Matching Items (8)
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- All Subjects: Cellular Biology
- Creators: Mehta, Shwetal
- Creators: Misra, Rajeev
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
The FOF1 ATP synthase is responsible for generating the majority of adenosine triphosphate (ATP) in almost all organisms on Earth. A major unresolved question is the mechanism of the FO motor that converts the transmembrane flow of protons into rotation that drives ATP synthesis. Using single-molecule gold nanorod experiments, rotation of individual FOF1 were observed to measure transient dwells (TDs). TDs occur when the FO momentarily halts the ATP hydrolysis rotation by the F1-ATPase. The work presented here showed increasing TDs with decreasing pH, with calculated pKa values of 5.6 and 7.5 for wild-type (WT) Escherichia coli (E. coli) subunit-a proton input and output half-channels, respectively. This is consistent with the conclusion that the periplasmic proton half-channel is more easily protonated than the cytoplasmic half-channel. Mutation in one proton half-channel affected the pKa values of both half-channels, suggesting that protons flow through the FO motor via the Grotthuss mechanism. The data revealed that 36° stepping of the E. coli FO subunit-c ring during ATP synthesis consists of an 11° step caused by proton translocations between subunit-a and the c-ring, and a 25° step caused by the electrostatic interaction between the unprotonated c-subunit and the aR210 residue in subunit-a. The occurrence of TDs fit to the sum of three Gaussian curves, which suggested that the asymmetry between the FO and F1 motors play a role in the mechanism behind the FOF1 rotation. Replacing the inner (N-terminal) helix of E. coli c10-ring with sequences derived from c8 to c17-ring sequences showed expression and full assembly of FOF1. Decrease in anticipated c-ring size resulted in increased ATP synthesis activity, while increase in c-ring size resulted in decreased ATP synthesis activity, loss of Δψ-dependence to synthesize ATP, decreased ATP hydrolysis activity, and decreased ACMA quenching activity. Low levels of ATP synthesis by the c12 and c15-ring chimeras are consistent with the role of the asymmetry between the FO and F1 motors that affects ATP synthesis rotation. Lack of a major trend in succinate-dependent growth rates of the chimeric E. coli suggest cellular mechanisms that compensates for the c-ring modification.
ContributorsYanagisawa, Seiga (Author) / Frasch, Wayne D (Thesis advisor) / Misra, Rajeev (Committee member) / Redding, Kevin (Committee member) / Singharoy, Abhishek (Committee member) / Wideman, Jeremy (Committee member) / Arizona State University (Publisher)
Created2023
Description
In this work, secretion of free fatty acids (FFAs) and ω-hydroxy FFAs wasachieved in the model cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis), and
FFAs were detected by a novel fluorescence assay. Current methods of detecting FFA
concentrations, including HPLC-based and GC-based methods or enzyme-based kits,
have hindered research advancement due to their laborious and/or expensive nature. The
work herein establishes a novel, rapid, fluorescence-based assay for detecting total FFA
concentrations secreted by Synechocystis FFA secretion strains. The novel FFA-detection
assay demonstrates the efficacy of using Nile Red as a fluorescent reporter for laurate or
palmitate at concentrations up to 500 µM in the presence of cationic surfactants. Total
FFA concentrations in Synechocystis supernatants quantified by the novel, Nile Red fluorescence-based assay are demonstrated herein to be highly correlative to total FFA
concentrations quantified by LC-MS; this correlation was seen in supernatant samples of
wild type Synechocystis and Synechocystis FFA secretion strains, both in 96-well plates
and 30-mL, aerated culture tubes.
This work also establishes the expression of a cytochrome P450 fusion enzyme,
CYP153A-CPRmut, or a monooxygenase system from Pseudomonas putida GPo1,
AlkBGT, in FFA secretion strains of Synechocystis for the generation of ω-hydroxy
laurate from laurate. After finding greatly increased ω-hydroxylation activity of
CYP153A-CPRmut with concurrent superoxide dismutase and catalase overexpression, 55
or 1.5 µM of ω-hydroxy laurate were produced over five days by Synechocystis strains
expressing CYP153A-CPRmut or AlkBGT, respectively. As further indication of the
presence of reactive oxygen species affecting ω-hydroxy laurate production with
Synechocystis strains expressing CYP153A-CPRmut, concentrations of ω-hydroxy laurate
in the supernatant increased over two-fold in the presence of 250 µM of the anti-oxidant,
methionine, in bench-scale cultures and in 96-well plate cultures. Additionally, a
mutation at the 55th amino acid position in AlkB (tryptophan to cysteine; AlkBW55C),
resulted in a more than two-fold shift in AlkB’s substrate preference from decanoate
towards the desired substrate, laurate. As a result, Synechocystis expressing AlkBW55C
could produce 5.9 µM ω-hydroxy laurate and 2.0 µM dodecanedioic acid over five days
of growth.
