SAMDI-MS, which stands for self-assembled monolayers (SAMs) for matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS), is a powerful tool that has enabled the development of novel high-throughput screening and experimentation methods for decades. SAMDI-MS works by immobilizing analytes to functionalized SAMs prior to MS analysis and is capable of...
Genetic studies have found variants in the protein-degrading autophagy-lysosomal pathway (ALP) to be among the most common risk factors for developing Parkinson’s disease (PD). Macroautophagy (MA) is the arm of this pathway which delivers cytosolic components to lysosomes for degradation and is essential for neuronal health. The defining pathological protein...
Proteins represent a critical class of biomolecules, universally employed by all living organisms to fulfill essential structural, functional, and enzymatic roles necessary to support life. In nature, these polymers are composed generally of twenty natural amino acid (AA) building blocks, which can be modified with covalent adducts known as post-translational...
Methanotrophs, bacteria that can metabolize methane, remain a promising solution to mitigating the effects of climate change by removing atmospheric methane and converting it to useful chemical precursors. However, a full understanding of the main enzyme they use to oxidize methane, particulate methane monooxygenase (pMMO), is critical for harnessing their...
The treatment of AML remains to be a challenge due to the high rates of resistance and relapse experienced by patients after initial therapy. The MAPK-interacting kinases 1 and 2 (MNK1/2) have generated increasing interest as therapeutic targets for AML due to their critical role in malignant hematopoietic transformation via...
Heat shock factor 1 (HSF1) is well known for its role in the heat shock response (HSR), where it drives a transcriptional program comprising heat shock protein (HSP) genes, and in tumorigenesis, where it drives a program comprising HSPs and many noncanonical target genes that support malignancy. Here, we find...
Transfer of information across membranes is fundamental to the function of all organisms and is primarily initiated by transmembrane receptors. This is an allosteric process and involves conformational coupling between ligand-binding domain and signaling domain of a receptor. This allosteric mechanism of activation is unclear for many receptors. Moreover, for...
MYC regulates multiple gene programs, raising questions about the potential selectivity and downstream transcriptional consequences of MYC inhibitors as cancer therapeutics. MYC func-tions to either globally amplify RNA production or selectively regulates genes by repression or ac-tivation. In models of MYC inhibition by small molecules the functionality of MYC is...
Breakthroughs in large-scale biological data collection have resulted in a wealth of -omics (genomics, metabolomics, etc.) datasets in the literature. However, the development of appropriate computational techniques for their analysis is lacking, yet crucial for fully extracting the rich information contained in these datasets. The work in this dissertation describes...
Stimulation of the cGAS-STING (cycle GMP-AMP synthase-Stimulator of Interferon Genes) pathway increases T cell activation and tracking into the tumor and reverses the immunosuppressive phenotype of myeloid cells. Direct targeting of the STING receptor using synthetic cyclic dinucleotide (CDN) ligands represents an attractive immunotherapeutic strategy for the treatment of lymphocyte-depleted...
The interaction of amyloid-β (Aβ) with endogenous metal ions is thought to play a role in the pathogenesis of Alzheimer’s disease (AD). However, limited tools exist to study and modulate Aβ-metal binding. The Meade lab has developed cobalt(III) Schiff base (Co(III)-sb) complexes as protein inhibitors that competitively displace metals from...
Methylation of histone 3 lysine 4 (H3K4) catalyzed by the COMPASS family of lysine methyltransferases is universally associated with eukaryotic transcription. However, despite thousands of published studies examining the deposition, dynamics, and genomic positions of this chromatin modification, there is no clear consensus as to the molecular function or biological...
This work combines the use of high-throughput mass spectrometry with peptide arrays for to monitor reactions on peptides. The Mrksich lab introduced a high-throughput, label-free, biochemical assay that relies on self-assembled monolayers on gold and matrix-assisted laser desorption/ionization mass spectrometry, termed SAMDI-MS. This dissertation introduces new applications of SAMDI-MS and...
