Biochemistry, Biophysics, and Structural Biology Commons^™

Investigating The Regulatory Circuitry Of Protein Kinases And Proteases In Apoptosis, Stephanie A. Zukowski

Electronic Thesis and Dissertation Repository

Apoptosis is a tightly regulated cellular process essential for normal development and tissue homeostasis. Perturbations to apoptotic signaling underscores numerous pathogenic processes emphasizing the importance of apoptotic regulation. During apoptosis, caspases orchestrate cellular degradation through proteolytic cleavage of key structural and enzymatic proteins. In a different manner, protein kinases regulate apoptosis by catalyzing the post-translational phosphorylation of substrate proteins to facilitate either pro- or anti-apoptotic signal transduction pathways. Emerging paradigms have indicated that bidirectional crosstalk between protein kinases and caspases serves to globally fine-tune the equilibrium between signals directing cell survival and cell death. In this regard, identifying points of …

Regulation Of Liver Mitochondrial Metabolism During Hibernation By Post-Translational Modification, Katherine E. Mathers

Electronic Thesis and Dissertation Repository

Hibernation, characterized by a seasonal reduction in metabolism and body temperature, allows animals to conserve energy when environmental conditions (e.g. temperature, food availability) are unfavourable. During hibernation, small mammals such as the 13-lined ground squirrel (Ictidomys tridecemlineatus) cycle between two distinct metabolic states: torpor, where metabolic rate is suppressed by >95% and body temperature falls to ~5 °C, and interbout euthermia (IBE), where metabolic rate and body temperature rapidly increase and are maintained at euthermic levels several hours. Suppression of metabolism during entrance into torpor is paralleled by rapid suppression of liver mitochondrial metabolism. In my thesis, I …

Role Of Asparagine As A Nitrogen Signal And Characterization Of A Nitrogen Responsive Glutamine Amidotransferase, Gat1_2.1 In Arabidopsis Thaliana, Shrikaar Kambhampati

Electronic Thesis and Dissertation Repository

Maintaining the proper balance between carbon (C) and nitrogen (N) metabolism is critical to the sustained growth of organisms. In plant leaves, this balance is achieved by photoperiod dependent cross-talk between the processes of photosynthesis, respiration, and amino acid metabolism. A crucial mechanism in maintaining C/N balance is the GS/GOGAT cycle, which is well known to serve as a cross-road between C and N metabolism. Importantly, non-photosynthetic tissues (e.g. roots, germinating seeds) lack a sufficient supply of carbon skeletons under high N conditions and hence may resort to other mechanisms, along with the GS/GOGAT cycle, to achieve proper C/N balance. …

Nbpmf: Novel Network-Based Inference Methods For Peptide Mass Fingerprinting, Zhewei Liang

Electronic Thesis and Dissertation Repository

Proteins are large, complex molecules that perform a vast array of functions in every living cell. A proteome is a set of proteins produced in an organism, and proteomics is the large-scale study of proteomes. Several high-throughput technologies have been developed in proteomics, where the most commonly applied are mass spectrometry (MS) based approaches. MS is an analytical technique for determining the composition of a sample. Recently it has become a primary tool for protein identification, quantification, and post translational modification (PTM) characterization in proteomics research. There are usually two different ways to identify proteins: top-down and bottom-up. Top-down approaches …

Effect On Pellicle Proteome Upon Adsorption Of Salivary Peptide On Hydroxyapatite And Their Effects On Enamel Demineralization, Rajesh Kr. Gupta

Electronic Thesis and Dissertation Repository

According to WHO, oral health is an important indicator of overall health. Poor oral health cause serious health problems (cardiovascular, stroke, diabetes, liver, and respiratory related), in different age groups, costing billions of dollars. So, proper oral health care management has assumed top priority. Salivary antimicrobial peptides (histatin and statherin), are part of enamel pellicle, required for the oral homeostasis. Current work focused to study the demineralization protection and pellicle proteome modulation capabilities of novel peptide constructs (single and tandem duplicate histatin constructs DR9, DR9-DR9 and histatin-statherin hybrid construct DR9-RR14). For the first time, we provided evidence that, these peptide …