ContributorsAshe, Christopher (Author) / Vermaas, Willem Fj (Thesis advisor, Committee member) / Wang, Xuan (Committee member) / Nielsen, David R (Committee member) / Misra, Rajeev (Committee member) / Arizona State University (Publisher)
Created2023
Description
The GGGGCC (G4C2) hexanucleotide repeat expansion (HRE) in the C9orf72 gene is the most common genetic abnormality associated with both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two devastatingly progressive neurodegenerative diseases. The discovery of this genetic link confirmed that ALS and FTD reside along a spectrum with clinical and pathological commonalities. Historically understood as diseases resulting in neuronal death, the role of non-neuronal cells like astrocytes is still wholly unresolved. With evidence of cortical neurodegeneration leading to cognitive impairments in C9orf72-ALS/FTD, there is a need to investigate the role of cortical astrocytes in this disease spectrum. Here, a patient-derived induced pluripotent stem cell (iPSC) cortical astrocyte model was developed to investigate consequences of C9orf72-HRE pathogenic features in this cell type. Although there were no significant C9orf72-HRE pathogenic features in cortical astrocytes, transcriptomic, proteomic and phosphoproteomic profiles elucidated global disease-related phenotypes. Specifically, aberrant expression of astrocytic-synapse proteins and secreted factors were identified. SPARCL1, a pro-synaptogenic secreted astrocyte factor was found to be selectively decreased in C9orf72-ALS/FTD iPSC-cortical astrocytes. This finding was further validated in human tissue analyses, indicating that cortical astrocytes in C9orf72-ALS/FTD exhibit a reactive transformation that is characterized by a decrease in SPARCL1 expression. Considering the evidence for substantial astrogliosis and synaptic failure leading to cognitive impairments in C9orf72-ALS/FTD, these findings represent a novel understanding of how cortical astrocytes may contribute to the cortical neurodegeneration in this disease spectrum.
ContributorsBustos, Lynette (Author) / Sattler, Rita (Thesis advisor) / Newbern, Jason (Committee member) / Zarnescu, Daniela (Committee member) / Brafman, David (Committee member) / Mehta, Shwetal (Committee member) / Arizona State University (Publisher)
Created2023
Description
Directed evolution using genetically diverse libraries is integral to advancing research in industrial microbial production and protein functionality enhancement. This process typically involves a step of sequence diversification and subsequent selection/screening steps for improved variants. While CRISPR-Cas9 systems are known to offer efficient and targeted modification of genes in vivo, concerns arise regarding off-target effects and the emergence of escaper cells evading Cas9 cleavage. This study investigated a strategy to leverage CRISPR-Cas9 counter-selection in Escherichia coli for targeted chromosomal mutagenesis. By designing gRNAs to target a desired region, the spontaneous mutations occurring at the targeted region will potentially disrupt Cas9 binding and thus allow the cell to avoid death caused by Cas9-induced double-stranded DNA breaks. This population of ‘escaper’ cells surviving the counter-selection will have mutations in the gRNA-targeting region at a higher frequency than their non-escaper counterparts. To optimize this counter-selection method, the design for the CRISPR-Cas9 expression system was improved, Cas9 variants with varied fidelities and activities were investigated, and the strategy of using truncated gRNAs for enhanced mutation selectivity was explored. Using the E. coli rpoB gene as a target for editing, the rifampicin-resistant mutation (caused by mutations in rpoB) frequency was increased by more than five orders of magnitude compared to the control E. coli strain without CRISPR targeting. Nanopore DNA sequencing of the mutants’ rpoB region confirmed the promising targeting efficacy of this approach. This study demonstrates a streamlined method for targeted genetic diversification in vivo, facilitating efficient protein engineering in bacterial systems.
