Molecular Biology Commons^™

A Pkcα-Mediated Growth Suppressive Mek-Erk Signaling Axis In Intestinal Epithelial Cells, Navneet Kaur

Theses & Dissertations

Members of the protein kinase C (PKC) family of serine/threonine kinases are involved in regulation of fundamental cellular functions, including proliferation, differentiation, survival, migration, and transformation. Increasing evidence points to anti-proliferative and tumor suppressive role of PKCs. Our laboratory and others have reported that the classical PKC isozyme, PKCαnegatively regulates proliferation and tumorigenesis in the intestinal epithelium. Our laboratory has further determined that PKCα signaling induces a program of cell cycle withdrawal in intestinal epithelial cells that involves downregulation of the pro-proliferative proteins, cyclin D1 and Id1, and upregulation of the cyclin dependent kinase (CDK) inhibitor, p21^Cip1. Unexpectedly, …

Molecular Mechanisms Of Aberrant Protein Glycosylation In Pancreatic Cancer Stemness And Metastasis, Frank Leon

Theses & Dissertations

A myriad of genetic and other abnormal changes underlies the aggressiveness and dissemination properties observed in pancreatic cancer (PC). Aberrant protein glycosylation is a commonly observed feature in PC. The modification of protein O-glycosylation is mediated by glycosyltransferases, which attach and sequentially elongate monosaccharides on Serine/Threonine (Ser/Thr) motifs. Aberrant glycosylation is recognized as an emerging hallmark of cancer where a disruption in normal glycosylation results in irregular O-glycans.

This dissertation research has investigated the consequences of aberrant protein glycosylation on stemness and enhancement of metastatic properties in pancreatic ductal adenocarcinoma (PDAC). Several publications have reported aberrant O-glycosylation increases in oncogenic …

Computationally Modeling Dynamic Biological Systems, Katherine Jarvis

Electronic Theses and Dissertations

Modeling biological systems furthers our understanding of dynamic relationships and helps us make predictions of the unknown properties of the system. The simple interplay between individual species in a dynamic environment over time can be modeled by equation-based modeling or agent- based modeling (ABM). Equation based modeling describes the change in species quantity using ordinary differential equations (ODE) and is dependent on the quantity of other species in the system as well as a predetermined rates of change. Unfortunately, this method of modeling does not model each individual agent in each species over time so individual dynamics are assumed to …

A Review Of Calcineurin Biophysics With Implications For Cardiac Physiology, Ryan B. Williams

Theses and Dissertations

Calmodulin is a prevalent calcium sensing protein found in all cells. Three genes exist for calmodulin and all three of these genes encode for the exact same protein sequence. Recently mutations in the amino acid sequence of calmodulin have been identified in living human patients. Thus far, patients harboring these mutations in the calmodulin sequence have only displayed an altered cardiac related phenotype. Calcineurin is involved in many key physiological processes and its activity is regulated by calcium and calmodulin. In order to assess whether or not calcineurin contributes to calmodulinopathy (a pathological state arising from dysfunctional calmodulin), a comprehensive …

Granulins In Norm And Neurodegenerative Pathologies, Anukool Bhopatkar

Dissertations

Granulins (GRNs) are small, cysteine-rich modules produced from the proteolytic cleavage of the precursor protein called progranulin (PGRN). GRNs are present in the form of seven tandem repeats within the precursor and are known to be produced in the extracellular and in lysosomal environments. In physiology, PGRN and GRNs plays pleiotropic roles such as neuronal growth and differentiation, immunomodulation, wound healing. Recent studies have implicated pathological role for PGRN in Alzheimer disease (AD) and frontotemporal dementia (FTD) but specific mechanism(s) remains unclear. However, potential interactions between GRNs and Ab42 and TDP-43 seem like a plausible underlying mechanism. Studies presented here …

Erecta Family Genes Regulate The Shoot Apical Meristem And Organ Formation, Daniel A. Degennaro

Doctoral Dissertations

Plants are sessile and must adjust their organ growth to their environments. A reservoir of stem cells in the shoot apical meristem (SAM) supplies cells for differentiation into organs. The SAM must balance organ production with stem cell maintenance. The ERECTA family (ERfs) encodes the leucine-rich repeat receptor-like kinases ERECTA (ER), ERECTA-LIKE 1 (ERL1), and ERL2. ERf signaling regulates organ initiation and stem cell maintenance. Results presented in this work include the following:

