Matching Items (196)
Description
the project led by Professor Emma Frow, researching of stem cell clinics focused on stem cell applications, adherence to FDA guidelines, and characterization of information available and physician credentials. Regenerative medicine clinics commonly offered stem cell therapy, but introduced platelet rich plasma (PRP) and prolotherapy as regenerative therapies.
PRP and Prolotherapy are individual treatments that were even suggested and used in combination with stem cell therapies. Prolotherapy predates PRP as a chemical irritant therapy originally used to sclerose tissues. Prolotherapy is meant to stimulate platelet derived growth factors release to improve tissue healing response. Prolotherapy shows negligible efficacy improvements over corticosteroids, but may have underlying side effects from being an irritant. PRP is a more modern therapy for improved healing. Speculations state initial use was in an open heart surgery to improve healing post-surgery. PRP is created via centrifugation of patient blood to isolate growth factors by removing serum and other biological components to increase platelet concentration. PRP is comparable to corticosteroid injections in efficacy, but as an autologous application, there are no side effects making it more advantageous. Growth factors induce healing response and reduce inflammation. Growth factors stimulate cell growth, proliferation, differentiation, and stimulate cellular response mechanism such as angiogenesis and mitogenesis. The growth factor stimulation of PRP and prolotherapy both assist stem cell proliferation. Additional research is needed to determine differential capacity to ensure multipotent stem cells regenerate the correct cell type from the increased differential capacity offered by growth factor recruitment. The application of combination therapy for stem cells is unsubstantiated and applications violate FDA ‘minimal manipulation’ guidelines.
PRP and Prolotherapy are individual treatments that were even suggested and used in combination with stem cell therapies. Prolotherapy predates PRP as a chemical irritant therapy originally used to sclerose tissues. Prolotherapy is meant to stimulate platelet derived growth factors release to improve tissue healing response. Prolotherapy shows negligible efficacy improvements over corticosteroids, but may have underlying side effects from being an irritant. PRP is a more modern therapy for improved healing. Speculations state initial use was in an open heart surgery to improve healing post-surgery. PRP is created via centrifugation of patient blood to isolate growth factors by removing serum and other biological components to increase platelet concentration. PRP is comparable to corticosteroid injections in efficacy, but as an autologous application, there are no side effects making it more advantageous. Growth factors induce healing response and reduce inflammation. Growth factors stimulate cell growth, proliferation, differentiation, and stimulate cellular response mechanism such as angiogenesis and mitogenesis. The growth factor stimulation of PRP and prolotherapy both assist stem cell proliferation. Additional research is needed to determine differential capacity to ensure multipotent stem cells regenerate the correct cell type from the increased differential capacity offered by growth factor recruitment. The application of combination therapy for stem cells is unsubstantiated and applications violate FDA ‘minimal manipulation’ guidelines.
ContributorsKrum, Logan (Author) / Frow, Emma (Thesis director) / Brafman, David (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
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
Current research into live-cell dynamics, particularly those relating to chromatin structure and remodeling, are limited. The tools that are used to detect state changes in chromatin, such as Chromatin Immunoprecipitation and qPCR, require that the cell be killed off. This limits the ability of researchers to pinpoint changes in live cells over a longer period of time. As such, there is a need for a live-cell sensor that can detect chromatin state changes. The Chromometer is a transgenic chromatin state sensor designed to better understand human cell fate and the chromatin changes that occur. HOXD11.12, a DNA sequence that attracts repressive Polycomb group (PCG) proteins, was placed upstream of a core promoter-driven fluorescent reporter (AmCyan fluorescent protein, CFP) to link chromatin repression to a CFP signal. The transgene was stably inserted at an ectopic site in U2-OS (osteosarcoma) cells. Expression of CFP should reflect the epigenetic state at the HOXD locus, where several genes are regulated by Polycomb to control cell differentiation. U2-OS cells were transfected with the transgene and grown under selective pressure. Twelve colonies were identified as having integrated parts from the transgene into their genomes. PCR testing verified 2 cell lines that contain the complete transgene. Flow cytometry indicated mono-modal and bimodal populations in all transgenic cell colonies. Further research must be done to determine the effectiveness of this device as a sensor for live cell state change detection.
ContributorsBarclay, David (Co-author) / Simper, Jan (Co-author) / Haynes, Karmella (Thesis director) / Brafman, David (Committee member) / School of Life Sciences (Contributor) / Harrington Bioengineering Program (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
Description
Microfluidic platforms have been exploited extensively as a tool for the separation of particles by electric field manipulation. Microfluidic devices can facilitate the manipulation of particles by dielectrophoresis. Separation of particles by size and type has been demonstrated by insulator-based dielectrophoresis in a microfluidic device. Thus, manipulating particles by size has been widely studied throughout the years. It has been shown that size-heterogeneity in organelles has been linked to multiple diseases from abnormal organelle size. Here, a mixture of two sizes of polystyrene beads (0.28 and 0.87 μm) was separated by a ratchet migration mechanism under a continuous flow (20 nL/min). Furthermore, to achieve high-throughput separation, different ratchet devices were designed to achieve high-volume separation. Recently, enormous efforts have been made to manipulate small size DNA and proteins. Here, a microfluidic device comprising of multiple valves acting as insulating constrictions when a potential is applied is presented. The tunability of the electric field gradient is evaluated by a COMSOL model, indicating that high electric field gradients can be reached by deflecting the valve at a certain distance. Experimentally, the tunability of the dynamic constriction was demonstrated by conducting a pressure study to estimate the gap distance between the valve and the substrate at different applied pressures. Finally, as a proof of principle, 0.87 μm polystyrene beads were manipulated by dielectrophoresis. These microfluidic platforms will aid in the understanding of size-heterogeneity of organelles for biomolecular assessment and achieve separation of nanometer-size DNA and proteins by dielectrophoresis.