ContributorsRick, Rachel Nicole (Author) / Wang, Xuan (Thesis advisor) / Nielsen, David (Committee member) / Misra, Rajeev (Committee member) / Arizona State University (Publisher)
Created2024
Description
Malignant brain tumors are devastating despite aggressive treatments such as surgical resection, chemotherapy and radiation therapy. The average life expectancy of patients with newly diagnosed glioblastoma is approximately 15 months. One novel therapeutic strategy involves using a ketogenic diet (KD) which increases circulating ketones and reduces circulating glucose. While the preclinical work has shown that the KD increases survival, enhances radiation and alters several pathways in malignant gliomas, its impact on the anti-tumor immune response has yet to be examined. This dissertation demonstrates that mice fed the KD had increased tumor-reactive innate and adaptive immune responses, including increased cytokine production and cytolysis via tumor-reactive CD8+ T cells. Additionally, we saw that mice maintained on the KD had increased CD4 infiltration, while T regulatory cell numbers stayed consistent. Lastly, mice fed the KD had a significant reduction in immune inhibitory receptor expression as well as decreased inhibitory ligand expression on glioma cells, namely programmed death receptor -1 (PD-1) and its ligand programmed death receptor ligand -1 (PD-L1). Further, it is demonstrated that the ketone body beta-hydroxybutyrate (BHB) reduces expression of PD-L1 on glioma cells in vitro suggesting it may be responsible in part for immune-related changes elicited by the KD. Finally this dissertation also shows that the KD increases the expression of microRNAs predicted to target PD-L1 suggesting a potential mechanism to explain the ability of the KD to modulate immune inhibitory checkpoint pathways. Taken together these studies shed important light on the mechanisms underlying the KD and provide additional support for its use an adjuvant therapy for malignant glioma.
ContributorsWoolf, Eric Christopher (Author) / Compton, Carolyn C. (Thesis advisor) / Scheck, Adrienne C (Committee member) / Preul, Mark C (Committee member) / Blattman, Joseph N (Committee member) / Mehta, Shwetal (Committee member) / Arizona State University (Publisher)
Created2018
Description
Cancer is a major health problem in the world today and is expected to become an even larger one in the future. Although cancer therapy has improved for many cancers in the last several decades, there is much room for further improvement. Mathematical modeling has the advantage of being able to test many theoretical therapies without having to perform clinical trials and experiments. Mathematical oncology will continue to be an important tool in the future regarding cancer therapies and management.
This dissertation is structured as a growing tumor. Chapters 2 and 3 consider spheroid models. These models are adept at describing 'early-time' tumors, before the tumor needs to co-opt blood vessels to continue sustained growth. I consider two partial differential equation (PDE) models for spheroid growth of glioblastoma. I compare these models to in vitro experimental data for glioblastoma tumor cell lines and other proposed models. Further, I investigate the conditions under which traveling wave solutions exist and confirm numerically.
As a tumor grows, it can no longer be approximated by a spheroid, and it becomes necessary to use in vivo data and more sophisticated modeling to model the growth and diffusion. In Chapter 4, I explore experimental data and computational models for describing growth and diffusion of glioblastoma in murine brains. I discuss not only how the data was obtained, but how the 3D brain geometry is created from Magnetic Resonance (MR) images. A 3D finite-difference code is used to model tumor growth using a basic reaction-diffusion equation. I formulate and test hypotheses as to why there are large differences between the final tumor sizes between the mice.
Once a tumor has reached a detectable size, it is diagnosed, and treatment begins. Chapter 5 considers modeling the treatment of prostate cancer. I consider a joint model with hormonal therapy as well as immunotherapy. I consider a timing study to determine whether changing the vaccine timing has any effect on the outcome of the patient. In addition, I perform basic analysis on the six-dimensional ordinary differential equation (ODE). I also consider the limiting case, and perform a full global analysis.
This dissertation is structured as a growing tumor. Chapters 2 and 3 consider spheroid models. These models are adept at describing 'early-time' tumors, before the tumor needs to co-opt blood vessels to continue sustained growth. I consider two partial differential equation (PDE) models for spheroid growth of glioblastoma. I compare these models to in vitro experimental data for glioblastoma tumor cell lines and other proposed models. Further, I investigate the conditions under which traveling wave solutions exist and confirm numerically.
As a tumor grows, it can no longer be approximated by a spheroid, and it becomes necessary to use in vivo data and more sophisticated modeling to model the growth and diffusion. In Chapter 4, I explore experimental data and computational models for describing growth and diffusion of glioblastoma in murine brains. I discuss not only how the data was obtained, but how the 3D brain geometry is created from Magnetic Resonance (MR) images. A 3D finite-difference code is used to model tumor growth using a basic reaction-diffusion equation. I formulate and test hypotheses as to why there are large differences between the final tumor sizes between the mice.
Once a tumor has reached a detectable size, it is diagnosed, and treatment begins. Chapter 5 considers modeling the treatment of prostate cancer. I consider a joint model with hormonal therapy as well as immunotherapy. I consider a timing study to determine whether changing the vaccine timing has any effect on the outcome of the patient. In addition, I perform basic analysis on the six-dimensional ordinary differential equation (ODE). I also consider the limiting case, and perform a full global analysis.
