Matching Items (198)
Filtering by
- Status: Published
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
Cancer is one of the leading causes of death globally according to the World Health Organization. Although improved treatments and early diagnoses have reduced cancer related mortalities, metastatic disease remains a major clinical challenge. The local tumor microenvironment plays a significant role in cancer metastasis, where tumor cells respond and adapt to a plethora of biochemical and biophysical signals from stromal cells and extracellular matrix (ECM) proteins. Due to these complexities, there is a critical need to understand molecular mechanisms underlying cancer metastasis to facilitate the discovery of more effective therapies. In the past few years, the integration of advanced biomaterials and microengineering approaches has initiated the development of innovative platform technologies for cancer research. These technologies enable the creation of biomimetic in vitro models with physiologically relevant (i.e. in vivo-like) characteristics to conduct studies ranging from fundamental cancer biology to high-throughput drug screening. In this review article, we discuss the biological significance of each step of the metastatic cascade and provide a broad overview on recent progress to recapitulate these stages using advanced biomaterials and microengineered technologies. In each section, we will highlight the advantages and shortcomings of each approach and provide our perspectives on future directions.
ContributorsPeela, Nitish (Author) / Nikkhah, Mehdi (Thesis director) / LaBaer, Joshua (Committee member) / Harrington Bioengineering Program (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
Description
Glioblastoma is the most aggressive and lethal brain tumor, due to its resistance to current conventional therapy. The resistance to chemo- and radiotherapy has been attributed to a special population of cells known as glioma stem cells. Previous literature has shown the importance of a Central Nervous System-restricted transcription factor OLIG2 in maintaining the tumor-propagating potential of these glioma stem cells. OLIG2's function was further elucidated, with its pro-mitogenic function due to its ability to negatively regulate the p53 pathway by suppressing the acetylation of the p53 protein's C terminal domain. Past work in our lab has confirmed that one of OLIG2's partner proteins is Histone Deacetylase 1 (HDAC1). In vitro experiments have also shown that targeting HDAC1 using hairpin RNA in glioma stem cells negatively impacts proliferation. In a survival study using a murine glioma model, targeting Hdac1 using hairpin RNA is shown to reduce tumor burden and increase survival. In this paper, we demonstrate that silencing Hdac1 expression reduces proliferation, increases cell death, likely a result of increased acetylation of p53. Olig2 expression levels seem to be unaffected in GSCs, demonstrating that the Hdac1 protein ablation is indeed lethal to GSCs. This work builds upon previously collected results, confirming that Hdac1 is a potential surrogate target for Olig2's pro-mitotic function in regulating the p53 pathway.
ContributorsLoo, Vincent You Wei (Author) / LaBaer, Joshua (Thesis director) / Mehta, Shwetal (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
Description
CREB3L1 has been previously shown to auto-acetylate itself when prepared from HeLa cell based in vitro protein expression lysates. To circumvent the concerns of the contamination of co-purified human proteins from HeLa lysates, the protein was purified through insect cell transfection in vitro. The objective of this study was to assay the auto-acetylation activity of CREB3L1 prepared from insect cells using the baculovirus expression vector system (BEVS). To this end, His-tagged CREB3L1 was affinity purified from Hi5 cells using an IMAC column and used for acetylation assay. Samples were taken different time points and auto-acetylation was by western using antibodies specific to acetylated lysines. Auto-acetylation activity was observed after overnight incubation. Future experiments will focus on the improvement of purification yield and the identification of the substrates and interacting proteins of CREB3L1 to better understand the biological functions of this novel acetyltransferase.
ContributorsSchwab, Anna (Author) / LaBaer, Joshua (Thesis director) / Qiu, Ji (Committee member) / Barrett, The Honors College (Contributor)
Created2017-05
Description
PD-L1 blockade has shown recent success in cancer therapy and cancer vaccine regimens. One approach for anti-PD-L1 antibodies has been their application as adjuvants for cancer vaccines. Given the disadvantages of such antibodies, including long half-life and adverse events related to their use, a novel strategy using synbodies in place of antibodies can be tested. Synbodies offer a variety of advantages, including shorter half-life, smaller size, and cheaper cost. Peptides that could bind PD-L1 were identified via peptide arrays and used to construct synbodies. These synbodies were tested with inhibition ELISA assays, SPR, and pull down assays. Additional flow cytometry analysis was done to determine the binding specificity of the synbodies to PD-L1 and the ability of those synbodies to inhibit the PD-L1/PD-1 interaction. Although analysis of permeabilized cells expressing PD-L1 indicated that the synbodies could successfully bind PD-L1, those results were not replicated in non-permeabilized cells. Further assays suggested that the binding of the synbodies was non-specific. Other tests were done to see if the synbodies could inhibit the PD-1/PD-L1 interaction. This assay did not yield any conclusive results and further experimentation is needed to determine the efficacy of the synbodies in inhibiting this interaction.
ContributorsMujahed, Tala (Author) / Johnston, Stephen (Thesis director) / Blattman, Joseph (Committee member) / Diehnelt, Chris (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
The devastating 2014 Ebola virus outbreak in Western Africa demonstrated the lack of therapeutic approaches available for the virus. Although monoclonal antibodies (mAb) and other molecules have been developed that bind the virus, no therapeutic has shown the efficacy needed for FDA approval. Here, a library of 50 peptide based ligands that bind the glycoprotein of the Zaire Ebola virus (GP) were developed. Using whole virus screening of vesicular stomatitis virus pseudotyped with GP, low affinity peptides were identified for ligand construction. In depth analysis showed that two of the peptide based molecules bound the Zaire GP with <100 nM KD. One of these two ligands was blocked by a known neutralizing mAb, 2G4, and showed cross-reactivity to the Sudan GP. This work presents ligands with promise for therapeutic applications across multiple variants of the Ebola virus.
ContributorsRabinowitz, Joshua Avraam (Author) / Diehnelt, Chris (Thesis director) / Johnston, Stephen (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
Both technological and scientific fields continue to revolutionize in a similar fashion; however, a major difference is that high-tech corporations have found models to continue progressions while still keeping product costs low. The main objective was to identify which, if any, components of certain technological models could be used with the vaccine and pharmaceutical markets to significantly lower their costs. Smartphones and computers were the two main items investigated while the two main items from the scientific standpoint were vaccines and pharmaceuticals. One concept had the ability to conceivably decrease the costs of vaccines and drugs and that was "market competition". If the United States were able to allow competition within the vaccine and drug companies, it would allow for the product prices to be best affected. It would only take a few small companies to generate generic versions of the drugs and decrease the prices. It would force the larger competition to most likely decrease their prices. Furthermore, the PC companies use a cumulative density function (CDF) to effectively divide their price setting in each product cycle. It was predicted that if this CDF model were applied to the vaccine and drug markets, the prices would no longer have to be extreme. The corporations would be able to set the highest price for the wealthiest consumers and then slowly begin to decrease the costs for the middle and lower class. Unfortunately, the problem within the vaccine and pharmaceutical markets was not the lack of innovation or business models. The problem lied with their liberty to choose product costs due to poor U.S. government regulations.
ContributorsCalderon, Gerardo (Author) / Johnston, Stephen (Thesis director) / Diehnelt, Chris (Committee member) / School of Molecular 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