ContributorsOrtiz, Ricardo (Author) / Ros, Alexandra (Thesis advisor) / Hayes, Mark (Committee member) / Borges, Chad (Committee member) / Arizona State University (Publisher)
Created2021
Description
Cardiovascular disease (CVD) remains the leading cause of mortality, resulting in 1 out of 4 deaths in the United States at the alarming rate of 1 death every 36 seconds, despite great efforts in ongoing research. In vitro research to study CVDs has had limited success, due to lack of biomimicry and structural complexity of 2D models. As such, there is a critical need to develop a 3D, biomimetic human cardiac tissue within precisely engineered in vitro platforms. This PhD dissertation involved development of an innovative anisotropic 3D human stem cell-derived cardiac tissue on-a-chip model (i.e., heart on-a-chip), with an enhanced maturation tissue state, as demonstrated through extensive biological assessments. To demonstrate the potential of the platform to study cardiac-specific diseases, the developed heart on-a-chip was used to model myocardial infarction (MI) due to exposure to hypoxia. The successful induction of MI on-a-chip (heart attack-on-a-chip) was evidenced through fibrotic tissue response, contractile dysregulation, and transcriptomic regulation of key pathways.This dissertation also described incorporation of CRISPR/Cas9 gene-editing to create a human induced pluripotent stem cell line (hiPSC) with a mutation in KCNH2, the gene implicated in Long QT Syndrome Type 2 (LQTS2). This novel stem cell line, combined with the developed heart on-a-chip technology, led to creation of a 3D human cardiac on-chip tissue model of LQTS2 disease.. Extensive mechanistic biological and electrophysiological characterizations were performed to elucidate the mechanism of R531W mutation in KCNH2, significantly adding to existing knowledge about LQTS2. In summary, this thesis described creation of a LQTS2 cardiac on-a-chip model, incorporated with gene-edited hiPSC-cardiomyocytes and hiPSC-cardiac fibroblasts, to study mechanisms of LQTS2.
Overall, this dissertation provides broad impact for fundamental studies toward cardiac biological studies as well as drug screening applications. Specifically, the developed heart on-a-chip from this dissertation provides a unique alternative platform to animal testing and 2D studies that recapitulates the human myocardium, with capabilities to model critical CVDs to study disease mechanisms, and/or ultimately lead to development of future therapeutic strategies.
ContributorsVeldhuizen, Jaimeson (Author) / Nikkhah, Mehdi (Thesis advisor) / Brafman, David (Committee member) / Ebrahimkhani, Mo (Committee member) / Migrino, Raymond Q (Committee member) / Plaisier, Christopher (Committee member) / Arizona State University (Publisher)
Created2021
Description
The TP53 tumor suppressor gene is the most frequently mutated gene in human cancers. In the highly aggressive triple negative breast cancer (TNBC), TP53 is mutated in 80% of cases. TNBC lacks viable drug targets, resulting in a low prognosis (12.2% 5 year survivability rate). As such, the discovery of druggable targets in TNBC would be beneficial. Mutated p53 protein typically occurs as a missense mutation and often endows cancer cells with gain of function (GOF) properties by dysregulating metabolic pathways. One of these frequently dysregulated pathways is the Hippo/Yes-associated protein-1 (YAP1)/WW Domain Containing Transcription Regulator 1 (TAZ) tumor suppressor pathway. This study therefore analyzed the involvement of the Hippo/YAP1/TAZ pathway in p53-mediated breast cancer cell invasion. From an RNA-seq screen in MCF10A cell lines harboring different TP53 missense mutations, each with a differing invasive phenotype, components of the Hippo pathway were found to correlate with cell invasion. To this end, the active and inactive forms of YAP1 and TAZ were studied. Phosphorylated (inactive) YAP1 and TAZ are retained in the cytoplasm and eventually degraded. Unphosphorylated (active) YAP1 and TAZ translocate to the nucleus to activate TEAD-family transcription factors, inducing cell survival and proliferation genes leading to increased cell invasion. Using quantitative western blot analysis, it was found that inactive TAZ expression was lower in the most invasive cell lines and higher in the least invasive cell lines (p = 0.003). Moreover, the ratio of inactive TAZ protein to total TAZ protein was also shown to be predominantly lower in the invasive cell lines compared to the non-invasive lines (p = 0.04). Finally, active TAZ expression was primarily higher in p53-mutant invasive cell lines and lower in non-invasive p53 mutant cells. Additionally, although YAP1 and TAZ are thought to be functionally redundant, the pattern seen in TAZ was not seen in the YAP1 protein. Taken together, the results demonstrated here suggest that TAZ holds a more dominant role in governing TNBC cell invasion compared to YAP1 and further highlights TAZ as a potential therapeutic target in TNBC.
