Molecular Biology Commons^™

Investigating Structures And Functions Of Apoptotic Caspases, Ishankumar V. Soni

Doctoral Dissertations

Caspases are cysteine aspartate proteases involved in various cellular pathways including apoptosis, inflammation, and neurodegeneration. Caspase-9 is classified as an initiator apoptotic caspase that is activated upon intrinsic stress. Caspase-9 is composed of two domains: an N- terminal CARD domain and a catalytic core domain. We have employed hydrogen deuterium exchange mass spectrometry (H/DX-MS) to determine the 1) dynamics of the full-length caspase- 9, 2) dynamic impacts on caspase-9 upon substrate-induced dimerization, and 3) regions involved in the CARD: catalytic core domains interactions. Upon intrinsic stress, caspase-9 activates executioners, procaspase-3 and -7 but not procaspase-6. We have employed site-directed mutagenesis …

Screening For Binding Partners And Protein-Protein Interactions Of A Fungal Transcription Factor- Xdr1, Nishadi Punsara Gallala Gamage

Masters Theses

Clarireedia spp. (formerly Sclerotinia homoeocarpaF.T. Bennett) is the causal agent dollar spot, the most economically important turfgrass disease impacting golf courses in North America. The most effective strategy for dollar spot control is repeated application of multiple classes of fungicides. However, reliance on chemical application has led to resistance to four classes of fungicides as well as multidrug resistance (MDR). Fungi are known to detoxify xenobiotics, like fungicides, through transcriptional regulation of three detoxification phases: modification, conjugation and secretion. Little is known, however, of the protein-protein interactions that facilitate these pathways. Following next-generation RNA sequencing of Clarireedia spp., a …

Utilizing Fluorescence Microscopy To Characterize The Subcellular Distribution Of The Novel Protein Acheron, Varun Sheel

Masters Theses

All cells carry the genetic machinery required to commit cell suicide; a process known as programmed cell death (PCD). While the ability to initiate PCD serves a number of useful purposes during development and homeostasis, misregulation of PCD is the underlying basis of most human diseases, including cancer, autoimmunity disorders and neurodegeneration. Using the tobacco hawkmoth Manduca sexta as a model organism, the Schwartz lab at UMass has demonstrated that PCD requires de novo gene expression and has cloned many death-associated genes. One of these genes encodes a novel protein that was named Acheron after one of the rivers of …

Pharmacological Chaperoning Of Human Lysosomal Neuraminidase 1, Di Chu

Doctoral Dissertations

Human lysosomal neuraminidase 1 (hNEU1) is an exo-a-sialidase which cleaves a(2-3) and a(2-6) linked sialic acids on glycoproteins in the lysosome. Deficiency of hNEU1 in the lysosome results in sialidosis, a lysosomal storage disease. Currently there is no effective treatment for sialidosis, which leads to a rising interest in discovering potential therapies. Here we presented a small molecule, α-D-N-acetylneuraminic acid (NANA), increases the protein amount and activity of both wild-type hNEU1 and three different hNEU1 mutations found in sialidosis patients in our mammalian cell system, suggesting that NANA works as a potential pharmacological chaperone for hNEU1 and provides …

The Shape Of U: Mapping Out Protective Elements In Mrna Escapees, Jacob Miles

Masters Theses

A crucial step of the viral life cycle of Kaposi’s Sarcoma Herpesvirus (KSHV) lytic infection is the triggering of a massive RNA decay event termed “Host Shutoff”. Host Shutoff is driven by the viral endonuclease SOX which leads to the destruction of over 70% of the total transcriptome. This process cripples cellular gene expression and allows for viral reprograming of the cell for the purpose of viral replication. Co-evolution has led to the host developing a multitude of antiviral defenses aimed at preserving certain cellular RNAs linked to antiviral responses. One such defense are RNA secondary structures located within the …

Probing Apoptotic Caspase Allostery And Exosite Interactions For Alternative Regulation, Derek J. Macpherson

Doctoral Dissertations

Programmed cell death, or apoptosis is a critical homeostatic pathway that monitors the balance of cell life and death. Apoptosis is regulated by a class of enzymes known as the cysteine aspartic proteases, or the caspases. The 12 human caspases that play important roles in the progression and regulation of apoptosis and inflammation. Caspases are tightly regulated by numerous factors including enzymatic activation, post-translational modifications, metal ligand binding, and protein modulation. Aberrant caspase activation and regulation has been implicated in the progression of numerous diseases such as proliferative diseases and neurodegeneration. The deeply entwined nature of caspases and apoptosis makes …

Feronia-Related Receptor Kinase 7 And Feronia And Their Role In Receiving And Transducing Signals, David Vyshedsky