1) WUSCHEL (WUS) and CLAVATA3 (CLV3) make up a negative feedback loop to maintain SAM size. WUS and CLV3 expression localization is critical for …

Liposomes: Production And Applications For Controlled Drug Delivery, Gamid Abatchev

Boise State University Theses and Dissertations

This dissertation recognizes the enormous potential presented by the ever-evolving development of liposomes as drug carriers and seeks to offer further investigation into their useful production and utilization. The first chapter presents the basic principles governing their formation by self-assembly in water solutions, briefly describes the most common production methods, and points out essential past advances that led to their use as drug carriers. Chapter two exemplifies production of liposomes by the traditional methods of extrusion and sonication, detailing passive and active loading, as well as physical characterization by Dynamic Light Scattering, microscopy imaging, and fluorescence spectroscopy. In the next …

Site-Specific Effects Of Lysine Acetylation On Aminoacyl-Trna Synthetase, Hao Chen

Graduate Theses and Dissertations

Aminoacyl-tRNA synthetases (AARSs) are an ancient and highly conserved family of enzymes which can catalyze a two-steps aminoacylation reaction to charge tRNAs with their cognate amino acids, thus playing crucial roles in ribosomal protein synthesis. Naturally, the accurate amino acids and tRNA recognition of these synthetases are essential to the fidelity of translation process. To assure the correct recognition, some of these synthetases have evolved with an editing function to help remove the mischarged tRNAs. In addition to these functions, AARSs are also involved in various biological processes ranging from transcription to translation. Currently, a series of proteomic studies have …

Engineering Fluorescently Labeled Human Fibroblast Growth Factor One Mutants And Characterizing Their Photophysics Properties Towards Designing Fret Assays, Mamello Mohale

Graduate Theses and Dissertations

Human fibroblast growth factor one (hFGF1) belongs to a family of 22 FGF members produced by fibroblast cells. Cell signaling during physiological processes of angiogenesis and wound healing occurs when hFGF1 binds to its receptor (FGFR). However, when heterogenous homeostasis is not maintained, fibroblast cells exhibit excessive proliferation which can lead to a myriad of cancers. smFRET is an ultrasensitive distant dependent (1-10 nm) technique capable of resolving such heterogeneity in structural dynamics and binding affinities (Kd). Therefore, we successfully designed and characterized fluorescently labeled hFGF1 tracers which span the visible light region of the electromagnetic spectrum for use in …

Deciphering The Role Of Hsp110 Chaperones In Diseases Of Protein Misfolding, Unekwu M. Yakubu

Dissertations & Theses (Open Access)

Molecular chaperones maintain protein homeostasis (proteostasis) by ensuring the proper folding of polypeptides. Loss of proteostasis has been linked to the onset of numerous neurodegenerative disorders including Alzheimer’s, Parkinson’s, and Huntington’s disease. Hsp110 is a member of the Hsp70 class of molecular chaperones and acts as a nucleotide exchange factor (NEF) for Hsp70, the preeminent Hsp70-family protein folding chaperone. Hsp110 promotes rapid cycling of ADP for ATP, allowing Hsp70 to properly fold nascent or unfolded polypeptides in iterative cycles. In addition to its NEF activity, Hsp110 possesses an Hsp70-like substrate binding domain (SBD) whose biological roles are undefined. Previous work …

Mechanisms Of Stress Survival In Gram Positive Bacterial Pathogens, Asia Poudel

Chemistry & Biochemistry Theses & Dissertations

Anaerobic Gram-positive bacteria are not a well-characterized group but include many human pathogens that are resilient against stresses caused by the human immune system or by antibiotic treatment. This dissertation investigated the survival mechanisms of two clinically relevant Gram-positive organisms, Clostridioides difficile and Cutibacterium acnes under extracellular stresses. The response of the opportunistic skin pathogen Cutibacterium acnes to nanosecond electric pulses is characterized and found that growth in a biofilm, which usually protects bacteria from stress, renders this species more killable by this treatment. In addition, the stringent response (SR), a conserved bacterial stress survival mechanism, is studied in the …

Impact Of Pank1 Deletion On Mitochondrial Acetylation And Cardiac Function During Pressure Overload., Timothy N. Audam

