Biochemistry, Biophysics, and Structural Biology Commons^™

Development Of A Genetic Over-Expression System For The Freshwater Sponge Ephydatia Muelleri, Joe Walsh

Honors Theses

The aim of this work is to develop an over-expression system for Ephydatia muelleri. Preliminary work by Klaske Schippers (Schippers, 2013) showed sponges were induced to transcribe reporter genes driven by the widely used cytomegalovirus promoter (CMV). Building off Schippers work, newly identified native Ephydatia promoters were identified from the genome and verified to drive expression ofreporter genes. Validated over-expression vectors and transfection technology will allow researchers around the world who are working with this organism to ask questions about cell specification, gene regulation and animal development.

Sestrin2, A Regulator Of Thermogenesis And Mitohormesis In Brown Adipose Tissue, Seung-Hyun Ro, Ian Semple, Allison Ho, Hwan-Woo Park, Jun Hee Lee

Department of Biochemistry: Faculty Publications

Sestrin2 is a stress-inducible protein that functions as an antioxidant and inhibitor of mTOR complex 1. In a recent study, we found that Sestrin2 overexpression in brown adipocytes interfered with normal metabolism by reducing mitochondrial respiration through the suppression of uncoupling protein 1 (UCP1) expression. The metabolic effects of Sestrin2 in brown adipocytes were dependent on its antioxidant activity, and chemical antioxidants produced similar effects in inhibiting UCP1-dependent thermogenesis. These observations suggest that low levels of reactive oxygen species (ROS) in brown adipocytes can actually be beneficial and necessary for proper metabolic homeostasis. In addition, considering that Sestrins are ROS …

Physiological Variation Of Garry Oak (Quercus Garryana) Seedlings To Drought Stress, Matthew A. Merz

All Master's Theses

The purpose of this study was to determine physiological differences in drought response among Garry oak (Quercus garryana Douglas ex Hook. [Fagaceae]) seedlings that grow in distinct populations in Washington state, in order to aid in restoration efforts by informing growers of the potential differences between the acorn collection sites. Acorns from six Washington populations east of the Cascades, as well as one population from Whidbey Island, were collected and grown in containers under controlled conditions. The plants were assessed with and without moderate and severe drought stress induced by withholding water. The most extreme differences in photosynthetic characteristics …

On The Protective Properties Of Glycine Based Osmolytes In A Thiol Reducing Environment, John Michael Hausman

Dissertations, Master's Theses and Master's Reports - Open

The protective properties of osmolytes have been studied intently for decades. Originally used to aid in the crystallization of proteins in x-ray diffraction studies, these cosolvents have been shown to reverse protein denaturation and aggregation. Osmolytes aid extremophiles in surviving harsh environments by preferentially excluding themselves from the surface of the protein, thus directing water molecules to the protein’s surface. These osmolytes are naturally found in many health foods and also in many daily use products such as shampoo. Due to their osmoprotective effect their use in everyday consumer product is increasing. Consumers also supplement their diets with thiol-based antioxidants …

Evaluation Of Inhibitors Of Histone Deacetylases As Potential Neurotrophic Agents, Surabhi Shukla

Electronic Theses and Dissertations

The inhibitors of histone deacetylases (HDACs) have shown promising neuroprotective and neuroactive properties. A set of pan- and isoform-specific HDAC inhibitors were evaluated for neurotrophic activity on Neuroscreen-1 (NS-1) cells, a subclone of PC-12 (rat pheochromocytoma) cells.

The HDAC inhibitors were tested alone and in combination with nerve growth factor (NGF). An in vitro method has been standardized that measures neurite outgrowth along with cytotoxicity of test compounds in a single assay. The neurotrophin signaling pathways were interrogated with selective inhibitors of MEK1/2 (PD98059/U0126) PI3K (LY294002) and TrkA (GW441756) and phosphorylation of target kinases. Associated factors namely, acetylation of histones …

Gadolinium Modifies The Cell Membrane To Inhibit Permeabilization By Nanosecond Electric Pulses, Elena C. Gianulis, Andrei G. Pakhomov

Bioelectrics Publications

Lanthanide ions are the only known blockers of permeabilization by electric pulses of nanosecond duration (nsEP), but the underlying mechanisms are unknown. We employed timed applications of Gd³⁺ before or after nsEP (600-ns, 20 kV/cm) to investigate the mechanism of inhibition, and measured the uptake of the membrane-impermeable YO-PRO-1 (YP) and propidium (Pr) dyes. Gd³⁺ inhibited dye uptake in a concentration-dependent manner. The inhibition of Pr uptake was always about 2-fold stronger. Gd³⁺ was effective when added after nsEP, as well as when it was present during nsEP exposure and removed afterward. Pores formed by nsEP in …

