Biochemistry Commons^™

Developing Regulated Crispr Systems To Control Bacterial Microbiomes, Gregory M. Pellegrino

Electronic Thesis and Dissertation Repository

Changes to the human microbiome’s composition and metabolome are associated with numerous diseases and alterations to xenobiotic metabolism. As such, targeting the human microbiome is an increasingly popular option for therapeutic interventions. However, traditional therapies that target the microbiome such as antibiotics lack specificity, which can affect the beneficial species of the microbiome and cause adverse health outcomes such as the rise of antimicrobial-resistant bacteria. Therefore, the research and development of specific, targeted antimicrobial therapies is crucial to effectively treating microbiome dysbioses.
CRISPR and CRISPRi provide easily modifiable, RNA-guided mechanisms mediated by the Cas9 or dCas9 enzymes to induce sequence-specific …

Acetylation Regulates Thioredoxin Reductase Activity And Oligomerization, David E. Wright

Electronic Thesis and Dissertation Repository

The Thioredoxin (Trx) system provides the cell with robust defense against oxidative stress and regulates the function of nearly every cellular process through the reduction-oxidation (redox) regulation of proteins. The Trx system is involved in the development of many diseases ranging from cancer to cardiovascular disorders. Thioredoxin reductase (TrxR) is the key enzyme in the Trx system and contains the 21^st genetically encoded amino acid, selenocysteine (Sec). There were multiple experimentally identified TrxR acetylation sites with an unknown effect on TrxR activity. My thesis tested the hypothesis that programmed protein acetylation will enhance the activity of TrxR1. I used …

Short-Term Versus Long-Term Effects Of Nitrogen Addition And Warming On Ecosystem N Dynamics In A Grass-Dominated Temperate Old Field, Benjamin F. A. Souriol

Electronic Thesis and Dissertation Repository

Increased atmospheric nitrogen (N) deposition and climate warming are both anticipated to influence the ecosystem N dynamics of northern temperate ecosystems substantially over the next century. Nevertheless, in field experiments with N addition and warming treatments, temporal scale can play an important role in determining the extent of treatment effects on N dynamics, and it is unclear to what extent the results of short-term studies can be extrapolated to responses over longer time scales. I compared the short-term versus long-term effects of N addition and warming on net N mineralization, N leaching, and N retention in a grass-dominated old field. …

Characterization Of Dna Regulatory Elements Of The Highly Abundant Secreted Protein 1 (Hasp1) Promoter And Analysis Of Transcriptomic Responses Under Phosphate Depletion In The Diatom Phaeodactylum Tricornutum, Mahsa Farmanbar

Electronic Thesis and Dissertation Repository

Phosphorus (P) is essential for all life. The bioavailability of phosphorus in oceans impacts diatoms like Phaeodactylum tricornutum (P. tricornutum). P. tricornutum is extensively used in transcriptomic studies to understand pathways involved in P-acquisition. However, activation mechanisms and roles of regulatory elements in P-acquisition responses remains unclear. Here, I deleted predicted phosphate regulatory sites in P.tricornutum’s HASP1 promoter to create different HASP1-eGFP constructs. Under P-depletion, two constructs showed increased eGFP secretion. Additionally, a HASP1 knockout strain was grown under different phosphorus sources to determine if HASP1 is a phytase. Cells grown in full phosphorus utilized organic …

Protein Stability In Solution And In The Gas Phase., Yousef Haidar

Electronic Thesis and Dissertation Repository

Electrospray Ionization mass spectrometry (ESI-MS) is widely used for probing proteins, yet many aspects of this technique remain elusive. Using MS, ion mobility spectrometry (IMS), and circular dichroism (CD) spectroscopy, this thesis sheds light on the stability differences of proteins in the gas phase and solution. After a general introduction (Chapter 1), Chapter 2 scrutinizes some aspects of native ESI. Our data highlight the significance of cone voltage in maintaining a native-like fold and show the advantage of using NH₄Ac in protein experiments. Chapter 3 focuses on hydrogen/deuterium exchange (HDX)-MS. Several studies have reported that D₂O …

State-Of-The-Art Approaches For Sequencing, Assembling And Annotating Naphthenic Acid Degrading Bacterial Metagenomes, Henry H. Say

