Biochemistry, Biophysics, and Structural Biology Commons^™

Conditional Cardiac-Specific Akap13 Knockout Induces Sex Dependent Biventricular Dilated Cardiomyopathy With Sarcomeric And Mitochondrial Defects, Kimberlyn M. Baig-Ward

Theses and Dissertations

Heart disease is a complex and heterogeneous disease. Notably, studies have demonstrated gender differences in the expression and types of cardiovascular disease, such as dilated cardiomyopathy (DCM), a major underlying cause of heart failure. Previously we showed that loss of A-Kinase Anchoring Protein 13 (Akap13), a unique proto-oncogene and estrogen receptor modulator, resulted in enlarged embryonic hearts, defective cardiac sarcomere formation, and embryonic lethality in mice. Data have also shown cAMP-dependent Protein Kinase A (PKA) to be involved in DCM pathophysiology. Given the established role of AKAP13 in cell signaling, its ability to bind and modulate ligand-activated nuclear hormone …

Role Of Vav2 In Podocyte Inflammasome Activation And Glomerular Injury During Hyperhomocysteinemia, Sabena Conley

Theses and Dissertations

Hyperhomocysteinemia (hHcys) is a widely known pathogenic factor in the progression of end-stage renal disease (ESRD) and it is also associated with an increased risk for injurious cardiovascular pathologies during ESRD. HHcys is linked to the formation and activation of the NOD-like receptor protein 3 (NLRP3) inflammasome, characterized as a critical early mechanism initiating the inflammatory response. NADPH oxidase (NOX)-derived reactive oxygen species (ROS) mediate the activation of the NLRP3 inflammasome in podocytes in response to elevated levels of homocysteine (Hcys) in vitro and in vivo. However, it remains unknown how NLRP3 inflammasome activation is triggered by NOX. The …

Atm Phosphorylates Subunit A Of Pp2a Resulting In Its Nuclear Export And Spatiotemporal Regulation Of The Dna Damage Response, Amrita D. Sule

Theses and Dissertations

Ataxia telangiectasia mutated (ATM) is a serine-threonine protein kinase and major regulator of the DNA damage response (DDR). One critical ATM target is protein phosphatase 2A (PP2A) known to regulate diverse cellular processes such as mitosis and cell growth as well as dephosphorylation of many proteins during the recovery from the DDR while returning the cell to normalcy. Interestingly, ATM and PP2A are known to form an auto-regulatory yin-yang kinase-phosphatase relationship. Herein, we show that the phosphorylation of the PP2A-Aα structural subunit at S401 by ATM results in nuclear export, which regulates the DDR at multiple levels and affects genomic …

Novel Cell Penetrating Peptide-Adaptors Effect Intracellular Delivery And Endosomal Escape Of Protein Cargos, John C. Salerno, Verra M. Ngwa, Scott J. Nowak, Carol A. Chrestensen, Allison N. Healey, Jonathan L. Mcmurry

Faculty Articles

The use of cell penetrating peptides (CPPs) as biomolecular delivery vehicles holds great promise for therapeutic and other applications, but development has been stymied by poor delivery and lack of endosomal escape. We have developed a CPP-adaptor system capable of efficient intracellular delivery and endosomal escape of user-defined protein cargos. The cell penetrating sequence of HIV transactivator of transcription was fused to calmodulin, which binds with subnanomolar affinity to proteins containing a calmodulin binding site. Our strategy has tremendous advantage over prior CPP technologies because it utilizes high affinity noncovalent, but reversible coupling between CPP and cargo. Three different cargo …

Artemisinin Production By Plant Hairy Root Cultures In Gas- And Liquid-Phase Bioreactors, Nivedita Patra, Ashok K. Srivastava

Articles

Key message

Alternative biotechnological protocol for large-scale artemisinin production was established. It featured enhanced growth and artemisinin production by cultivation of hairy roots in nutrient mist bioreactor (NMB) coupled with novel cultivation strategies.