The work in this thesis focuses on computational methods for the identification of novel enzymatic pathways. In particular this work focuses on the utilization of the Biological Network Integrated Computational Explorer (BNICE) software suite to predict de novo enzymatic pathways for the production of commercially relevant compounds and on improvements...
DNA double-strand breaks (DSBs) are caused by either endogenous agents or exogenous ionizing radiation and chemicals. Incorrect DSB repair can lead to undesired genome rearrangements. Homologous recombination (HR) and non-homologous end-joining (NHEJ) pathways are two major DNA repair pathways that repair DSBs and maintain genome integrity. When homologous DNA is...
G protein-coupled receptors (GPCRs) are the largest family of membrane receptors in humans and play a role in nearly all physiological processes. Among GPCRs, metabotropic glutamate receptors (mGluRs) are a structurally distinct family of synaptic receptors that are essential in regulating neurotransmission and synaptic plasticity. Due to their important regulatory...
Nuclear receptors (NRs) are an important family of transcription factors that often regulate genes in response to ligands and by way of direct interactions with coactivator proteins. Many NR-coactivator pairs have been identified that cooperate to regulate transcription but fully understanding how NRs recruit specific coactivators involves learning which of...
Cobalt(III) Schiff-base complexes (Co(III)-sb) have been utilized in the literature as antibacterial, antiviral, and inhibitory agents. Recent work has utilized their ability to displace endogenous metals from metalloproteins that exhibit aberrant gain of function pathologies in human disease. Specifically, in this dissertation Co(III)-sb has been applied as inhibitors of pathogenic...
SAMDI-MS (Self-Assembled Monolayers for MALDI-TOF Mass Spectrometry) couples the use of chemically-defined self-assembled monolayers of alkane thiolates on gold surfaces with MALDI-TOF mass spectrometry for rapid characterization of the surfaces. Reactions performed on the surfaces can be quantified directly by detection of the reaction substrates and products. This rapid detection...
Epigenetics is the study of chromatin-based events that regulate gene expression without the change of DNA sequence, including DNA methylation, histone modification and chromatin remodeling. Epigenetic regulators are encoded to modify chromatin in a highly regulated and dynamic manner. A growing number of studies have suggested the dysregulation of epigenetic...
Meiosis is a highly regulated process necessary for proper chromosome division. Zincfluxes regulate mammalian meiosis; between prophase I and metaphase II, total intracellular zinc
increases by 50%, while 20% of zinc is released in “zinc sparks” following fertilization. Although
zinc fluxes had been shown to be conserved in mammals, it...
Melanin is a functional biopolymer most commonly associated with human skin pigmentation, where it serves as a radiation protection agent, shielding us from the harmful effects of UV radiation. However, melanin is also present in human ears, eyes, hair, and brains, serving a variety of functions. In fact, melanin...
Natural products from fungi have had an immeasurable societal impact as both friends and foes to human health. This includes numerous therapeutics that have had widespread clinical success, mycotoxins that are implicated in fungal pathogenicity, and various agrochemicals. Recent genome sequencing efforts have revealed that fungal genomes contain the capacity...
The opposing activities of phosphatases and kinases determine the phosphorylation status of proteins, yet kinases have received disproportionate attention in studies of cellular processes, with the roles of phosphatases remaining less understood. This dissertation describes the use of self-assembled monolayer laser desorption/ionization mass spectrometry (SAMDI-MS) together with peptide arrays to...
Pediatric high-grade gliomas (pHGGs) are aggressive pediatric CNS tumors and an important subset are characterized by mutations in H3F3A, the gene that encodes Histone H3.3 (H3.3). Substitution of Glycine at position 34 of H3.3 with either Arginine or Valine (H3.3G34R/V), was recently described and characterized in a large cohort...
More than half of proteins in humans are modified with carbohydrates in a process called glycosylation, yet this process remains poorly understood even though approximately 1% of the expressed human genome encodes biosynthetic machinery for glycosylation. Unlike genomics and proteomics where high throughput tools are now routinely used to generate...
The eukaryotic genome is packaged into chromatin. The nucleosome, the basic unit of chromatin, is composed of DNA coiled around a histone octamer. Histones are among the longest-lived protein species in mammalian cells, due to their thermodynamic stability and their associations with DNA and histone chaperones. Histone metabolism plays an...