Electronic Theses and Dissertations

Recent studies have associated elevated protein acetylation levels with heart failure in humans. Although mechanisms promoting elevated acetylation levels are not fully known, excess acetyl-CoA may drive enzyme-independent acetylation of cardiac proteins. Accumulation of acetyl-CoA depends on the availability of sufficient CoA, whose production is regulated by pantothenate kinases in the CoA biosynthetic pathway. We show that cardiac proteins are hyperacetylated during heart failure in humans and tested in mice whether limiting CoA abundance would improve ventricular remodeling during pressure overload-induced hypertrophy. We limited cardiac CoA levels by deleting the rate-limiting enzyme in CoA biosynthesis, Pank1 (one of three PANK-encoding …

Understanding How Camkii Holoenzyme Dynamics Facilities Activation-Triggered Subunit Exchange, Ana P. Torres-Ocampo

Doctoral Dissertations

Long-term memory and learning are still poorly understood from a molecular and cellular standpoint. Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is an oligomeric kinase that is involved in this remarkable process. However, the molecular details of its specific roles in these processes remains elusive. CaMKII activation-triggered subunit exchange presents a novel possible mechanism involved in long-term memory and learning by exchanging active subunits with other CaMKIIs. CaMKII subunit exchange also shows that exchanged CaMKIIs spread their phosphorylation state to newly synthesized CaMKIIs. This provides a long-lasting signal that might possibly be involved in long-term memory by escaping a cell’s …

Hsp70 Phosphorylation: A Case Study Of Serine Residues 385 And 400, Sashrika Saini

Masters Theses

Molecular chaperones play a key role in maintaining a healthy cellular proteome by performing protein quality control. Heat shock protein 70s (Hsp70s) are a diverse class of evolutionarily conserved chaperones that interact with short hydrophobic sequences presented in unfolded proteins, promoting productive folding, and preventing proteins from aggregation. Most of the extensive research on chaperone examines mechanism, substrate promiscuity, and engagement with many co-chaperones. Only recently were chaperones recognized to be frequent targets of post-translational modifications (PTMs). Despite the recent rise in PTMs identified, the impact of these modifications on chaperone function, whether singular or in concert with other modifications, …

Exploring Knockdown Phenotypes And Interactions Between Atad3 Proteins In Arabidopsis Thaliana, Eli S. Gordon

Masters Theses

Mitochondria are required for a diverse array of cellular functions and processes. ATAD3 (ATPase family AAA domain containing protein 3) proteins are newly discovered mitochondrial membrane proteins in Arabidopsis thaliana. Homologous to ATAD3A in metazoans, Co-Immunoprecipitation/Mass spectrometry and genomic analysis identified a four ATAD3A homologues in A. thaliana. The four A. thaliana proteins are referred to as ATAD3A1 (At3g03060), ATAD3A2 (At5g16930), ATAD3B1 (At2g18330), and ATAD3B2 (At4g36580). Studies in metazoans indicate that ATAD3A localizes to Mitochondria-ER contact sites and is involved in a variety of processes required for proper mitochondrial function, but ATAD3A proteins are poorly defined in plants. …

Dual Control Of One Component Signaling: Mechanistic And Structural Insights Into El222 Active States, Uthama Phani R. Edupuganti

Dissertations, Theses, and Capstone Projects

Photoreceptors play a crucial role in signal transduction as specialized proteins which sense light as environmental stimuli and transduce the signal to control of downstream functions. Here we focus our attention on one class of these proteins, the Light-Oxygen-Voltage (LOV) domain, which is sensitive to blue light via an internally-bound flavin chromophore. Since their initial discovery in plant phototropins, many details of their photochemistry, chromophore interactions, and use with a diverse set of functional effectors have been described. However, several key details, especially a comprehensive understanding of signaling mechanism and its regulation, still remain elusive due in part to the …

Oxydifficidin-Producing Bacillus Presents Novel Antimicrobial Activity Against Neisseria Gonorrhoeae Involving The Deda Protein, Jingbo Kan

Dissertations, Theses, and Capstone Projects

Bacterial human pathogens cause severe infectious diseases which are the second most common cause of death next to cancer and cardiovascular diseases in the world, especially in developing countries. Gonorrhea particularly, is the second most common sexually transmitted infection (STI) which is caused by the microorganism Neisseria gonorrhoeae (GC). Centers for Disease Control and Prevention (CDC) estimates that more than 1.6 million new gonorrhea cases emerged in USA in 2018 (“Detailed STD Facts - Gonorrhea” n.d.). Also, the WHO (World Health Organization) shows that gonorrhea is the most antibiotic resistant STI (“PAHO/WHO | Gonorrhea” n.d.), highlighting the shortage of efficient …