ContributorsGrief, Dustin (Author) / LaBaer, Joshua (Thesis advisor) / Anderson, Karen (Committee member) / Nikkhah, Mehdi (Committee member) / Arizona State University (Publisher)
Created2022
Description
Glioblastoma (GBM), the most common and aggressive primary brain tumor affecting adults, is characterized by an aberrant yet druggable epigenetic landscape. The Histone Deacetylases (HDACs), a major family of epigenetic regulators, favor transcriptional repression by mediating chromatin compaction and are frequently overexpressed in human cancers, including GBM. Hence, over the last decade there has been considerable interest in using HDAC inhibitors (HDACi) for the treatment of malignant primary brain tumors. However, to date most HDACi tested in clinical trials have failed to provide significant therapeutic benefit to patients with GBM. This is because current HDACi have poor or unknown pharmacokinetic profiles, lack selectivity towards the different HDAC isoforms, and have narrow therapeutic windows. Isoform selectivity for HDACi is important given that broad inhibition of all HDACs results in widespread toxicity across different organs. Moreover, the functional roles of individual HDAC isoforms in GBM are still not well understood. Here, I demonstrate that HDAC1 expression increases with brain tumor grade and is correlated with decreased survival in GBM. I find that HDAC1 is the essential HDAC isoform in glioma stem cells and its loss is not compensated for by its paralogue HDAC2 or other members of the HDAC family. Loss of HDAC1 alone has profound effects on the glioma stem cell phenotype in a p53-dependent manner and leads to significant suppression of tumor growth in vivo. While no HDAC isoform-selective inhibitors are currently available, the second-generation HDACi quisinostat harbors high specificity for HDAC1. I show that quisinostat exhibits potent growth inhibition in multiple patient-derived glioma stem cells. Using a pharmacokinetics- and pharmacodynamics-driven approach, I demonstrate that quisinostat is a brain-penetrant molecule that reduces tumor burden in flank and orthotopic models of GBM and significantly extends survival both alone and in combination with radiotherapy. The work presented in this thesis thereby unveils the non-redundant functions of HDAC1 in therapy- resistant glioma stem cells and identifies a brain-penetrant HDACi with higher selectivity towards HDAC1 as a potent radiosensitizer in preclinical models of GBM. Together, these results provide a rationale for developing quisinostat as a potential adjuvant therapy for the treatment of GBM.
ContributorsLo Cascio, Costanza (Author) / LaBaer, Joshua (Thesis advisor) / Mehta, Shwetal (Committee member) / Mirzadeh, Zaman (Committee member) / Mangone, Marco (Committee member) / Paek, Andrew (Committee member) / Arizona State University (Publisher)
Created2022
Description
In cold chain tracking systems, accuracy and flexibility across different temperatures ranges plays an integral role in monitoring biospecimen integrity. However, while two common cold chain tracking systems are currently available (electronic and physics/chemical), there is not an affordable cold chain tracking mechanism that can be applied to a variety of temperatures while maintaining accuracy for individual vials. Hence, our lab implemented our understanding of biochemical reaction kinetics to develop a new cold chain tracking mechanism using the permanganate/oxalic acid reaction. The permanganate/oxalic acid reaction is characterized by the reduction of permanganate (MnVII) to Mn(II) with Mn(II)-autocatalyzed oxidation of oxalate to CO2, resulting in a pink to colorless visual indicator change when the reaction system is not in the solid state (i.e., frozen or vitrified). Throughout our research, we demonstrate, (i) Improved reaction consistency and accuracy along with extended run times with the implementation of a nitric acid-based labware washing protocol, (ii) Simulated reaction kinetics for the maximum length reaction and 60-minute reaction based on previously developed MATLAB scripts (iii) Experimental reaction kinetics to verify the simulated MATLAB maximum and 60-minute reactions times (iv) Long-term stability of the permanganate/oxalic acid reaction with water or eutectic solutions of sodium perchlorate and magnesium perchlorate at -80°C (v) Reaction kinetics with eutectic solvents, sodium perchlorate and magnesium perchlorate, at 25°C, 4°C, and -8°C (vi) Accelerated reaction kinetics after the addition of varying concentrations of manganese perchlorate (vii) Reaction kinetics of higher concentration reaction systems (5x and 10x; for darker colors), at 25°C (viii) Long-term stability of the 10x higher concentration reaction at -80°C.
ContributorsLjungberg, Emil (Author) / Borges, Chad (Thesis director) / Levitus, Marcia (Committee member) / Williams, Peter (Committee member) / Barrett, The Honors College (Contributor) / School of Molecular Sciences (Contributor) / Department of Psychology (Contributor)
Created2022-12