Cross-Talk Between The Tumor Suppressors Par-4 And P53, Tripti Shrestha Bhattarai

Theses and Dissertations--Toxicology and Cancer Biology

This work describes the fascinating interplay between two tumor suppressors Prostate apoptosis response-4 (Par-4) and p53. The guardian of the genome, p53, is frequently mutated in human cancers, and may contribute to therapeutic resistance. However, p53 is intact and functional in normal tissues, and we observed that specific activation of p53 in normal fibroblasts could induce apoptosis selectively in p53-deficient cancer cells. This paracrine apoptotic effect was executed by Par-4 secreted in response to p53 activation. Accordingly, activation of p53 in wild-type mice, but not in p53^-/- or Par-4^-/- mice, caused systemic elevation of Par-4 that induced apoptosis …

Viscoelastic Effects On Electrokinetic Particle Focusing In A Constricted Microchannel, Xinyu Lu, John Dubose, Sang Woo Joo, Shizhi Qian, Xiangchun Xuan

Mechanical & Aerospace Engineering Faculty Publications

Focusing suspended particles in a fluid into a single file is often necessary prior to continuous-flow detection, analysis, and separation. Electrokinetic particle focusing has been demonstrated in constricted microchannels by the use of the constriction-induced dielectrophoresis. However, previous studies on this subject have been limited to Newtonian fluids only. We report in this paper an experimental investigation of the viscoelastic effects on electrokinetic particle focusing in non-Newtonian polyethylene oxide solutions through a constricted microchannel. The width of the focused particle stream is found NOT to decrease with the increase in DC electric field, which is different from that in Newtonian …

Altered Connexin 43 Expression Underlies Age-Dependent Decrease Of Regulatory T Cell Suppressor Function In Nonobese Diabetic Mice, Michel Kuczma, Cong-Yi Wang, Leszek Ignatowicz, Robert Gourdi, Piotr Kraj

Biological Sciences Faculty Publications

Type 1 diabetes is one of the most extensively studied autoimmune diseases, but the cellular and molecular mechanisms leading to T cell–mediated destruction of insulin-producing β cells are still not well understood. In this study, we show that regulatory T cells (T_regs) in NOD mice undergo age-dependent loss of suppressor functions exacerbated by the decreased ability of activated effector T cells to upregulate Foxp3 and generate T_regs in the peripheral organs. This age-dependent loss is associated with reduced intercellular communication mediated by gap junctions, which is caused by impaired upregulation and decreased expression of connexin 43. Regulatory …

Parental Rna Interference Of Genes Involved In Embryonic Development Of The Western Corn Rootworm, Diabrotica Virgifera Virgifera Leconte, Chitvan Khajuria, Ana Maria Vélez, Murugesan Rangasamy, Haichuan Wang, Elane Fishilevich, Meghan L.F. Frey, Newton Portilho Carneiro, Premchand Gandra, Kenneth E. Narva, B. D. Siegfried

Department of Entomology: Faculty Publications

RNA interference (RNAi) is being developed as a potential tool for insect pest management and one of the most likely target pest species for transgenic plants that express double stranded RNA (dsRNA) is the western corn rootworm. Thus far, most genes proposed as targets for RNAi in rootworm cause lethality in the larval stage. In this study, we describe RNAi-mediated knockdown of two developmental genes, hunchback (hb) and brahma (brm), in the western corn rootworm delivered via dsRNA fed to adult females. dsRNA feeding caused a significant decrease in hb and brm transcripts in the adult …

Interactions Between Phospholipids And Polymers With Time Resolved Fluorescence Spectroscopy, Zachary E. Hier

Williams Honors College, Honors Research Projects

Interactions between macromolecule species and fluorescently labeled phospholipids were explored in supported lipid bilayers (SLBs). The concentration and the lateral mobility of the labeled phospholipids within the confocal volume were determined via fluorescence correlation spectroscopy. Low molecular weight polystyrene was used for the neutral charged macromolecule. Three separate concentrations of polystyrene were used and a significant change in lateral mobility of phospholipids was found for the SLB doped with all three concentration of styrene from polystyrene (molecular weight = 1000). Polyester with mimic lysine pendant groups was used for the cationic polymer at physiological pH. A large decrease in the …

The Optimization Of Mitoneet Synthesis, Purification And Mutation Stability In The [2fe-2s] Cluster, Alexandria Defabio

Williams Honors College, Honors Research Projects

MitoNEET, a mitochondrial membrane protein with an unsymmetrical Fe-S cluster coordination, was compared to similar proteins within the Rieske and Ferredoxin families. Point mutations were introduced via site-directed mutagenesis to induce stability, greater retention of the cluster, and to uncover structural limitations of the protein. Mutations were made within the cluster coordination and within the hydrophobic core of the protein. Some of the cluster mutations displayed a natural affinity for nickel during immobilized metal-ion affinity chromatography. It is possible that these mutations aid in the formation of a chelate when exposed to nickel. Spectroscopic monitoring of changes in cluster stability …