Electronic Thesis and Dissertation Repository

Naphthenic acids (NAs) are the main toxic component of oil refinery wastewater and require special processes to be removed. Harnessing bacterial biodegradation for NA removal has the potential to be effective, yet NA-degrading bacteria and pathways are poorly understood and uncharacterized. To improve our understanding of NA degradation, I characterize the metagenomes of novel NA-degrading bacterial communities seeded in NA-enriched granulated activated carbon (GAC) filters. I demonstrate methods that maximize the throughput of extraction, sequencing, and annotation of novel metagenomes - producing 72 MAGs and other 5432 circular contigs - 226 of which were putative phages. I also include state-of-the-art …

The Effects Of Resistance Exercise Training On Insulin Resistance Development In Female Rodents With Type 1 Diabetes, Mitchell James Sammut

Electronic Thesis and Dissertation Repository

The etiology of insulin resistance (IR) development in type 1 diabetes mellitus (T1DM) remains unclear; however, impaired skeletal muscle metabolism may play a role. While IR development has been established in male T1DM rodents, female rodents have yet to be examined in this context. Resistance exercise training (RT) has been shown to improve IR and is associated with a lower risk of hypoglycemia onset in T1DM compared to aerobic exercise. Additionally, the molecular mechanisms mediating RT-induced improvements in insulin sensitivity remain unclear. Therefore, the purpose of this study was to investigate the effects of RT on IR development in female …

Selective Activation Of Thrombin Activatable Fibrinolysis Inhibitor (Tafi) Attenuates Metastatic And Angiogenic Capabilities Of Melanoma And Lung Carcinoma In Vitro, Jacklyn Krizsan

Electronic Thesis and Dissertation Repository

Metastasis and angiogenesis are hallmarks of aggressive cancers, both depending on degradation of extracellular matrix by proteases such as plasmin. Plasmin activation is inhibited by thrombin-activatable fibrinolysis inhibitor (TAFI)-mediated cleavage of terminal lysine residues on plasminogen receptors. Activation of TAFI is most effectively done in complex with thrombomodulin (TM). TM is known to have anti-cancer properties, but it is not known if this is due to TAFI activation or an alternative substrate protein C (PC). We hypothesize that specific promotion of TAFI activation with TM treatment will attenuate metastatic and angiogenic capabilities of tumour cells.

Melanoma and lung carcinoma cells …

The Inner Workings Of Thrombin-Activatable Fibrinolysis Inhibitor: A Study Of The Tm-Mediated Activation Of Tafi And Inactivation Of Tafia, Haley D N Marier

Electronic Thesis and Dissertation Repository

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a key regulator of hemostasis and inflammation. TAFI is activated by thrombin proteolysis, most effectively when in complex with cofactor thrombomodulin (TM). Soluble TM, encompassing EGF-like domains 3 through 6 is sufficient to promote TAFI activation, though the role of EGF 4 has not been assessed. Through rearrangement of the soluble TM domains, we determined that EGF 4 was acting as a spacer to promote TAFI activation. Activated TAFI (TAFIa) is a metastable enzyme, with a half-life of only 8-15 min at 37^oC. The mobile loop of TAFIa was expected to modulate stability; …

Advances In Phaeodactylum Tricornutum Nuclear Engineering, Mark Pampuch

Electronic Thesis and Dissertation Repository

The marine diatom Phaeodactylum tricornutum has the potential to become an excellent platform for the sustainable production of valuable compounds and pharmaceuticals, but currently large-scale engineering of this organism remains a challenge due factors like inefficient genetic transformation protocols and a lack of accurate genomic data. This thesis addresses these two bottlenecks by (i) optimizing an electroporation protocol to P. tricornutum and (ii) remapping genomic data from a scaffolded genome assembly to a telomere-to-telomere genome assembly. An optimized transformation protocol was developed that could consistently transform blunt-ended and DNA with overhangs and yielded up to 1000+ colony forming units per …

Elucidating The Biomechanics Of Mertk-Mediated Efferocytosis, Brandon Hayato Dickson

