mitochondrial function followed by GC-MS and LC-MS analysis to evaluate mitochondrial metabolism. 13C metabolic flux analysis was then used to trace the metabolic flux of the Krebs cycle. The results of the CCK-8 assay and fluorescence microscopy showed that EGCG helps attenuate the decreased viability of PC12 cells as well as the morphology changes induced by MPP+. The Seahorse and GC-MS assay found that the it also helps prevent impaired mitochondrial respiration caused by MPP+. The impaired mitochondrial respiration manifested as alterations to the Krebs cycle and glycolysis. In addition, 13C metabolic flux analysis revealed significant increases in Krebs cycle activity in MPP+-induced PC12 cells if treated with EGCG beforehand. Moreover, LC-MS showed a distinct metabolite profile for each group and identified 26 potent biomarkers. In conclusion, this study demonstrated that EGCG exerts a neuroprotective effect on PC12 cells and helps maintain mitochondrial metabolic balance in the presence of MPP+.
ContributorsLawrence, Kent Alexander (Author) / Gu, Haiwei (Thesis director) / Lake, Douglas (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
In the development of personalized medicine and many other clinical studies, biospecimen integrity serves as the prerequisite for not only the accurate derivation of patient- and disease-specific molecular data from biological specimens but the meaningful downstream validation of biomarkers. However, a large number of preanalytical variables may influence the quality of biospecimens in an undesired way and ultimately render the samples unsuitable for molecular analysis. The limited ability to directly reduce discrepancies caused by preanalytical variables gives rise to the need for development and retrospective application of appropriate tests for assessment of biospecimen integrity. Nevertheless, the most standard approaches to assessing biospecimen integrity involve nontrivial procedures. Thus, the need for quality control tools or tests that are readily applicable and can produce results in a straightforward way becomes critical. As one of the major ex vivo biomolecular degradation mechanisms, oxidation that occurs when blood plasma and serum samples are exposed to thawed states during storage and processing is hard to forestall and detect. In an attempt to easily detect and monitor the degree of oxidation, the technique of Fluorescence Resonance Energy Transfer (FRET) was examined to determine whether this concept could be employed to monitor exposure of samples to thawed conditions when controlled by spontaneous oxidative disulfide bonding. The intended mode of usage was envisioned as a fluorescence liquid being stored in a separate compartment but within the same test tube as archived plasma and serum samples. This would allow the assessment of sample integrity by direct visualization of fluorescence under a hand-held black light. The fluorescent dynamic range as well as kinetic control of the reaction were studied. While the addition of Cu(II) proved to facilitate excellent dynamic range with regard to fluorescence quenching, the kinetics of the reaction were too rapid for practical use. Further investigation revealed that the fluorescence quenching mechanism might have actually occurred via Intramolecular Charge Transfer (ICT) rather than FRET mediated by oxidative disulfide bond formation. Introduction of Cu(II) via copper metal slowed fluorescence quenching to the point of practical utility; facilitating demonstration that storing at room temperature, refrigerating or freezing the samples delayed fluorescence quenching to different extents. To establish better kinetic control, future works will focus on establishing controlled, thoroughly understood kinetic release of Cu(II) from copper metal.
ContributorsZhang, Zihan (Author) / Borges, Chad (Thesis director) / Emady, Heather (Committee member) / Williams, Peter (Committee member) / Chemical Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2018-12
Description
Programmed cell death ligand-1 (PD-L1) is an overexpressed protein on many tumor cell types. PD-L1 is involved in normal immune regulation, playing an important role in self-tolerance and controlling autoimmunity. However, ligation of PD-L1 to PD-1 on activated T cells leads to tumor-mediated T cell suppression. Inhibiting the PD-1/PD-L1 pathway has emerged as an effective target for anti-tumor immunotherapies. Monoclonal antibodies (mAbs) targeting tumor-associated antigens such as PD-L1 have proven to be effective checkpoint blockades, improving therapeutic outcomes for cancer patients and receiving FDA approval as first line therapies for some cancers. A single chain variable fragment (scFv) is composed of the variable heavy and light chain regions of a mAb, connected by a flexible linker. We hypothesized that scFv proteins based on the published anti-PD-L1 monoclonal antibody sequences of atezolizumab and avelumab would bind to cell surface PD-L1. Four single chain variable fragments (scFvs) were constructed based on the sequences of these mAbs. PCR was used to assemble, construct, and amplify DNA fragments encoding the scFvs which were subsequently ligated into a eukaryotic expression vector. Mammalian cells were transfected with the scFv and scFv-IgG plasmids. The scFvs were tested for binding to PD-L1 on tumor cell lysates by western blot and to whole tumor cells by staining and flow cytometry analysis. DNA sequence analysis demonstrated that the scFv constructs were successfully amplified and cloned into the expression vectors and recombinant scFvs were produced. The binding capabilities of the scFvs constucts to PD-L1 protein were confirmed by western blot and flow cytometry analysis. This lead to the idea of constructing a CAR T cell engineered to target PD-L1, providing a possible adoptive T cell immunotherapy.
