Biochemistry, Biophysics, and Structural Biology Commons^™

Incorporating Genomics And Bioinformatics Across The Life Sciences Curriculum, Jayna L. Ditty, Christopher A. Kvaal, Brad Goodner, Sharyn K. Freyermuth, Cheryl Bailey, Robert A. Britton, Stuart G. Gordon, Sabine Heinhorst, Kelynne Reed, Zhaohui Xu, Erin R. Sanders-Lorenz, Seth Axen, Edwin Kim, Mitrick Johns, Kathleen Scott, Cheryl A. Kerfeld

Department of Biochemistry: Faculty Publications

Undergraduate life sciences education needs an overhaul, as clearly described in the National Research Council of the National Academies’ publication BIO 2010: Transforming Undergraduate Education for Future Research Biologists. Among BIO 2010’s top recommendations is the need to involve students in working with real data and tools that reflect the nature of life sciences research in the 21st century [1]. Education research studies support the importance of utilizing primary literature, designing and implementing experiments, and analyzing results in the context of a bona fide scientific question [1–12] in cultivating the analytical skills necessary to become a scientist. Incorporating these …

Evolution Of Lactate Dehydrogenase Genes In Primates, With Special Consideration Of Nucleotide Organization In Mammalian Promoters, Zack Papper

Wayne State University Dissertations

Concomitant with an increase in brain volume and mass, the allocation of energetic resources to the brain increased during stem anthropoid evolution, leading to humans. One mechanism by which this allocation may have occurred is through greater use of lactate as a neuronal fuel. Both the production of lactate, and conversion to pyruvate for use in aerobic metabolism, are catalyzed, in part, by the tetrameric enzyme lactate dehydrogenase (LDH). The two primary LDH genes, LDHA and LDHB, confer different rates of substrate turnover to the LDH enzyme, and these rates lend to the argument that LDHA supports anaerobic while LDHB …

Characterization Of Arsd: An Arsenic Chaperone For The Arsab As(Iii)-Translocating Atpase, Jianbo Yang

Wayne State University Dissertations

Arsenic is a metalloid toxicant that is widely distributed throughout the earth's crust and causes a variety of health and environment problems. As an adaptation to arsenic-contaminated environments, organisms have developed resistance systems. In bacteria and archaea various ars operons encode ArsAB ATPases that pump the trivalent metalloids As(III) or Sb(III) out of cells. In these operons, an arsD gene is almost always adjacent to the arsA gene, suggesting a related function. ArsA is the catalytic subunit of the pump that hydrolyzes ATP in the presence of arsenite or antimonite. ArsB is a membrane protein which containing arsenite-conducting pathway. ArsA …

Regulatory And Functional Aspects Of Foxo3a Transcription Factor And Their Implications In Prostate Cancer, Melissa Elise Dobson

Wayne State University Dissertations

The P13K/Akt pathway is a critical mediator of growth factor signaling involving many cellular functions. The deregulation of this pathway has been shown to be involved in the development of various cancers. One of the main targets of this pathway is FoxO3a, a transcription factor whose target genes are involved in important cellular processes such as apoptosis, cell cycle control, and glucose metabolism. FoxO3a is regulated by various post translational modifications including acetylation, ubiquitination and phosphorylation. The transcription factor is directly phosphorylated by Akt on 3 residues: Threonine 32, Serine 253 and Serine 315. Phosphorylation by Akt generates binding sites …

Single Molecule Studies Of Spliceosomal Snrnas U2-U6, Zhuojun Guo

Wayne State University Dissertations

Spliceosomes catalyze the maturation of precursor mRNAs in organisms ranging

from yeast to humans. Their catalytic core comprises three small nuclear RNAs (U2, U5

and U6) involved in substrate positioning and catalysis. It has been postulated, but never

shown experimentally, that the U2-U6 complex adopts at least two conformations that

reflect different activation states. We have used single-molecule fluorescence to probe the

structural dynamics of a protein-free RNA complex modeling U2-U6 from yeast and

mutants of highly conserved regions of U2-U6. Our data show the presence of at least

three distinct conformations in equilibrium. The minimal folding pathway consists of …

Development Of Ultra-Sensitive Fluorescence Photoamplification Assays For The Detection Of Molecular Recognition Events, Tiffany Priscilla Gustafson

Electronic Theses and Dissertations

During the course of this research a novel method which couples the molecular recognition-triggered photoamplification chain in diaryl ketone adducts of dithiane with a "turn-off" or "turn-on" fluorescence-based assay for the detection of biological targets and ligands, regardless of their nature, through a molecular recognition event has been developed. This research has included several key steps, the most significant being: (1) the design of fluorophore adducts or dyads which recover fluorescence upon photocleavage for a "turn-on" assay and the identification of fluorophores which are quenched upon the photochemical release of a quencher for a "turn off" assay; (2) Optimization of …

Animal Models Of Alzheimer's Disease, Gemma Casadesus, Gary Arendash, Frank Laferla, Mike Mcdonald

Molecular Biosciences Faculty Publications

No abstract provided.