Electronic Thesis and Dissertation Repository

Macrophages are key mediators of efferocytosis – the phagocytic engulfment and removal of apoptotic cells. During engulfment, the coordinated activity of efferocytic receptors induces the remodeling of the actin cytoskeleton, which facilitates the envelopment of the cell by the plasma membrane. Mer receptor tyrosine kinase (MERTK) is a crucial efferocytic receptor, but its role during actin remodeling is not well understood. Previously, our lab showed that MERTK is an activator of β₂ integrins – which are comprised of receptors known to induce the actin polymerization that is required for engulfment. We hypothesized that MERTK is an indirect stimulator of …

Functionalizing Conjugative Systems To Deliver Crispr Nucleases For Targeted Bacterial Killing, Thomas A. Hamilton

Electronic Thesis and Dissertation Repository

The interactions between humans and microbes are intimately important to human health, with both commensal and pathogenic bacteria affecting homeostasis and disease. Increasing concern over antibiotic resistance in bacterial pathogens represents a significant threat to human health, and use of traditional antibiotics to treat infections can be detrimental to commensal bacteria as well as pathogens, demonstrating a need for more specific antibacterial reagents. RNA-guided CRISPR nucleases, which can target and cleave genomes of interest, are a potential tool for specific bacterial targeting. A key limitation to the use of CRISPR antimicrobials is effective and robust delivery to the target bacteria. …

The Synthesis And Characterization Studies Of Modified Nucleobase In Pna And Dna, Gyeongsu Park

Electronic Thesis and Dissertation Repository

Nucleic acids have been extensively studied not only for their importance in biological systems as the medium of genetic information but also for their potential uses in therapeutic, diagnostic and other biological applications. As such, modified oligonucleotides and oligonucleotide analogues have drawn the attention of researchers from various disciplines. Modification of oligonucleotides can enhance their desired characteristics and engender unique properties, such as fluorescence, giving rise to a variety of applications. Peptide nucleic acid (PNA) is an oligonucleotide mimic with a pseudo-peptide backbone based on N-(2-aminoethyl)glycine that is renowned for high target binding affinity and resistance to enzyme degradation. …

Conformational Dynamics And Aggregation Of Thermally Stressed Proteins Studied By Hydrogen/Deuterium Exchange Mass Spectrometry, Nastaran Nosrat Tajoddin

Electronic Thesis and Dissertation Repository

Proteins perform various biological functions, e.g., as enzymes or transporters. In addition to naturally occurring proteins, the use of protein therapeutic drugs for treating cancer and other diseases is a rapidly growing area. A thorough biophysical characterization of proteins and protein therapeutics opens the door to a more comprehensive understanding of their role in health and disease. This dissertation aims to expand the capabilities of an existing technique (Hydrogen Deuterium Exchange Mass Spectrometry, HDX-MS), which is widely used for probing protein structure and dynamics. Conventionally, HDX-MS experiments are performed as a function of labelling time. Here we aim to establish …

Rhamm As A Biomarker And Therapeutic Target In Triple-Negative Breast Cancer, Britney Messam

Electronic Thesis and Dissertation Repository

Triple-negative breast cancer (TNBC) is a heterogeneous group of tumours characterized by early metastases and poor prognosis. Discovering novel biomarkers and therapeutic targets is necessary to improve TNBC patient outcomes as resistance to chemotherapy, the main therapeutic approach for TNBC, is common. In my study, RHAMM promoted proliferation of TNBC MDA-MB-231 tumour cells. RHAMM expression increased sensitivity to doxorubicin (p=0.0002) and strongly increased sensitivity to the FDA-approved MEK1/2 inhibitor trametinib (p≤0.0001). Doxorubicin and trametinib selectively killed RHAMM^+/+ MDA-MB-231 tumour cells grown as co-cultures with RHAMM^-/- MDA-MB-231 tumour cells. RHAMM-loss or trametinib decreased phosphorylated ERK1/2 protein levels and …