ContributorsPfeffer, Kirsten M. (Author) / Lake, Douglas (Thesis director) / Ho, Thai (Committee member) / Hastings, Karen (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Exosomes have been known to secrete an increased amount of miRNA and noncoding genes that are abnormally expressed in various cancer subtypes. Thus, they may be an early marker for pediatric cancer types that are more difficult to diagnosis without invasive techniques, and may also help identify progression of the disease. In the project, six types of pediatric cancer cell lines, along with their extracted exosomes, were analyzed and tested for different monoclonal antibodies through western blot analysis. The genes EWS-FLI1 and FGFR4 were also identified in some cancer cell lines through Reverse-Transcriptase Polymerase Chain Reaction analysis (RT-PCR). The results were indicative of similar protein markers being found in both the originating cells and their corresponding exosomes.
ContributorsKaur Bhinder, Harsimran (Author) / Lake, Douglas (Thesis director) / Azorsa, David (Committee member) / Barrett, The Honors College (Contributor)
Created2017-12
Description
Biomarkers are the cornerstone of modern-day medicine. They are defined as any biological substance in or outside the body that gives insight to the body's condition. Doctors and researchers can measure specific biomarkers to diagnose and treat patients, such as the concentration of hemoglobin Alc and its connection to diabetes. There are a variety of methods, or assays, to detect biomarkers, but the most common assay is enzyme-linked immunosorbent assay (ELISA). A new-generation assay termed mass spectrometric immunoassay (MSIA) can measure proteoforms, the different chemical variations of proteins, and their relative abundance. ELISA on the other hand measures the overall concentration of protein in the sample. Measuring each of the proteoforms of a protein is important because only one or two variations could be biologically significant and/or cause diseases. However, running MSIA is expensive. For this reason, an alternative plate-based MSIA technique was tested for its ability to detect the proteoforms of a protein called apolipoprotein C-III (ApoC-III). This technique combines the protein capturing procedure of ELISA to isolate the protein with detection in a mass spectrometer. A larger amount of ApoC-III present in the body indicates a considerable risk for coronary heart disease. The precision of the assay is determined on the coefficient of variation (CV). A CV value is the ratio of standard deviation in relation to the mean, represented as a percentage. The smaller the percentage, the less variation the assay has, and therefore the more ability it has to detect subtle changes in the biomarker. An accepted CV would be less than 10% for single-day tests (intra-day) and less than 15% for multi-day tests (inter-day). The plate-based MSIA was started by first coating a 96-well round bottom plate with 2.5 micrograms of ApoC-III antibody. Next, a series of steps were conducted: a buffer wash, then the sample incubation, followed by another buffer wash and two consecutive water washes. After the final wash, the wells were filled with a MALDI matrix, then spotted onto a gold plate to dry. The dry gold target was then placed into a MALDI-TOF mass spectrometer to produce mass spectra for each spot. The mass spectra were calibrated and the area underneath each of the four peaks representing the ApoC-III proteoforms was exported as an Excel file. The intra-day CV values were found by dividing the standard deviation by the average relative abundance of each peak. After repeating the same procedure for three more days, the inter-day CVs were found using the same method. After completing the experiment, the CV values were all within the acceptable guidelines. Therefore, the plate-based MSIA is a viable alternative for finding proteoforms than the more expensive MSIA tips. To further validate this, additional tests will need to be conducted with different proteins and number of samples to determine assay flexibility.