Identification Of Novel Binding Partners And Substrates Of Histone H3k4 Specific Demethylase Kdm5b/Jarid1b, Qi Fang

Electronic Thesis and Dissertation Repository

It has been 50 years since the first histone methylation was identified. From then, numerous studies have verified that histone methylation neutralizes the positive charges of lysine, alters chromatin architectures, and recruits other DNA binding complexes. In this study, we examined the molecular basis of a histone demethylase – KDM5B in both its substrate and binding machineries. We hypothesized that KDM5B has alternative binding partners and substrates besides its known target H3K4me3. By utilizing biochemical approaches such as in vitro pull-down assays and fluorescence polarization assays, we were able to identify potential KDM5B binding partners – H2BK43me0/2. Also, we reinvestigated …

The Mystery Of Nuclear Localization Of Arogenate Dehydratase5 From Arabidopsis Thaliana, Sara Abolhassani Rad

Electronic Thesis and Dissertation Repository

Arogenate dehydratases (ADTs) have been identified to catalyze the last step of phenylalanine (Phe) biosynthesis in plants. All ADTs have a transit peptide sequence that targets them into the chloroplasts where the biosynthesis of Phe happens. Subcellular localization studies using fluorescently tagged Arabidopsis thaliana ADTs demonstrated that all six ADTs localize to chloroplast stromules (stroma filled tubules). However, one member of this family, ADT5, was also detected in the nucleus. As dual targeting of proteins to different cell compartments is an indication of multifunctionality, ADT5 nuclear localization suggests that this member of the ADT protein family is a moonlighting protein …

An Investigation Of The Ck2-Dependent Phosphoproteome Using Inhibitor Refractory Ck2-Alpha, Edward Cruise

Electronic Thesis and Dissertation Repository

Protein kinase CK2 is a constitutively active serine/threonine kinase that is overexpressed in several human cancers, and by virtue of the vast number of putative substrates in the phosphoproteome, is implicated in the regulation of numerous cellular processes. Consequently, CK2 is an emerging therapeutic target with many CK2 inhibitors having been developed. An example of one such inhibitor is the clinical stage compound CX-4945. Although highly selective for CK2, the ATP competitive CX-4945 has demonstrated affinity for other kinases. Unique features of the catalytic pocket of CK2 have allowed for the development of inhibitor refractory mutants, which have since been …

Regulation Of C-Raf Stability By The Ranbpm/Ctlh Complex, Christina J. Mctavish

Electronic Thesis and Dissertation Repository

RanBPM is an evolutionarily conserved multi-domain protein that has been implicated in the regulation of several cellular process, including protein stability, cell migration, gene transcription, and apoptosis. RanBPM is identified as a key member of the CTLH complex, an orthologous complex to a yeast E3 ubiquitin ligase complex, the exact function of which remains unknown. Previously, our laboratory identified RanBPM as an inhibitor of the ERK1/2 pathway through the modulation of C-RAF protein levels. This study shows that RanBPM-mediated degradation of C-RAF occurs through the proteasome and the entire CRA domain of RanBPM is necessary for direct interaction with C-RAF …

Deciphering Sulfur Amino Acid Metabolism In Developing Seeds Of Common Bean, Jaya Joshi

Electronic Thesis and Dissertation Repository

With increasing food insecurity in the populated world, the number of people affected by chronic undernourishment is also increasing. Alone, protein energy malnutrition is linked to 6 million deaths annually. Despite being a good source of protein and dietary fibre, the quality of bean protein is limited because of sub optimal levels of essential sulfur amino acids: methionine and cysteine. Levels of cysteine and methionine in developing seeds have an inverse relationship with the non-protein sulfur amino acid S-methyl-cysteine (S-methylCys) and dipeptide g-glutamyl-S-methyl-cysteine (g-Glu-S-methylCys).