Transcription of nucleic acids (DNA and RNA) is a hallmark of life, taking the information stored within genomic nucleic acids and converting it into a form that is useful for producing the proteins necessary for cellular and organismal function. In eukaryotes, transcription of DNA into messenger RNA (mRNA) requires the...
Although centrosomes nucleate and organize microtubules in mitotically-dividing cells, spindles in female reproductive cells (oocytes) form in their absence. In some organisms acentrosomal spindle assembly is mediated by acentriolar microtubule organizing centers (MTOCs) that are thought to functionally replace centrosomes. However, spindle assembly in human oocytes does not require MTOCs;...
The literature has established glucokinase (GCK) to be the principal hexokinase (HK) in the liver, operating as a glucose sensor to regulate glucose metabolism and lipid homeostasis. We have recently proposed Hexokinase Domain Containing-1 (HKDC1) to be a novel 5th HK with expression in the liver. Here, we reveal HKDC1...
One of the grand challenges in science is development of soft materials that mimic living organisms to optimize the way we use energy, translate or morph reversibly or sense their envi- ronment and respond in a useful fashion. Using the insights from studying biological structures, we hope to design soft...
Eukaryotic genomes are organized into chromatin, which acts to regulate access to the organism’s genetic material. A large and diverse class of proteins, known as chromatin modifiers and remodelers, are responsible for regulating the composition and structure of chromatin by monitoring nucleosomes. Chromatin remodelers are involved in multiple cellular processes,...
Heart failure due to genetic cardiomyopathy is associated with a range of phenotypic expression. The studies in this body of work interrogated the role of noncoding variation in modifying cardiomyopathy phenotypes. We used cap analysis of gene expression in heathy and failed left ventricles to define the regulatory environment of...
Nucleic acid drugs promise to revolutionize the development of therapeutics. They offer a platform for digital medicine, where systematic changes to the nucleic acid sequence can be utilized to target the entire human genome. However, nucleic acids suffer from a number of drawbacks, such as negligible cellular uptake and rapid...
Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated Cas proteins provide an immune-like response in many prokaryotes against extraneous nucleic acids. CRISPR-Cas systems are classified into different classes and types that vary widely in composition, target recognition, and overall mechanism. The main division of CRISPR-Cas systems occurs between...
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease, affecting children and adults. Chemotherapy regimens show high response rates but have debilitating effects and carry risk of relapse. Until now, no targeted therapy has been approved. In addition, 40% of patients will relapse and their treatment options are limited because...
Proper partitioning of mitochondria and mtDNA is critical for cellular health. Investigations into mitochondrial inheritance, specifically how mtDNA inheritance is coupled with the inheritance mitochondrial compartment, are still in the early stages. We use budding yeast as a model polarized cell system to study a mitochondrial Myo2-adaptor protein, Mmr1, in...
The worldwide community of patients affected by Basal Cell Carcinoma of the skin (BCC) is larger than that of any other cancer. While BCC is rarely lethal, currently available treatment strategies often leave patients with disfiguring scars on their faces, heads, and necks. Moreover, the high recurrence rates of BCC...
This thesis proposes a robust multi-pronged approach to study the effect of nanoparticles on cells. In the first place, this work is focused on investigation of the protein corona that accumulates on the surface of nanoparticles internalized by the cells and their poly-pathway effects on protein availability and messenger RNA...
Harnessing the metabolic potential of methanotrophic bacteria is a compelling strategy for the bioremediation of environmentally harmful methane gas. Methanotrophs can activate a 105 kcal/mol C-H bond in methane at ambient conditions using metalloenzymes called methane monooxygenases (MMOs). Particulate methane monooxygenase (pMMO) is a copper-dependent, membrane-bound enzyme that is the...
Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed in the heart and central nervous system where they regulate membrane excitability. Recently, inhibiting HCN channels in the hippocampus has been identified as a possible therapeutic intervention for the treatment of Major Depressive Disorder (MDD). However, based on the high expression level of...