ContributorsTieu, Luc (Author) / Borges, Chad (Thesis director) / Nedelkov, Dobrin (Committee member) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2017-12
Description
Brown adipose tissue (BAT) is thought to be important in combating obesity as it can expend energy in the form of heat, e.g. thermogenesis. The goal of this study was to study the effect of injected norepinephrine (NE) on the activation of BAT in rats that were fed a high fat diet (HFD). A dose of 0.25 mg/kg NE was used to elicit a temperature response that was measured using transponders inserted subcutaneously over the BAT and lower back and intraperitoneally to measure the core temperature. The results found that the thermic effect of the BAT increased after the transition from low fat diet to a high fat diet (LFD) yet, after prolonged exposure to the HFD, the effects resembled levels found with the LFD. This suggests that while a HFD may stimulate the effect of BAT, long term exposure may have adverse effects on BAT activity. This may be due to internal factors that will need to be examined further.
ContributorsSion, Paul William (Author) / Herman, Richard (Thesis director) / Borges, Chad (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
Description
Valley Fever, also known as coccidioidomycosis, is a respiratory disease that affects 10,000 people annually, primarily in Arizona and California. Due to a lack of gene annotation, diagnosis and treatment of Valley Fever is severely limited. In turn, gene annotation efforts are also hampered by incomplete genome sequencing. We intend to use proteogenomic analysis to reannotate the Coccidioides posadasii str. Silveira genome from protein-level data. Protein samples extracted from both phases of Silveira were fragmented into peptides, sequenced, and compared against databases of known and predicted proteins sequences, as well as a de novo six-frame translation of the genome. 288 unique peptides were located that did not match a known Silveira annotation, and of those 169 were associated with another Coccidioides strain. Additionally, 17 peptides were found at the boundary of, or outside of, the current gene annotation comprising four distinct clusters. For one of these clusters, we were able to calculate a lower bound and an estimate for the size of the gap between two Silveira contigs using the Coccidioides immitis RS transcript associated with that cluster's peptides \u2014 these predictions were consistent with the current annotation's scaffold structure. Three peptides were associated with an actively translated transposon, and a putative active site was located within an intact LTR retrotransposon. We note that gene annotation is necessarily hindered by the quality and level of detail in prior genome sequencing efforts, and recommend that future studies involving reannotation include additional sequencing as well as gene annotation via proteogenomics or other methods.
ContributorsSherrard, Andrew (Author) / Lake, Douglas (Thesis director) / Grys, Thomas (Committee member) / Mitchell, Natalie (Committee member) / Computing and Informatics Program (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
Almost every form of cancer deregulates the expression and activity of anabolic glycosyltransferase (GT) enzymes, which incorporate particular monosaccharides in a donor acceptor as well as linkage- and anomer-specific manner to assemble complex and diverse glycans that significantly affect numerous cellular events, including tumorigenesis and metastasis. Because glycosylation is not template-driven, GT deregulation yields heterogeneous arrays of aberrant intact glycan products, some in undetectable quantities in clinical bio-fluids (e.g., blood plasma). Numerous glycan features (e.g., 6 sialylation, β-1,6-branching, and core fucosylation) stem from approximately 25 glycan “nodes:” unique linkage specific monosaccharides at particular glycan branch points that collectively confer distinguishing features upon glycan products. For each node, changes in normalized abundance (Figure 1) may serve as nearly 1:1 surrogate measure of activity for culpable GTs and may correlate with particular stages of carcinogenesis. Complementary to traditional top down glycomics, the novel bottom-up technique applied herein condenses each glycan node and feature into a single analytical signal, quantified by two GC-MS instruments: GCT (time-of-flight analyzer) and GCMSD (transmission quadrupole analyzers). Bottom-up analysis of stage 3 and 4 breast cancer cases revealed better overall precision for GCMSD yet comparable clinical performance of both GC MS instruments and identified two downregulated glycan nodes as excellent breast cancer biomarker candidates: t-Gal and 4,6-GlcNAc (ROC AUC ≈ 0.80, p < 0.05). Resulting from the activity of multiple GTs, t-Gal had the highest ROC AUC (0.88) and lowest ROC p‑value (0.001) among all analyzed nodes. Representing core-fucosylation, glycan node 4,6-GlcNAc is a nearly 1:1 molecular surrogate for the activity of α-(1,6)-fucosyltransferase—a potential target for cancer therapy. To validate these results, future projects can analyze larger sample sets, find correlations between breast cancer stage and changes in t-Gal and 4,6-GlcNAc levels, gauge the specificity of these nodes for breast cancer and their potential role in other cancer types, and develop clinical tests for reliable breast cancer diagnosis and treatment monitoring based on t-Gal and 4,6-GlcNAc.