One of the strategies to improve protein quality in …

Amelioration Of Prenatal Alcohol Effects By Environmental Enrichment In A Mouse Model Of Fasd, Aniruddho Chokroborty-Hoque

Electronic Thesis and Dissertation Repository

Maternal alcohol consumption during pregnancy results in a spectrum of behavioural and cognitive deficits collectively known as Fetal Alcohol Spectrum Disorders (FASD). Currently, little is know about if and how the external environment may modulate these deficits. I have used C57BL/6 mice to study this interaction between prenatal alcohol exposure and the postnatal environment. Alcohol exposure during synaptogenesis produces high levels of anxiety-like traits and decreased memory performance. Alcohol-exposed mice (and matched unexposed controls) were put in 'environmentally-enriched' conditions of voluntary exercise, physical activities and cognitive stimulation to ascertain the effects of a positive postnatal environment. The results show that …

Genetic Engineering Studies Of Escherichia Coli And Microalgae For Expression Of Hydrolytic Enzymes And Development Of High Throughput Screening Technique, Shreyas S. Yedahalli

Electronic Thesis and Dissertation Repository

The field of biochemical engineering has made substantial progress through major advances in genetic and metabolic engineering with applications in various sectors such as energy, food science, pharmaceuticals, etc. The hosts used for this work are constantly broadening. A host particularly important for energy applications are microalgae. The potential to enhance microalgae genetically for energy applications is not well explored and was therefore investigated in this thesis. Non-photosynthetic micro-organisms and photosynthetic microalgae offer a potential approach to enhance sustainable biochemical production. In this study expression vectors for Escherichia coli (E. coli) and Chlorella vulgaris (C. vulgaris) …

Structural And Functional Characterization Of Non-Homologous End Joining Factors, Huasheng Wang

Electronic Thesis and Dissertation Repository

DNA double strand breaks represent the most toxic form of DNA damage. In mammals, non-homologous end-joining (NHEJ) is the primary DNA repair pathway for such damage, preventing both carcinogenesis and accelerated aging. Structural understanding of this repair pathway has received considerable attention, but has been significantly limited by the inability to obtain structures of higher order nucleoprotein complexes. A main obstacle in this respect has been difficulty in obtaining highly purified proteins, sufficient for structural determination. Improved protein expression and purification methods developed in this thesis permitted several NHEJ complexes to be selected for structural studies. Among these, Ku70-DNA and …

Expression, Characterization And Metallation Studies Of Human Metallothionein Isoform 2a Using Electrospray Ionization Mass Spectrometry, Devika P. Jayawardena

Electronic Thesis and Dissertation Repository

Maintenance of the homeostasis of zinc (Zn) is very important in regulating bodily functions. There are over 300 Zn dependent enzymes identified, where Zn plays a structural or catalytic role. However, excess of Zn in a cell is toxic and free Zn ions are tightly controlled. Metallothioneins (MTs) are small cysteine rich proteins, which can bind up to seven Zn ions and act as a Zn reservoir. The MT2a isoform is predominantly found in the liver. My research focused on the overexpression of human MT2a in Escherichia coli and the investigation of Zn binding pathways of MT2a in vitro. …

Gmmyb176 Interactome And Regulation Of Isoflavonoid Biosynthesis In Soybean, Arun Kumaran Anguraj Vadivel

Electronic Thesis and Dissertation Repository

MYB transcription factors are one of the largest transcription factor families characterized in plants. They are classified into four types: R1 MYB, R2R3 MYB, R3 MYB and R4 MYB. GmMYB176 is an R1MYB transcription factor that regulates Chalcone synthase (CHS8) gene expression and isoflavonoid biosynthesis in soybean. Silencing of GmMYB176 suppressed the expression of the GmCHS8 gene and reduced the accumulation of isoflavonoids in soybean hairy roots. However, overexpression of GmMYB176 does not alter either GmCHS8 gene expression or isoflavonoid levels suggesting that GmMYB176 alone is not sufficient for GmCHS8 gene regulation. I hypothesized that GmMYB176 acts cooperatively with another …