Artemisinin is used for the treatment of cerebral malaria. Presently, its main source is from seasonal plant Artemisia annua. This study featured investigation of growth and artemisinin production by A. annua hairy roots (induced by Agrobacterium rhizogenes-mediated genetic transformation of explants) in three bioreactor configurations—bubble column reactor, NMB and modified NMB particularly to establish their suitability for commercial production. It was observed that cultivation of …

Quantitative Comparison Of A Nanoengineered Alumina Coated Cnt Arrays To Sio2 Coated Cnts And Solution Based Delivery System, Shree Aier

Maseeh Summer Undergraduate Research Experience

To meet the growing need for nanoengineered biocompatible materials to serve as drug delivery platforms, in this research, carbon nanotube arrays were fabricated by chemical vapor deposition, followed by an alumina coating by the high yielding, tightly controlled atomic layer deposition. This nanoengineered vertically aligned alumina nanowire array serves as a platform for delivering antigens, which act as cancer adjuvants. The physicochemical characteristics of the nanowires (NWs) can significantly influence the delivery of a biomolecule to immune cells. To investigate the material characteristics, the delivery efficiency of the antigen using NWs was quantitatively assessed by flow cytometry. Further, the mechanism …

Molecular Basis Of Sex Differences In Cardiac Hypertrophy And Heart Failure, Maya James

Online Theses and Dissertations

Transforming growth factor beta ligands and receptors are known to be pro-hypertrophic and pro-fibrotic factors in the heart, and are known to contribute to the development of cardiac hypertrophy and heart failure. It is well established that premenopausal females possess a lower incidence of these pathologies. We and others have observed a greater level of fibrosis in male hearts compared to female hearts in rodent models of cardiac hypertrophy. It is well established that estrogen is cardioprotective in that it can prevent the development of cardiac hypertrophy, as well as abrogate the development of heart failure following sustained cardiac hypertrophy. …

Enzyme Analysis Of Oxidatively Modified Proteins Post-Tbi, Brittany Rice

Online Theses and Dissertations

The brain is one of the most important organs in the body. It functions as a control center by regulating and coordinating actions and reactions, which is facilitated via signal transduction pathways. Its function is primarily dependent upon sufficient supply of glucose for energy metabolism. The dysfunction of the brain resulting from an external force is known as traumatic brain injury (TBI). Symptoms range from physical to psychological and effects can be mild, moderate, or severe depending on the extent of injury. TBI is associated with oxidative damage, the overproduction of reactive oxygen/nitrogen species. Reduced energy metabolism is a consequence …

A Novel Forensic Dna Profiling Method Based On Molecular Beacons Without Dna Purification, Benjamin C. Shaw

Online Theses and Dissertations

Analysis of polymorphisms in nucleic acid sequences provides the basis for identification of individuals and their genetic deficiencies. Currently, the accepted method of analysis for profiling is Short Tandem Repeat (STR) profiling. This is a lengthy process, typically taking up to 3 days. The time necessary to generate an STR profile, along with the ever-increasing reliance on DNA to solve crimes, has led to a large DNA sample backlog, with violent crime turnaround taking an average of 103 days. The time and resource investment required for STR analysis is significant, and not all samples generate useful profiles.

The current methods …

Recruitment Of Monocytes To The Pre-Ovulatory Ovary, Alex Paige Whitaker

Online Theses and Dissertations

Ovulation is a recurring biological process that involves inflammatory reactions that degrade and restructure tissue. It requires the chemotaxis of leukocytes to the ovary to help regulate and promote these events. Hormonal signals and chemokines that are released during the peri-ovulatory period initiate the release of specific leukocyte populations, possibly from the spleen, that infiltrate the ovary and facilitate the release of oocytes from ovarian follicles. These studies utilized a super-ovulation protocol to initiate ovulation in immature balb/c and cd1 mice. Ovaries were collected at several times post human chorionic gonadotropin (hCG) injection and analyzed by multi-color flow cytometry. Oviducts …

Efficiency Of The Targetron Gene Knockout System As A Transformative Protocol For The Mutagenesis Of Listeria Monocytogenes, Hilaree Noele Frazier

Online Theses and Dissertations

The process of using electroporation to introduce plasmid DNA into host cells is a valuable molecular technique that is increasingly employed in labs worldwide. Electroporators are generally small and relatively inexpensive, making them attractive systems to use for a variety of purposes. Electroporation protocols are numerous in the published literature and encompass all cell types, from prokaryotic bacterial cells to eukaryotic human cells. The TargeTron Gene Knockout System by Sigma-Aldrich is an affordable option for the electroporation of numerous bacterial species. However, its use in Listeria monocytogenes has not been extensively characterized. Here we sought to discuss the effectiveness of …