ContributorsZaare, Sahba (Author) / Borges, Chad (Thesis director) / LaBaer, Joshua (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Aberrant glycosylation has been shown to be linked to specific cancers, and using this idea, it was proposed that the levels of glycans in the blood could predict stage I adenocarcinoma. To track this glycosylation, glycan were broken down into glycan nodes via methylation analysis. This analysis utilized information from N-, O-, and lipid linked glycans detected from gas chromatography-mass spectrometry. The resulting glycan node-ratios represent the initial quantitative data that were used in this experiment.
For this experiment, two Sets of 50 µl blood plasma samples were provided by NYU Medical School. These samples were then analyzed by Dr. Borges’s lab so that they contained normalized biomarker levels from patients with stage 1 adenocarcinoma and control patients with matched age, smoking status, and gender were examined. An ROC curve was constructed under individual and paired conditions and AUC calculated in Wolfram Mathematica 10.2. Methods such as increasing size of training set, using hard vs. soft margins, and processing biomarkers together and individually were used in order to increase the AUC. Using a soft margin for this particular data set was proved to be most useful compared to the initial set hard margin, raising the AUC from 0.6013 to 0.6585. In regards to which biomarkers yielded the better value, 6-Glc/6-Man and 3,6-Gal glycan node ratios had the best with 0.7687 AUC and a sensitivity of .7684 and specificity of .6051. While this is not enough accuracy to become a primary diagnostic tool for diagnosing stage I adenocarcinoma, the methods examined in the paper should be evaluated further. . By comparison, the current clinical standard blood test for prostate cancer that has an AUC of only 0.67.
For this experiment, two Sets of 50 µl blood plasma samples were provided by NYU Medical School. These samples were then analyzed by Dr. Borges’s lab so that they contained normalized biomarker levels from patients with stage 1 adenocarcinoma and control patients with matched age, smoking status, and gender were examined. An ROC curve was constructed under individual and paired conditions and AUC calculated in Wolfram Mathematica 10.2. Methods such as increasing size of training set, using hard vs. soft margins, and processing biomarkers together and individually were used in order to increase the AUC. Using a soft margin for this particular data set was proved to be most useful compared to the initial set hard margin, raising the AUC from 0.6013 to 0.6585. In regards to which biomarkers yielded the better value, 6-Glc/6-Man and 3,6-Gal glycan node ratios had the best with 0.7687 AUC and a sensitivity of .7684 and specificity of .6051. While this is not enough accuracy to become a primary diagnostic tool for diagnosing stage I adenocarcinoma, the methods examined in the paper should be evaluated further. . By comparison, the current clinical standard blood test for prostate cancer that has an AUC of only 0.67.
ContributorsDe Jesus, Celine Spicer (Author) / Taylor, Thomas (Thesis director) / Borges, Chad (Committee member) / School of Mathematical and Statistical Sciences (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Disturbances in the protein interactome often play a large role in cancer progression. Investigation of protein-protein interactions (PPI) can increase our understanding of cancer pathways and will disclose unknown targets involved in cancer disease biology. Although numerous methods are available to study protein interactions, most platforms suffer from drawbacks including high false positive rates, low throughput, and lack of quantification. Moreover, most methods are not compatible for use in a clinical setting. To address these limitations, we have developed a multiplexed, in-solution protein microarray (MISPA) platform with broad applications in proteomics. MISPA can be used to quantitatively profile PPIs and as a robust technology for early detection of cancers. This method utilizes unique DNA barcoding of individual proteins coupled with next generation sequencing to quantitatively assess interactions via barcode enrichment. We have tested the feasibility of this technology in the detection of patient immune responses to oropharyngeal carcinomas and in the discovery of novel PPIs in the B-cell receptor (BCR) pathway. To achieve this goal, 96 human papillomavirus (HPV) antigen genes were cloned into pJFT7-cHalo (99% success) and pJFT7-n3xFlag-Halo (100% success) expression vectors. These libraries were expressed via a cell-free in vitro transcription-translation system with 93% and 96% success, respectively. A small-scale study of patient serum interactions with barcoded HPV16 antigens was performed and a HPV proteome-wide study will follow using additional patient samples. In addition, 15 query proteins were cloned into pJFT7_nGST expression vectors, expressed, and purified with 93% success to probe a library of 100 BCR pathway proteins and detect novel PPIs.
ContributorsRinaldi, Capria Lakshmi (Author) / LaBaer, Joshua (Thesis director) / Mangone, Marco (Committee member) / Borges, Chad (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12