The Structural And Functional Role Of Photosensing In Rgs-Lov Proteins, Zaynab Jaber

Dissertations, Theses, and Capstone Projects

Light provides organisms with energy and spatiotemporal information. To survive and adapt, organisms have developed the ability to sense light to drive biochemical effects that underlie vision, entrainment of circadian rhythm, stress response, virulence, and many other important molecularly driven responses. Blue-light sensing Light-Oxygen-Voltage (LOV) domains are ubiquitous across multiple kingdoms of life and modulate various physiological events via diverse effector domains. Using a small molecule flavin chromophore, the LOV domain undergoes light-dependent structural changes leading to activation or repression of these catalytic and non-catalytic effectors. In silico analyses of high-throughput genomic sequencing data has led to the marked expansion …

Insights Into Halophilic Microbial Adaptation: Analysis Of Integrons And Associated Genomic Structures And Characterization Of A Nitrilase In Hypersaline Environments, Sarah Sonbol

Theses and Dissertations

Hypersaline environments are extreme habitats that can be exploited as biotechnological resources. Here, we characterized a nitrilase (NitraS-ATII) isolated from Atlantis II Deep brine pool. It showed higher thermal stability and heavy metal tolerance compared to a closely related nitrilase.

We also studied integrons in halophiles and hypersaline environments. Integrons are genetic platforms in which an integron integrase (IntI) mediates the excision and integration of gene cassettes at specific recombination sites. In order to search for integrons in halophiles and hypersaline metagenomes, we used a PCR-based approach, in addition to different bioinformatics tools, mainly IntegronFinder.

We found that integrons and …

Rna Polymerase Binding Protein A (Rbpa) Regulation Of Mycobacteria Transcription And Sensitivity To Fidaxomicin, Jerome Prusa

Arts & Sciences Electronic Theses and Dissertations

Mycobacterium tuberculosis is the causative agent of the disease tuberculosis (TB) and remains one of the deadliest microorganisms on the planet. The effort to eradicate M. tuberculosis would benefit from the development of novel therapeutics, which requires a detailed understanding of M. tuberculosis physiology. Like all living organisms, M. tuberculosis gene expression requires transcription. Transcription in the phylum Actinobacteria, which includes mycobacteria, is unique because it includes RNA Polymerase Binding Protein A (RbpA) that is essential in both M. tuberculosis and the nonpathogenic model organism Mycobacterium smegmatis. RbpA increases the housekeeping A and housekeeping like B interactions with the RNA …

Fibroblast Growth Factor Receptor 4 And R4-Icd Increased Proliferation, Cell Survival And Metastasis In Cholangiocarcinoma, Yamnah Hafeji

Theses & Dissertations

Fibroblast growth factor receptor 4 (FGFR4) is over-expressed in many cancers, including cholangiocarcinoma (CCA). FGFR4 is activated by fibroblast growth factor ligand 19 (FGF19) and plays a critical role in CCA progression. An intracellular cleaved product of FGFR4, referred as R4-ICD (FGFR4 intracellular domain) is also overexpressed in CCA. However, the specific role of R4-ICD in CCA is unknown. In this study, we hypothesized that FGFR4 and R4-ICD play a role in cell proliferation, cell survival and metastasis in CCA. To test this, FGFR4 and R4-ICD were cloned into a cholangiocarcinoma cell line (HuCCT-1) that does not endogenously express FGFR4. …

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. …

Modulation Of Glucose Homeostasis By Nucleotide P2y2 Receptor And Biological Sex, Hailee Anne Marino

MSU Graduate Theses

Recent insights into the pathological role of Nucleotide P2Y₂ receptor suggest P2Y₂R involvement in high fat diet-induced obesity and potentiates insulin resistance. However, these recent insights do not demonstrate how P2Y₂R modulates glucose homeostasis under physiological conditions. Further, it remains unknown how sex biological factors influence P2Y₂R receptor signaling in the regulation of glucose homeostasis. The research objective for the present study is to elucidate the novel roles of P2Y₂ in fasting blood glucose and glucose tolerance (basal insulin sensitivity) under resting conditions in males and females. We expected that under physiological …

Characterizing Multivalent Interactions Between Folded Protein Domains And Intrinsically Disordered Regions Or Peptide Substrates, Tongyin Zheng