Comparing Models Of Evolution For Ordered And Disordered Proteins, Celeste J. Brown, Audra K. Johnson, Gary W. Daughdrill

Molecular Biosciences Faculty Publications

Most models of protein evolution are based upon proteins that form relatively rigid 3D structures. A significant fraction of proteins, the so-called disordered proteins, do not form rigid 3D structures and sample a broad conformational ensemble. Disordered proteins do not typically maintain long-range interactions, so the constraints on their evolution should be different than ordered proteins. To test this hypothesis, we developed and compared models of evolution for disordered and ordered proteins. Substitution matrices were constructed using the sequences of putative homologs for sets of experimentally characterized disordered and ordered proteins. Separate matrices, at three levels of sequence similarity ( …

Biochemical Characterization Of Suppressor Of Ikk-Ε And Nak-Associated Protein 1, Jonathan Forbes

Theses and Dissertations

Innate immunity provides the first line of defense against invading pathogen by recognizing and mounting a response to the pathogenic challenge. Among the cellular mechanisms of the innate immune response, Toll-like receptor 3 (TLR3) recognizes viral dsRNA and signals subsequent production of type-I interferon. The TLR3:interferon signaling cascades contains a kinase complex composed of two kinases and a scaffold protein, NAK-associated protein 1 (NAP1). The role of NAP1 in modulating kinase activation or regulation is unknown. A key inhibitory protein identified in the TLR3:interferon pathway, silencer of inhibitor of κBα kinase ε (SIKE), blocks the activity of this kinase complex …

Cell-Cell Junction Signaling Regulating Dna Double-Strand Break Repair In Breast Cells, Sinduja Ethiraj

Theses and Dissertations

Genomic instability and acquisition of invasiveness through the basement membrane extracellular matrix (ECM) are two major processes for epithelial cell malignancy in breast cancer. DNA double-strand break repair (DSBR) is one of the processes that get misregulated during breast cancer progression. In addition, radiation induced breaks such as those induced during radiation therapy to treat breast cancer patients are repaired by DSBR, rendering this pathway relevant for therapy as well. DSBR can occur either by homologous recombination (HR) or non-homologous end-joining (NHEJ). HR is accepted as the more error-free pathway. HR is regulated by the cell cycle status such that …

P63 And Vdr Are Regulated By Vitamin D (Vd3) And Uv Signaling, Andrew J. Whitlatch

Browse all Theses and Dissertations

Skin cancers, such as squamous cell carcinoma (SCC), develop from accumulated mutations as a result of excessive exposure to Ultraviolet B (UVB) radiation. Intriguingly, UVB also catalyzes the synthesis of 1alpha, 25-dihydroxy Vitamin D3 (VD3), the hormonally active form of Vitamin D. Downstream VD3 signaling has been associated with promoting the inhibition of cell cycle progression, regulating calcium homeostasis, and inducing differentiation and apoptosis. VD3 mediates these processes via genomic mechanisms through interaction with its cognate receptor, the Vitamin D Receptor, (VDR). In addition, it was recently discovered that VD3 reduces UVB-mediated phosphorylation of the SAPK/c-Jun N-terminal kinase (JNK), which …

Inhibition Of Cell Invasion By Targeting Pld, Terry C. Farkaly

Browse all Theses and Dissertations

Phospholipase D (PLD) is a crucial signaling enzyme involved in many cellular processes. The catalytic activity of PLD is essential for the production of Phosphatidic Acid (PA), a critical second messenger in cell signaling cascades downstream. Using the highly invasive rat mammary adenocarcinoma cell line mTLn3 as a metastatic model, we investigated the proficiency of these cells to invade using matrigels that mimic the basement membrane of the extracellular matrix (ECM), their activity through PLD enzymatic assays, as well as the potency of our potential inhibitors to inhibit PLD-mediated cell invasion and lipase activity. This study reveals that PLD-mediated cell …

Genetic Studies Of Genes Involved In The Initiation Of Dna Replication In The Fission Yeast Schizosaccharomyces Pombe, Zhuo Wang

Browse all Theses and Dissertations

The initiation of DNA replication is a highly conserved process in all eukaryotes. However, the underlying mechanism is not well understood. Genetic studies in the fission yeast S. pombe have contributed greatly to and will continue to provide insights to our understanding of this important biological process.