Pore-Lining Residues And Intracellular Magnesium Concentration Influence Connexin50 Gap Junction Unitary Channel Conductance, Mary Grace M. Tejada

Electronic Thesis and Dissertation Repository

Gap junction (GJ) channels mediate direct intercellular communication. Each GJ channel consists of two hemichannels and each hemichannel is a hexamer of connexins. GJs formed by different connexins display different unitary channel conductance (γ_j) and intracellular magnesium modulation. The underlying mechanisms are not fully clear. The present study investigates the effect of mutating putative pore-lining residues (G8, G46, and V53 individually or together) into glutamate in Cx50 on homotypic GJ channel properties. Expression of the triple and individual mutants in GJ-deficient N2A cells resulted in the formation of functional GJ channels similar to that of Cx50 GJs. However, …

Investigation Of The Interactions Between Elg1 And Pcna Involved In The Dna Damage Response, Jingwei Yan

Electronic Thesis and Dissertation Repository

Elg1, the major component of an alternative PCNA loader, is vital in maintaining chromosomal stability. PCNA is an essential coordinator for DNA replication and the damage response through protein-protein interactions. The N-terminus of Elg1 interacts with PCNA and preferentially SUMOylated PCNA (sPCNA) and contains five putative motifs for PCNA and SUMO binding. How these motifs contribute to PCNA interactions is unclear. In this study, we biochemically characterized the interactions between Elg1 and PCNA and found that i) two PCNA-interacting protein boxes (PIP boxes) participate in the interaction with PCNA, which may be interfered by the SUMO-interacting motifs (SIMs); ii) the …

Characterizing The Cofactor Specificity Of Nqo2, Shahed Al Massri

Electronic Thesis and Dissertation Repository

Quinone Reductase 2 (NQO2) is a part of an enzyme family implicated in detoxification of the cell. This enzyme differs from its highly similar sister protein Quinone Reductase 1 (NQO1) by its unique cofactor preference for dihydro-nicotinamide riboside (NRH) instead of NADH. Cellular levels of NRH have not been characterized, contributing to the mystery surrounding the physiological role of NQO2. This project focused on identifying what determines the cofactor specificity of NQO2, and engineering it to use NADH instead of NRH. The investigation included optimization of protein purification, an in silico analysis that revealed the different stereochemistry of NQO2/1 from …

Evolutionary Genetic Aspects Of Host Association In Generalist Ectoparasites, Benoit Talbot

Electronic Thesis and Dissertation Repository

Despite the use of the host for dispersal by most parasite species, the extremely loose relationship typical between highly mobile hosts and generalist ectoparasites may lead to very different gene flow patterns between the two, leading in turn to different spatial genetic structure, and potentially different demographic history. I examined how similar gene flow patterns are between Cimex adjunctus, a generalist ectoparasite of bats present throughout North America, and two of its key bat hosts. I first analyzed the continent-scale genetic structure and demographic history of C. adjunctus and compared it to that of two of its hosts, the …

The Cdk-Resistant Prb-E2f1 Complex Recruits Chromatin-Organizing Proteins To Repetitive Dna Sequences, Charles A. Ishak

Electronic Thesis and Dissertation Repository

This thesis investigates mechanistic links between genome integrity and the recruitment of chromatin organizing proteins to repetitive DNA sequences mediated by the retinoblastoma tumor suppressor protein (pRB). I demonstrate that a CDK-resistant interaction between the pRB C-terminus and the E2F1 coiled-coil marked box domain establishes a scaffold that facilitates recruitment of multiple chromatin-organizing proteins to repetitive sequences across the genome throughout the cell cycle. Specifically, pRB recruits the enhancer-of-zeste-homologue 2 (EZH2) histone methyltransferase to establish repressive facultative heterochromatin at repetitive sequences, and the Condensin II complex to ensure proper DNA replication and mitotic progression. To disrupt the CDK-resistant pRB-E2F1 interaction …