Characterization Of A Phosphonate-Specific Cytidylyltransferase, Kissa Batul

Theses and Dissertations (Comprehensive)

Antibiotic resistance is a major global health concern that requires new therapeutic approaches. Furthermore, a lack of narrow spectrum antibiotics on the market produces unintended consequences with respect to changes in our microbial make up. Phosphonates are reduced versions of phosphates that possess a C-P bond which is more resistant to enzymatic and chemical degradation. The role of phosphonate containing macromolecules (e.g. cell surface polysaccharides) remains enigmatic, however their presence suggests that they may confer an advantage. The biosynthesis of phosphonate-containing macromolecules is unknown, but a pathway is proposed involving aLicC type cytidylyltransferase-catalyzed conjugation to a phosphonate followed by a …

Effect Of Two-Year Caloric Restriction On Bone Metabolism And Bone Mineral Density In Non-Obese Younger Adults: A Randomized Clinical Trial, Dt Villareal, L Fontana, Sk Das, Et Al.

Faculty, Staff and Students Publications

Although caloric restriction (CR) could delay biologic aging in humans, it is unclear if this would occur at the cost of significant bone loss. We evaluated the effect of prolonged CR on bone metabolism and bone mineral density (BMD) in healthy younger adults. Two-hundred eighteen non-obese (body mass index [BMI] 25.1?±?1.7?kg/m(2) ), younger (age 37.9?±?7.2 years) adults were randomly assigned to 25% CR (CR group, n?=?143) or ad libitum (AL group, n?=?75) for 2 years. Main outcomes were BMD and markers of bone turnover. Other outcomes included body composition, bone-active hormones, nutrient intake, and physical activity. Body weight (-7.5?±?0.4 versus …

Identification Of A Novel Gene On 10q221 Causing Autosomal Dominant Retinitis Pigmentosa (Adrp), Stephen P Daiger, Lori S Sullivan, Sara J Bowne, Daniel C Koboldt, Susan H Blanton, Dianna K Wheaton, Cheryl E Avery, Elizabeth D Cadena, Robert K Koenekoop, Robert S Fulton, Richard K Wilson, George M Weinstock, Richard A Lewis, David G Birch

Faculty, Staff and Students Publications

Whole-genome linkage mapping identified a region on chromosome 10q21.3-q22.1 with a maximum LOD score of 3.0 at 0 % recombination in a six-generation family with autosomal dominant retinitis pigmentosa (adRP). All known adRP genes and X-linked RP genes were excluded in the family by a combination of methods. Whole-exome next-generation sequencing revealed a missense mutation in hexokinase 1, HK1 c.2539G > A, p.Glu847Lys, tracking with disease in all affected family members. One severely-affected male is homozygous for this region by linkage analysis and has two copies of the mutation. No other potential mutations were detected in the linkage region nor were …

Local Corticotropin Releasing Hormone (Crh) Signals To Its Receptor Crhr1 During Postnatal Development Of The Mouse Olfactory Bulb, Isabella Garcia, Paramjit K Bhullar, Burak Tepe, Joshua Ortiz-Guzman, Longwen Huang, Alexander M Herman, Lesley Chaboub, Benjamin Deneen, Nicholas J Justice, Benjamin R Arenkiel

Faculty, Staff and Students Publications

Neuropeptides play important physiological functions during distinct behaviors such as arousal, learning, memory, and reproduction. However, the role of local, extrahypothalamic neuropeptide signaling in shaping synapse formation and neuronal plasticity in the brain is not well understood. Here, we characterize the spatiotemporal expression profile of the neuropeptide corticotropin-releasing hormone (CRH) and its receptor CRHR1 in the mouse OB throughout development. We found that CRH-expressing interneurons are present in the external plexiform layer, that its cognate receptor is expressed by granule cells, and show that both CRH and CRHR1 expression enriches in the postnatal period when olfaction becomes important towards olfactory-related …

Molecular Dynamics Simulation Reveals Correlated Inter-Lobe Motion In Protein Lysine Methyltransferase Smyd2, Nicholas Spellmon, Xiaonan Sun, Nualpun Sirinupong, Brian Fp Edwards, Chunying Li, Zhe Yang