Dual Functions Of Interstrand Crosslink Repair Nuclease Snm1a, Ryan Grainger

Electronic Thesis and Dissertation Repository

Interstrand crosslinks (ICL) are a highly cytotoxic form of DNA damage, covalently linking opposing strands of DNA. ICLs disrupt essential cellular processes requiring strand separation, including transcription and replication. Consequently, lesion recognition and removal are critical to prevent chromosomal aberrations, mitotic catastrophe and apoptosis. ICL repair requires the coordination of a complex network of nucleases necessary for remodelling, unhooking and resolving repair intermediates. While many nucleases participate, little is known about where and when each nuclease acts. SNM1A is a dual-function exonuclease and endonuclease necessary for ICL repair. Where SNM1A is absent, cells accumulate irreparable double-strand breaks and exhibit reduced …

Perseverance Of Protein Homeostasis Despite Mistranslation, Farah Hasan

Electronic Thesis and Dissertation Repository

Transfer RNAs (tRNAs) are essential for protein synthesis and translation fidelity. Some human tRNA variants may cause amino acid misincorporation: tRNA^Gly variants (tRNA^Gly_CCC, tRNA^Gly_GCC) have mutations that generate an alanine tRNA identity element (G3:U70), likely causing mis-aminoacylation of glycine tRNAs with alanine, while the tRNA^Ala_AGC G35C (tRNA^Ala_ACC) variant may function similarly to mis-incorporate Ala at Gly codons by generating a Gly anticodon. I propose that these mistranslating tRNAs will disrupt protein homeostasis in mammalian cells. Although the tRNA^Gly and tRNA^Ala variants did not affect protein synthesis …

Towards More Complete Metagenomic Analyses Through Circularized Genomes And Conjugative Elements, Benjamin R. Joris

Electronic Thesis and Dissertation Repository

Advancements in sequencing technologies have revolutionized biological sciences and led to the emergence of a number of fields of research. One such field of research is metagenomics, which is the study of the genomic content of complex communities of bacteria. The goal of this thesis was to contribute computational methodology that can maximize the data generated in these studies and to apply these protocols human and environmental metagenomic samples.

Standard metagenomic analyses include a step for binning of assembled contigs, which has previously been shown to exclude mobile genetic elements, and I demonstrated that this phenomenon extends to all conjugative …

Global Regulation Of Mirna And Mrna Homeostasis By Terminal Uridylyltransferases Tent3a/B, Pengcheng Zhang

Electronic Thesis and Dissertation Repository

The terminal nucleotidyltransferases TENT3A and TENT3B (TENT3A/B) regulate microRNA (miRNA) and messenger RNA (mRNA) stability by 3' end uridylation, leading to degradation by the U-specific exonuclease DIS3L2. Uridylation dependent decay is thought to be a highly specific process, and the prevalence and impact of this pathway on the transcriptome is unexplored. I investigate the prevalence of uridylation dependent decay by transcriptionally profiling HEK 293T cells lacking TENT3A/B. I found while TENT3A/B target a variety of miRNAs, the let-7 family of miRNAs were the most impacted (increased) in TENT3A/B deleted cells. Consequently, let-7 mRNA targets are decreased in abundance in TENT3A/B …

The Effect Of Breathing Patterns Common To Competitive Swimming On Gas Exchange And Muscle Deoxygenation During Heavy-Intensity Fartlek Exercise, Kevin J. Grossman

Electronic Thesis and Dissertation Repository

The purpose of this study was to compare the respiratory and muscle deoxygenation (HHb) responses of regulated breathing versus free-breathing, during continuous exercise (CONLD) and intermittent 5s breath holds (BH) (CONLD-BH), intermittent 5s sprint (FLK) and combined 5s BH and sprint (FLK-BH) followed by 25s of free-breathing. Oxygen uptake(V̇O₂)was unchanged between CONLD (2.12±0.35L/min) and CONLD-BH (2.15±0.42L/min; p=0.116), and FLK (2.24±0.40L/min) and FLK-BH (2.20±0.45L/min; p=0.861). Δ[Hb_tot]: CONLD (3.3±1.6µM) > CONLD-BH (-2.5±1.2µM; ∆177%; p<0.001), but unchanged between FLK (2.0±1.6µM) and FLK-BH (0.82±1.4µM; p=0.979). Δ[HHb]: CONLD (7.3±1.8µM) > CONLD-BH (7.0±2.0µM; ∆4%; p=0.011), and FLK (6.7±1.8µM) < FLK-BH (8.7±2.4µM; p<0.001). It is suggested that the unchanged V̇O₂ between CONLD and CONLD-BH was supported by increased deoxygenation, reflected by decreased ∆[Hb_tot] and blunted …