Metalation And Structural Properties Of Apo-Metallothioneins, Gordon W. Irvine

Electronic Thesis and Dissertation Repository

Metals are required by a quarter of all proteins to achieve their biological function, whether in an active site involved in catalytic chemistry or in a structural capacity. Metals are tightly regulated at the cellular level due to their propensity to cause unwanted side reactions and to be scavenged for use by pathogens. One of the proteins involved in this regulation of metal homeostasis is metallothionein (MT) which is a small, cysteine rich protein primarily involved in the regulation of zinc and copper homeostasis and heavy metal detoxification. MT is unique in its high cysteine content (~30% of the residues), …

Autoinhibition And Activation Of Parkin, Jacob D. Aguirre

Electronic Thesis and Dissertation Repository

Mutations in the gene encoding parkin, an E3-ubiquitin ligase, result in 50% of Autosomal Recessive Juvenile Parkinsonism cases. Parkin has been identified as a key mediator of mitochondrial regeneration following oxidative stress, and pathogenic mutations have been shown to impair its ubiquitin ligase activity. Neurodegeneration of dopamine-producing neurons appears to be a downstream consequence of parkin loss-of-function, resulting in early-onset forms of Parkinson’s disease. Although ubiquitination activity is essential for its neuroprotective function, parkin is autoinhibited in its native state by various mechanisms, including its N-terminal ubiquitin-like (UBL) domain. Therefore, the overarching objective of this thesis was to structurally characterize …

Investigating E2f Independent Cell Cycle Control And Tumor Suppression By Prb, Michael J. Thwaites

Electronic Thesis and Dissertation Repository

Cellular division is primarily controlled at the G1 to S-phase transition of the cell cycle by the retinoblastoma tumor-suppressor protein (pRB). The ability of pRB to restrict S-phase entry is primarily attributed to the repression of E2F transcription factors required to upregulate cell cycle target genes necessary for cellular division. Interestingly, while pRB is disrupted in the vast majority of human cancers, mutations typically target upstream regulators of pRB leading to inactivation through hyperphosphorylation. The rarity of direct pRB mutations suggests that the regulation of the cell cycle by pRB may involve additional mechanisms outside of E2F repression, as this …

Building Muscle: The Role Of Insulin-Like Growth Factor Binding Protein-6 And Insulin-Like Growth Factors In The Differentiation Of Placental Mesenchymal Stem Cells Into Skeletal Muscle, Doaa Aboalola

Electronic Thesis and Dissertation Repository

Human placenta is a readily available source for isolation of adult mesenchymal stem cell (MSC), for potential use in regenerative therapies. MSC fate is influenced by the microenvironment in vivo, and insulin-like growth factors (IGFs) are critical components of the stem cell niche, as they regulate proliferation and differentiation into different lineages including bone, fat, and skeletal muscle. Insulin-like growth factor binding protein-6 (IGFBP-6), relative to other IGFBPs, has high affinity for IGF2 and is believed to be the main modulator of IGF-2 function. However, the role of IGFBP-6 in muscle development has not been clearly defined. In this …

Assessing The Role Of Ku70 Vwa Domain Phosphorylation In The Inhibition Of Aurora B And Activation Of The Dna Damage Response, Elizabeth A. Walden

Electronic Thesis and Dissertation Repository

Ku is a key component of the Non-Homologous End Joining DNA repair pathway. Recently, a function for Ku in DNA damage response (DDR) signalling was identified through studies exploring a Ku70 S155D phosphomimetic mutant. We hypothesize that Ku70 S155D mimics phosphorylation of Ku70 in response to DNA damage, and that Ku S155 phosphorylation inhibits Aurora B and causes sustained DDR activation. In this study we show that the S155D mutant is competent for heterodimerization, and its expression does not induce DNA damage. Phosphorylated Ku70 associates with Aurora B by co-immunoprecipitation and this association was demonstrated in situ with Ku70 S155D. …