Biochemistry and Molecular Biology Faculty Publications

SMYD proteins are an exciting field of study as they are linked to many types of cancer- related pathways. Cardiac and skeletal muscle development and function also depend on SMYD proteins opening a possible avenue for cardiac-related treatment. Previous crystal structure studies have revealed that this special class of protein lysine methyltransferases have a bilobal structure, and an open–closed motion may regulate substrate specificity. Here we use the molecular dynamics simulation to investigate the still-poorly-understood SMYD2 dynamics. Cross-correlation analysis reveals that SMYD2 exhibits a negative cor- related inter-lobe motion. Principle component analysis suggests that this correlated dynamic is contributed to …

Phylogenetic Investigation Of Enteric Bovine Coronavirus In Ireland Reveals Partitioning Between European And Global Strains, Lynda Gunn, P. J. Collins, M. J. O'Connell, Helen O'Shea

Department of Biological Sciences Publications

Background

Bovine coronavirus is a primary cause of neonatal calf diarrhea worldwide, and is also associated with acute diarrhea in adult cattle during the winter season. There are no reports on molecular characterization of bovine coronavirus in Ireland, and little data exists apart from serological studies.

Findings

In this study, 11 neonatal (mean age 9 days) calf BCoV strains from the south of Ireland were collected over a one year period and characterized using molecular methods. The spike gene which encodes a protein involved in viral entry, infectivity and immune response shows the most variability amongst the isolates and was …

Alternative Use Of Dna Binding Domains By The Neurospora White Collar Complex Dictates Circadian Regulation And Light Responses, Bin Wang, Xiaoying Zhou, Jennifer J. Loros, Jay C. Dunlap

Dartmouth Scholarship

In the Neurospora circadian system, the White Collar complex (WCC) of WC-1 and WC-2 drives transcription of the circadian pacemaker gene frequency (frq), whose gene product, FRQ, as a part of the FRQ-FRH complex (FFC), inhibits its own expression. The WCC is also the principal Neurospora photoreceptor; WCC-mediated light induction of frq resets the clock, and all acute light induction is triggered by WCC binding to promoters of light-induced genes. However, not all acutely light-induced genes are also clock regulated, and conversely, not all clock-regulated direct targets of WCC are light induced; the structural determinants governing the shift …

Period-1 Encodes An Atp-Dependent Rna Helicase That Influences Nutritional Compensation Of The Neurospora Circadian Clock, Jillian M. Emerson, Bradley M. Bartholomai, Carol S. Ringelberg, Scott E. Baker, Jennifer Loros, Jay Dunlap

Dartmouth Scholarship

Mutants in the period-1 (prd-1) gene, characterized by a recessive allele, display a reduced growth rate and period lengthening of the developmental cycle controlled by the circadian clock. We refined the genetic location of prd-1 and used whole genome sequencing to find the mutation defining it, confirming the identity of prd-1 by rescuing the mutant circadian phenotype via transformation. PRD-1 is an RNA helicase whose orthologs, DDX5 [DEAD (Asp-Glu-Ala-Asp) Box Helicase 5] and DDX17 in humans and DBP2 (Dead Box Protein 2) in yeast, are implicated in various processes, including transcriptional regulation, elongation, and termination, ribosome biogenesis, and mRNA decay. …

Characterization Of Cytidylyltransferase Enzyme Activity Through High Performance Liquid Chromatography, James Brault

Theses and Dissertations

The cytidylyltransferases are a family of enzymes that utilize cytidine 5â?? triphosphate (CTP) to synthesize molecules that are precursors to membrane phospholipids. There are four well known enzymes: CTP: phosphoethanolamine cytidylyltransferase (ECT), CTP: glycerol-3-phosphate cytidylyltransferase (GCT), 2-C-methyl-D-erythritol-4-phosphate cytidylyltransferase synthetase (CMS), and CTP: phosphocholine cytidylyltransferase (CCT). Previously, a radioisotope tagging method was employed to study cytidylyltransferase catalysis. Using CCT as a model, a method utilizing high performance liquid chromatography (HPLC) was developed to replace the radioisotope scintillation technique. The development of this new HPLC method is cheaper, more efficient, and more accurate than the previously established method.

The targets of separation …