Biochemistry Of Trehalose Accumulation In The Spring Field Cricket, Gryllus Veletis, Alyssa R. Stephens

Electronic Thesis and Dissertation Repository

The freeze tolerant spring field cricket, Gryllus veletis, accumulates trehalose in the blood and tissues during cold acclimation. Trehalose is the main blood sugar in insects, thus its blood concentration is tightly regulated, and trehalose is readily metabolized. How do crickets modify their metabolism to accumulate trehalose in their hemolymph and tissues? I hypothesized that trehalose production, transport, and consumption were modified during the cold acclimation to facilitate trehalose accumulation. Trehalose and the trehalose-specific transporter, TRET-1, are distributed among all tissues, and trehalose accumulates in the hemolymph, fat body, Malpighian tubules, and gut. Trehalose production increases during cold acclimation …

Kinetic And Structural Influences Of Acetylation On Ubiquitin Processing, Rachel E. Lacoursiere

Electronic Thesis and Dissertation Repository

Ubiquitin (Ub) is a small modifying protein abundant in cells where it serves numerous regulatory roles including immune signaling, transcriptional regulation, and proteostasis. To exert its function, Ub covalently interacts with a series of E1, E2, and E3 enzymes before final substrate modification. Dysregulation of Ub signaling has implications in human maladies such as cancer, autoimmune disorders, and neurodegenerative diseases. In these diseases and associated in cellulo models, modifications to Ub serve an additional role in Ub regulation. Post-translational modifications like acetylation or phosphorylation modulate protein-protein interactions and Ub signaling. To understand how acetylation of Ub alters the central E2 …

Illuminating Transfer Rna Variants As Genetic Modifiers In Models Of Human Disease, Jeremy T. Lant

Electronic Thesis and Dissertation Repository

Transfer RNAs (tRNAs) physically link the genetic code to an amino acid sequence, by recruiting amino acids to three-nucleotide codons in messenger RNAs. To ensure that the genetic code is translated as intended, tRNAs must be accurately aminoacylated and faithfully recognize codons in the ribosome during protein synthesis. Given the critical function of tRNAs, it has often been assumed that mutations in human tRNA genes would be either lethal to cells or not significantly impair tRNA function. My goal was to rigorously test this assumption in mammalian cell models, prompted by the recent discovery of unprecedented variation in human tRNA …

Investigating The Role Of Pkc And Its Mechanisms In Regulation Of Igf-I Bioavailability In Fetal Growth Restriction, Allan W. Chen

Electronic Thesis and Dissertation Repository

Fetal growth restriction (FGR) is associated with decreased nutrient availability and reduced insulin-like growth factor (IGF)-I bioavailability via increased IGF binding protein (IGFBP)-1 phosphorylation. While protein kinase C (PKC) is implicated in IGFBP-1 hyperphosphorylation in nutrient deprivation, the mechanisms remain unclear. I hypothesized that the interaction of PKCα with protein kinase CK2β and activation of PKCα under leucine deprivation (L0) mediate fetal hepatic IGFBP-1 hyperphosphorylation. Parallel Reaction Monitoring Mass Spectrometry (PRM-MS) followed by PKCα knockdown demonstrated the PKCα isoform interacts with IGFBP-1 and CK2β under L0. Pharmacological PKCα activation with phorbol 12-myristate 13-acetate (PMA) increased whereas inhibition with bisindolylmaleimide II …

The Study Of The Structure And Dynamics Of Parkin Activation, Elaine Aisha Freeman

Electronic Thesis and Dissertation Repository

Parkin is an RBR E3 ubiquitin ligase that has been implicated in both sporadic and familial Parkinson’s disease. Upon mitochondrial damage, parkin is activated step-wise to recruit and ligate ubiquitin to a substrate on the outer mitochondrial membrane. Disruption of this activation and ligation cascade is hypothesized to result in neuronal death related to Parkinson’s disease.