The Effects Of Acetylenic Tricyclic Bis-(Cyano Enone) On Cell Migration, Eddie Chan

Electronic Thesis and Dissertation Repository

Although cancer survival rates have significantly improved over the past few decades, the improvements are primarily due to early diagnosis and inhibiting cancer growth. Limited progress has been made in the treatment of cancer metastasis, which contributes to 90% of cancer related deaths, and therapeutic agents targeting the various aspects of metastasis are lacking. One potential approach is to utilize small pharmacological compounds to inhibit tumour cell motility, as a strategy against tumour cell migration, invasion, and metastasis. The acetylenic tricyclic bis-(cyano enone), TBE-31, has been shown to be a promising chemopreventative compound. However, its effects on cell migration are …

Cytosolic Acetyl-Coa Promotes Histone H3 Lysine 27 Acetylation In Arabidopsis, Chen Chen

Electronic Thesis and Dissertation Repository

Acetyl-coenzyme A (acetyl-CoA) serves as a central metabolite in energy metabolism and biosynthesis. High level of acetyl-CoA can fuel the tricarboxylic acid (TCA) cycle to generate energy and store excess energy in fatty acids. Meanwhile, it also provides acetyl groups for protein acetylation, which normally occurs at the lysine or arginine residues. Acetylation regulates protein functions largely due to the change of total charges. Acetylation of histones, for example, can lead to loss of the interaction between histone and DNA, thus relaxing chromatin structure and potentially promoting gene expression. However, whether and how acetyl-CoA regulates plant chromatin remains unexplored. Here, …

Quantitative Proteomic Characterization Of Cx-4945, A Clinical Stage Inhibitor Of Protein Kinase Ck2, Adam J. Rabalski

Electronic Thesis and Dissertation Repository

Protein phosphorylation is controlled by protein kinases, and represents a critical signaling mechanism involved in the regulation of fundamental biological processes. Furthermore, the aberrant regulation of kinase activity is implicated in diseases such as cancer and has resulted in efforts to target kinases therapeutically. Protein kinase CK2, although frequently considered constitutively active, has emerged as a clinical target on the basis of its altered expression in different types of human cancers and its regulatory participation in multiple biological processes. In fact, CX-4945, a small molecule ATP-competitive inhibitor of CK2 has advanced to clinical trial and has been widely used to …

First-Order Statistical Speckle Models Improve Robustness And Reproducibility Of Contrast-Enhanced Ultrasound Perfusion Estimates, Matthew R. Lowerison

Electronic Thesis and Dissertation Repository

Contrast-enhanced ultrasound (CEUS) permits the quantification and monitoring of adaptive tumor responses in the face of anti-angiogenic treatment, with the goal of informing targeted therapy. However, conventional CEUS image analysis relies on mean signal intensity as an estimate of tracer concentration in indicator-dilution modeling. This discounts additional information that may be available from the first-order speckle statistics in a CEUS image. Heterogeneous vascular networks, typical of tumor-induced angiogenesis, lead to heterogeneous contrast enhancement of the imaged tumor cross-section.

To address this, a linear (B-mode) processing approach was developed to quantify the change in the first-order speckle statistics of B-mode cine …

Dual-Active Genome-Editing Reagents, Jason M. Wolfs

Electronic Thesis and Dissertation Repository

Manipulation of complex genomes has many beneficial downstream applications in agriculture and human gene therapy. Precise genome-editing requires the introduction of a specific DNA double-stand break at a locus of interest, in turn inducing host DNA repair pathways to cause gene knockout through non-homologous end-joining or gene repair using homologous recombination and donor template. No matter the application, the field has depended on a few reagents to introduce precise double-strand breaks in host genomes. LAGLIDADG homing endonucleases or meganucleases harness the natural properties of these rare-cutting enzymes to target precise sequences in a complex genome. Other successful reagents are derived …