Dissertations - ALL

Protein-protein interactions (PPIs) play central roles in most biological processes. Studying PPIs is a fundamental step in understanding the molecular basis of cellular processes such as cell-cell contact, enzyme activity, and transient assembly of signaling complexes or cellular structures. In this work, we employed a combination of biophysical and biochemical methods to characterize PPIs, with a focus on interactions between structured domains and intrinsically disordered regions or peptide substrates.The subject of Chapter two is prolyl isomerase Ess1, which is an essential enzyme found in Saccharomyces cerevisiae. Ess1 regulates the transcription and co-transcriptional RNA processing by catalyzing the isomerization of serine-proline …

Evaluating Bioenergetics And Mitochondrial Dynamics In Patient Fibroblasts With Pathogenic Mitochondrial Dna Mutations Causing Leigh Syndrome, Ajibola Bakare

Graduate Theses and Dissertations

Leigh syndrome (LS) is a rare fatal mitochondrial disorder of infants caused by pathogenic mutations in the nuclear (nDNA) or mitochondrial DNA (mtDNA) leading to mitochondrial dysfunction. The extent to which pathogenic mtDNA variants regulate disease severity in LS is not well understood. The heterogeneous nature of this disorder, based in part by complex mitochondrial genetics, and the nuclear and mitochondrial cross-talk has made it particularly challenging to investigate and develop therapies for treating LS . While the prognosis is poor, several studies are underway to understand the pathophysiology of LS. This dissertation provides a comprehensive structural and functional analysis …

Novel Approaches Towards Improved Purity In High Yield Transcription Reactions, Elvan Cavac

Doctoral Dissertations

High yields of RNA (e.g., mRNA, gRNA, lncRNA) are routinely prepared following a two-step approach: high yield in vitro transcription using T7 RNA polymerase, followed by extensive purification using gel or chromatic methods. In high yield transcription reactions, as RNA accumulates in solution, T7 RNA polymerase rebinds and extends the encoded RNA (using the RNA as a template), resulting in a product pool contaminated with longer than desired, (partially) double stranded impurities. Current purification methods often fail to fully eliminate these impurities which, if present in therapeutics, can stimulate the innate immune response with potentially fatal consequences. This study establishes …

The Role Of Native Lens Α-Crystallin In Amyloid Suppression Using Β-Amyloid As A Model Amyloid Client, Leilani Lopes

University Honors Theses

Cataracts are a result of age-related protein aggregate formation in the eye lens, and the leading cause of blindness worldwide. α-crystallin, a small heat shock protein, acts a molecular chaperone that serves as the primary defense mechanism against protein aggregate formation in the lens. Recent evidence suggest amyloid formation in the lens may contribute to cataract formation, and that β-amyloid is present in lens epithelia of age-related cataracts. Previous studies have shown that αB-crystallin in other parts of the body, such as the brain, is protective against neurodegenerative diseases such as Alzheimer’s disease that are associated with plaque deposits containing …

Molecular Dynamics Simulations Provide Insight Into Stability Of Hyperthermophilic Endoglucanases, Logan E. Sheffield

Electronic Theses and Dissertations

Endoglucanases play a key role in the industrial production of bioethanol, but the most efficient method requires the utilization of high temperatures and is currently limited by the thermostability of endoglucanases. For this reason, it would be beneficial to discover more high-efficiency, thermostable enzymes to utilize in the hydrolytic process. In this study molecular dynamics simulations were performed on structurally similar endoglucanases with varying levels of thermostability to gain insight on what factors contribute to thermostability in endoglucanases. RMSD, RMSF, PCA, hydrogen bonding and salt bridges were analyzed. Finally, protein energy networks were constructed from nonbonded interaction potentials and analysis …

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 …

Determining The Role Of Methylglyoxal (Mgo) And The Trpa1 Channel In Inducing Astrocyte Senescence And Neurodegeneration, Natalie Hill

Natural Sciences and Mathematics | Biological Sciences Master's Theses

Aging is the largest risk factor for the development of Alzheimer’s disease (AD) and related dementias. A recently proposed driver of age-related pathologies is cellular senescence, a phenotype that consists of cell-cycle arrest and an inflammatory response known as the senescence-associated secretory phenotype (SASP). Although there is a link between the accumulation of senescent cells and neurodegeneration, much remains unknown about how senescent cells arise in the brain. Astrocytes are the most abundant cell type in the brain that serve important roles like supporting neurons and proliferating in response to stress. Methylglyoxal (MGO) is a glycolytic byproduct that can react …