In the first chapter, we have used a complementary method to test three recently identified human replication proteins DUE-B, Ticrr/Treslin, and GEMC1 as the candidate functional homologue of Sld3 in S. pombe. Sld3 is an essential replication initiation protein discovered in yeasts. Since no apparent sequence similarity can be found, its homologue in …

Characterization Of Three Mutations In Conserved Domain Of Subunit Iii Of Cytochrome C Oxidase From Rhodobacter Sphaeroides, Rachel Omolewu

Browse all Theses and Dissertations

Cytochrome c oxidase (COX) is the final electron acceptor in mitochondrial respiratory chain and in many bacterial species including Rhodobacter sphaeroides. Electron transfer is coupled with the pumping of protons across the membrane. Previous work has shown that reaction of beef COX with dicyclohexylcarbodiimide (DCCD) resulted in an inhibition of proton translocation by covalently binding to the conserved amino acid residue E90 located in a nonpolar region of subunit III (SIII). E90 is involved in a bonding pair with another conserved residue H212, possibly connected by a salt bridge or a hydrogen bond in the three dimensional structure of SIII. …

Purification And Properties Of Amycolatopsis Mediterranei Dsm 43304 Lipase And Its Potential In Flavour Ester Synthesis, Dharmendra Dheeman, Gary Henehan, Jesus Maria Frias

Articles

An extracellular thermostable lipase from Amycolatopsis mediterranei DSM 43304 has been purified to homogeneity using ammonium sulphate precipitation followed by anion exchange chromatography and hydrophobic interaction chromatography. This protocol resulted in 398 fold purification with 36% final recovery. The purified A. mediterranei DSM 43304 lipase (AML) has an apparent molecular mass of 33 kDa. The N-terminal sequence, AANPYERGPDPTTASIEATR, showed highest similarity to a lipase from Streptomyces exfoliatus. The values of and for p-nitrophenyl palmitate (p-NPP) under optimal temperature (60°C) and pH (8.0) conditions were 0.10 ± 0.01 mM and 2.53 ± 0.06 mmol/minmg, respectively. The purified …

Expression, Purification, And Analysis Of Unknown Translation Factors From Escherichia Coli: A Synthesis Approach, Justin D. Walter, Peter Littlefield, Scott P. Delbecq, Gerry Prody, P. Clint Spiegel

Chemistry Faculty and Staff Publications

New approaches are currently being developed to expose biochemistry and molecular biology undergraduates to a more interactive learning environment. Here, we propose a unique project-based laboratory module, which incorporates exposure to biophysical chemistry approaches to address problems in protein chemistry. Each of the experiments described herein contributes to the stepwise process of isolating, identifying, and analyzing a protein involved in a central biological process, prokaryotic translation. Students are provided with expression plasmids that harbor an unknown translation factor, and it is their charge to complete a series of experiments that will allow them to develop hypotheses for discovering the identity …

Protein Folding, Aggregation And Unfolding In Monte Carlo Simulations, Sandipan Mohanty, Anders Irbäck, Simon Mitternacht, Giorgio Favrin, U. H.E. Hansmann

Michigan Tech Publications

An implicit water all-atom model is used to study folding, aggregation and mechanical unfolding of small proteins. Physically reasonable results obtained for a variety of applications indicate healthy global properties of the interaction potential.

Patterns Of Apoptotic Poly(Adp-Ribose) Polymerase Cleavage Induced By Laromustine And Its Analogs, Adam N. Paine

Honors Theses

The anticancer prodrug Laromustine (VNP40101M) has produced promising remission rates in clinical trials among leukemic patients relative to currently available chemotherapeutics. Such improvements demand that the agent’s mechanism of action be elucidated. This study aimed to determine the role of poly(ADP-ribose) polymerase (PARP) in Laromustine-induced cell death. Previous studies indicated that the methyl isocyanate produced upon activation of Laromustine is largely responsible for its effective induction of apoptosis. Results reported herein strongly support the activation of a PARP-dependent apoptotic pathway by Laromustine’s carbamoylating and chlorethylating subspecies. Furthermore, it is evident that the Laromustine-induced PARP-dependent apoptosis is primarily attributable to methyl …

Influence Of Retinal States On The Development And Maintenance Of Retinofugal Projections, Duncan Morhardt

Theses and Dissertations

Vision provides a critical interface with the physical world. This work examines visual development and vision loss in mice to glean the influence of the retinal state on visual connections. I first assessed the impact of retinal activity on the eye-specific segregation of retinal afferents in the lateral geniculate nucleus (LGN) of young Gβ5 -/- mice. Gβ5 is the fifth member of the β subfamily of heterotrimeric G proteins. Gβ5 binds and stabilizes the R7 family of regulators of G-protein signaling (RGS), which accelerate Gi/o GTP hydrolysis. Gβ5 -/- mice, which lack R7RGS activity, have malformed synapses in the outer …

Group Ii Intron Dynamics In Heterologous Hosts, Venkata Raghavendra Aditya Chalamcharla

Legacy Theses & Dissertations (2009 - 2024)

Group II introns are ribozymes with an innate ability to self-splice. They are found predominantly in bacterial and bacterial-derived organellar genomes, but not in the nuclear genomes of eukaryotes. In bacteria, group II introns often behave as mobile retroelements, invading host DNA and exploiting its machinery to complete the retromobility process. The object of my studies is the group II intron found in the Lactococcus lactis relaxase gene. To determine the nature of the group II intron-host relationship, we performed a genetic screen and identified several host factors that affect group II intron retromobility in Escherichia coli, which provides a …