Masters Theses

Receptor kinases (RKs) are transmembrane proteins that have been shown to regulate an array of important processes in A. thaliana, including polar cell growth, plant reproduction, and many other plant growth processes. In this thesis, I examine RECEPTOR KINASE 7 (RK7) and FERONIA (FER), two closely related transmembrane RKs, and their effects on plant reproduction. The RK7 gene when knocked out (rk7) in conjunction with FER resulted in delayed plant growth, decreased seed yield, and a lower percentage of the seeds germinating as compared to the single FER knockout. Transgenic plants with GUS reporter driven by RK7 promoter …

The Role Of The Metallochaperone Hypa In The Acid Survival And Activities Of Nickel Enzymes In Helicobacter Pylori, Heidi Hu

Doctoral Dissertations

Helicobacter pylori is a bacterium that has colonized the human gastric mucosa of over 50% of the world population. Persistent infection can cause gastritis, peptic ulcers, and cancers. The ability of H. pylori to colonize the acidic environment of the human stomach is dependent on the activity of the nickel containing enzymes, urease and NiFe-hydrogenase. The nickel metallochaperone, HypA, was previously shown to be required for the full activity of both enzymes. In addition to a Ni-binding site, HypA also contains a structural Zn site, which has been characterized to alter its averaged structure depending on pH and the presence …

Modifications Of Myofilament Structure And Function During Global Myocardial Ischemia, Mike K. Woodward

Masters Theses

Cardiac arrest is a prevalent condition with a poor prognosis, attributable in part to persistent myocardial dysfunction following resuscitation. The molecular basis of this dysfunction remains unclear. We induced cardiac arrest in a porcine model of acute sudden death and assessed the impact of ischemia and reperfusion on the molecular function of isolated cardiac contractile proteins. Cardiac arrest was electrically induced, left untreated for 12 min, and followed by a resuscitation protocol. With successful resuscitations, the heart was reperfused for 2 h (IR2) and the muscle harvested. In failed resuscitations, tissue samples were taken following the failed efforts (IDNR). Actin …

Expression And Purification Of Human Lysosomal Β-Galactosidase From Pichia Pastoris, Sarah E. Tarullo

Masters Theses

Lysosomal storage diseases are genetically inherited diseases caused by the dysfunction of lysosomal enzymes. In a normal cell, lysosomal enzymes cleave specific macromolecules as they are transported to the lysosome. However, in diseased cells, these lysosomal enzymes are either absent or malfunctioning, causing macromolecular substrates to accumulate, becoming toxic to the cell. Over fifty lysosomal storage diseases have been identified, collectively occurring in one out of 7,700 live births. We investigated the lysosomal enzyme β-galactosidase (β-gal). In order to study the biochemistry and enzymology of this protein a robust expression system was needed. The GLB1 gene has been inserted into …

Pharmacological Chaperoning In Fabry Disease, Jerome Rogich

Masters Theses 1911 - February 2014

Fabry Disease is an X-­‐linked lysosomal storage disorder characterized by a variety of symptoms including hypohydrosis, seizures, cardiac abnormalities, skin lesions, and chronic pain. These symptoms stem from a lack of functional endogenous α-­‐ Galactosidase A (α-­GAL), which leads to an accrual of its natural substrate. The severity of the disease symptoms can be directly correlated with the amount of residual enzyme activity. It has been shown that an imino sugar, 1-deoxygalactonojirimycin (DGJ), can increase enzymatic activity and clear excess substrate. This pH-­‐dependent chaperoning phenomenon is believed to arise from the presence of aspartic acid 170 in the active site. …

Novel Adaptor-Dependent Domains Promote Processive Degradation By Clpxp, Keith L. Rood

Masters Theses 1911 - February 2014

Protein degradation by ATP dependent proteases is a universally conserved process. Recognition of substrates by such proteases commonly occurs via direct interaction or with the aid of a regulatory adaptor protein. An example of this regulation is found in Caulobacter crescentus, where key regulatory proteins are proteolysed in a cell-cycle dependent fashion. Substrates include essential transcription factors, structural proteins, and second messenger metabolism components. In this study, we explore sequence and structural requirements for regulated adaptor mediated degradation of PdeA, an important regulator of cyclic-di-GMP levels.

Robust degradation of PdeA is dependent on the response regulator CpdR in vivo …

Chemical Information Based Elastic Network Model: A Novel Way To Identification Of Vibration Frequencies In Proteins., Sharad K. Raj

Masters Theses 1911 - February 2014

A novel method of analysis of macromolecules has been worked upon through this research. In an effort to understand the dynamics of macromolecules and to further our knowledge, pertaining specifically to the low frequency domain and also to elucidate certain important biological functions associated with it, a theoretical technique of chemical information based Normal Mode Analysis has been developed. These simulations render users with the ability to generate animations of modeshapes as well as key insight on the associated vibration frequencies. Harmonic analysis using atomistic details is performed taking into account appropriate values of masses of constituent atoms of a …