While structures of parkin for a number of these activation states exist, it is important to note they are not of full-length human parkin. These structures are often truncated and come from various non-human species to eliminate important, yet hard to quantify structural elements. …

Development Of 3d Bioartificial Human Tissue Models Of Periprosthetic Shoulder Joint Infection, Tony B. Huang

Electronic Thesis and Dissertation Repository

Periprosthetic joint infection (PJI) is a devastating and costly post-surgical complication that is not well understood due to the scarcity of physiologically representative experimental models. This thesis outlines the development of two 3D bioartificial human tissue models designed to study the cellular and biochemical interactions between primary fibroblasts from the shoulder capsule (SC) and infectious microorganisms. Using the Fibroblast-Bacteria Co-culture in 3D Collagen model, we demonstrated a global gene repression of metabolic and homeostatic processes in SC fibroblasts following 48 hours of co-culture with Cutibacterium acnes – the most common microbial cause of PJI in the shoulder. These cellular changes …

The Regulation Of Pannexin1 And Pannexin2 In The Skin In Health And Disease, Rafael E. Sanchez Pupo

Electronic Thesis and Dissertation Repository

Pannexins (PANX1, 2, 3) are a family of channel-forming glycoproteins that mediate intracellular and paracrine signaling. In contrast to PANX2, PANX1 has been extensively investigated in the skin, modulating cell differentiation, wound healing, and melanoma development. PANX1 and PANX2 can co-exist in the same cell and form mixed channels where their glycosylation seems to regulate their intermixing. N-glycosylation and caspase cleavage have been proposed as modulators of the function of PANX1, but their effects on PANX2 are unknown. We explored the PANX2 expression in mouse skin and showed that a Panx2 splice variant (PANX2-202) is continuously expressed throughout aging skin. …

Mapping Ku70 Protein Interactions Using Proximity-Dependent Biotin Identification, Sanna Abbasi

Electronic Thesis and Dissertation Repository

The Ku heterodimer, composed of subunits Ku70 and Ku80, is a highly abundant protein complex, known for its affinity for double-stranded DNA ends. Accordingly, Ku is most well-studied for repairing double-stranded DNA breaks through the non-homologous end-joining (NHEJ) DNA repair pathway. Aside from NHEJ, Ku has also been implicated and studied in various other cellular processes including V(D)J recombination and telomere maintenance.

Numerous protein interactions have been mapped to the C-terminal region of Ku70, although few have been mapped to its N-terminal von Willebrand A-like (vWA) domain. Here, we used the high-throughput proteomic technique, proximity-dependent biotin identification (BioID, BioID2) to …

New Perspectives On Phosphorylation State In The Parkin Ubiquitination Cascade, Karen Dunkerley

Electronic Thesis and Dissertation Repository

The RBR E3 ligase parkin is recruited to the outer mitochondrial membrane (OMM) during oxidative stress where it becomes activated and ubiquitinates numerous proteins. Parkin activation involves binding of a phosphorylated ubiquitin (pUb), followed by phosphorylation of parkin itself, both mediated by the OMM kinase, PINK1. However, targeted mitochondrial proteins have little structural or sequence similarity, with the commonality between substrates being proximity to the OMM. The objective of this thesis was to identify the molecular consequences of parkin phosphorylation, interaction with pUb and how this promotes ubiquitination activity of known Ub-acceptor proteins and parkin itself.

The three-dimensional structure of …

Characterization And Modulatory Influence Of Pyruvate Kinase Muscle Isoforms 1 And 2 Within The Murine Pluripotent Continuum, Joshua G. Dierolf

Electronic Thesis and Dissertation Repository

Mouse embryonic stem cells (mESCs) and mouse epiblast stem cells (mEpiSCs) represent opposite ends of a pluripotency continuum, respectively referred to as naïve and primed pluripotent states. A third, recently discovered intermediate state has been described as the ‘formative state’. Metabolism has been traditionally regarded as a by-product of cell fate; however, recent evidence now supports metabolism as promoting stem cell fate. Pyruvate kinase muscle isoforms 1 and 2 (PKM1 and PKM2) catalyze the final, rate limiting step of glycolysis generating adenosine triphosphate (ATP) and pyruvate; however, the precise role(s) of these isozymes in naïve, formative, and primed pluripotency is …