ContributorsRutter, Erica Marie (Author) / Kuang, Yang (Thesis advisor) / Kostelich, Eric J (Thesis advisor) / Frakes, David (Committee member) / Gardner, Carl (Committee member) / Jackiewicz, Zdzislaw (Committee member) / Arizona State University (Publisher)
Created2016
Description
Development of the cerebral cortex requires the complex integration of extracellular stimuli to affect changes in gene expression. Trophic stimulation activates specialized intracellular signaling cascades to instruct processes necessary for the elaborate cellular diversity, architecture, and function of the cortex. The canonical RAS/RAF/MEK/ERK (ERK/MAPK) cascade is a ubiquitously expressed kinase pathway that regulates crucial aspects of neurodevelopment. Mutations in the ERK/MAPK pathway or its regulators give rise to neurodevelopmental syndromes termed the “RASopathies.” RASopathy individuals present with neurological symptoms that include intellectual disability, ADHD, and seizures. The precise cellular mechanisms that drive neurological impairments in RASopathy individuals remain unclear. In this thesis, I aimed to 1) address how RASopathy mutations affect neurodevelopment, 2) elucidate fundamental requirements of ERK/MAPK in GABAergic circuits, and 3) determine how aberrant ERK/MAPK signaling disrupts GABAergic development.
Here, I show that a Noonan Syndrome-linked gain-of-function mutation Raf1L613V, drives modest changes in astrocyte and oligodendrocyte progenitor cell (OPC) density in the mouse cortex and hippocampus. Raf1L613V mutant mice exhibited enhanced performance in hippocampal-dependent spatial reference and working memory and amygdala-dependent fear learning tasks. However, we observed normal perineuronal net (PNN) accumulation around mutant parvalbumin-expressing (PV) interneurons. Though PV-interneurons were minimally affected by the Raf1L613V mutation, other RASopathy mutations converge on aberrant GABAergic development as a mediator of neurological dysfunction.
I therefore hypothesized interneuron expression of the constitutively active Mek1S217/221E (caMek1) mutation would be sufficient to perturb GABAergic circuit development. Interestingly, the caMek1 mutation selectively disrupted crucial PV-interneuron developmental processes. During embryogenesis, I detected expression of cleaved-caspase 3 (CC3) in the medial ganglionic eminence (MGE). Interestingly, adult mutant cortices displayed a selective 50% reduction in PV-expressing interneurons, but not other interneuron subtypes. PV-interneuron loss was associated with seizure-like activity in mutants and coincided with reduced perisomatic synapses. Mature mutant PV-interneurons exhibited somal hypertrophy and a substantial increase in PNN accumulation. Aberrant GABAergic development culminated in reduced behavioral response inhibition, a process linked to ADHD-like behaviors. Collectively, these data provide insight into the mechanistic underpinnings of RASopathy neuropathology and suggest that modulation of GABAergic circuits may be an effective therapeutic option for RASopathy individuals.
Here, I show that a Noonan Syndrome-linked gain-of-function mutation Raf1L613V, drives modest changes in astrocyte and oligodendrocyte progenitor cell (OPC) density in the mouse cortex and hippocampus. Raf1L613V mutant mice exhibited enhanced performance in hippocampal-dependent spatial reference and working memory and amygdala-dependent fear learning tasks. However, we observed normal perineuronal net (PNN) accumulation around mutant parvalbumin-expressing (PV) interneurons. Though PV-interneurons were minimally affected by the Raf1L613V mutation, other RASopathy mutations converge on aberrant GABAergic development as a mediator of neurological dysfunction.
I therefore hypothesized interneuron expression of the constitutively active Mek1S217/221E (caMek1) mutation would be sufficient to perturb GABAergic circuit development. Interestingly, the caMek1 mutation selectively disrupted crucial PV-interneuron developmental processes. During embryogenesis, I detected expression of cleaved-caspase 3 (CC3) in the medial ganglionic eminence (MGE). Interestingly, adult mutant cortices displayed a selective 50% reduction in PV-expressing interneurons, but not other interneuron subtypes. PV-interneuron loss was associated with seizure-like activity in mutants and coincided with reduced perisomatic synapses. Mature mutant PV-interneurons exhibited somal hypertrophy and a substantial increase in PNN accumulation. Aberrant GABAergic development culminated in reduced behavioral response inhibition, a process linked to ADHD-like behaviors. Collectively, these data provide insight into the mechanistic underpinnings of RASopathy neuropathology and suggest that modulation of GABAergic circuits may be an effective therapeutic option for RASopathy individuals.
ContributorsHolter, Michael (Author) / Newbern, Jason (Thesis advisor) / Anderson, Trent (Committee member) / Mehta, Shwetal (Committee member) / Neisewander, Janet (Committee member) / Arizona State University (Publisher)
Created2019