Histological And Immunohistochemical Analyses Used To Study Craniosynostosis In Pediatric Patients, Palvir Kaur Baadh

Williams Honors College, Honors Research Projects

Craniosynostosis is a condition in which one or more of the sutures of the skull grow together (fuse) earlier than normal in infants. Sutures are large gaps located at the bony plates or joints of the head. Craniosynostosis causes the skull to expand and grow in the direction of any normal open suture, creating craniofacial complications, such as drooping eyelids and abnormal intracranial pressure, head shape, or brain morphology. This premature fusion or ossification of sutures affects approximately 300-500 live births in 1,000,000 (Kolpakova-Hart et al., 2008) with considerable variation in phenotype, depending on which suture(s) is involved. Corrective …

Evaluation Of Small Molecules On The Excitatory Amino Acid Transporter 3 (Eaat3) For The Treatment Of Ocd, Emmanuel Zubia

Open Access Theses & Dissertations

We seek to discover new drug candidates for the excitatory amino acid transporter 3 (EAAT3) for the treatment of Obsessive Compulsive Disorder (OCD). We hypothesize that decreasing the glutamatergic transmission via EAAT3 intervention will create an amelioration of the symptoms of OCD. For this we have prepared in-silico binding calculations to identify a better compound that can stimulate EAAT3 and decrease glutamatergic transmission. This research will help us further understand and elucidate the role of glutamate in the neural mechanisms of this illness, which to date remain inconclusive. Those areas of study are significant because, until now, there is no …

The Mir-17-92 Cluster Contributes To Mll Leukemia Development Through The Repression Of The Meis1 Competitor Pknox1, Yousaf Anwar Mian

Dissertations

Mixed lineage leukemias have a relatively poor prognosis and arise as a result of translocations between the MLL gene and one of multiple partner genes. Downstream targets of MLL are aberrantly upregulated and include the developmentally important HOX genes and MEIS1, as well as multiple miRNAs, including the miR-17-92 cluster and miR-196b. Here I utilize custom anti-miRNA oligonucleotides to examine the contribution of specific miRNAs to MLL leukemias both as individual miRNAs and in cooperation with other miRNAs. Combinatorial treatment with antagomirs against miR-17 and miR-19a of the miR-17-92 cluster dramatically reduces colony forming ability of MLL-fusion containing cell lines …

Identifying The Amino Acids Important For Hiv Rev-Tubulin Interactions, Bruce E. Dukes Ii

Browse all Theses and Dissertations

The Rev protein is Human Immunodeficiency virus's "switch" from events occurring early in infection to later events. Early in infection, the 13 KDa Rev protein begins to accumulate in the host cell nucleus. Once enough Rev is produced, Rev stimulates a switch in viral gene expression by multimerizing onto nuclear viral RNAs and stimulating their export into the cytoplasm. Multimerization occurs on an RNA structure called the Rev Response Element (RRE). Several Rev monomers bind the RRE and once that threshold is met the Rev-RRE complex is exported out of the nucleus. Once out of the nucleus the Rev-RRE complex …

Membrane Protein Spatial Organization In Mdck Cell Cultures, Rachel E. Neugebauer

Williams Honors College, Honors Research Projects

Studies on the organization of membrane proteins such as GPCRs in live cells help to draw important conclusions regarding cellular communication, and have become a significant topic in drug therapy development. Observing proteins in MDCK cells, which are able to form three-dimensional cultures, provides an intermediate step to better understanding the organization and behavior of these proteins in a tissue-like culture. In this study, fluorescent stains were used on fixed cells to show the morphological differences between MDCK cells in monolayer cultures and three-dimensional cultures, which form hollow, spherical cysts of tightly packed cells. Monolayer cells were transfected to view …

Evaluation Of The Signature Molecular Descriptor With Blosum62 And An All-Atom Description For Use In Sequence Alignment Of Proteins, Lindsay M. Aichinger

Williams Honors College, Honors Research Projects

This Honors Project focused on a few aspects of this topic. The second is comparing the molecular signature kernels to three of the BLOSUM matrices (30, 62, and 90) to test the accuracy of the mathematical model. The kernel matrix was manipulated in order to improve the relationship by focusing on side groups and also by changing how the structure was represented in the matrix by increasing the initial height distance from the central atom (Height 1 and Height 2 included).

There were multiple design constraints for this project. The first was the comparison with the BLOSUM matrices (30, 62, …

Drosophila Cyclin J And The Somatic Pirna Pathway Cooperate To Regulate Germline Stem Cells, Paul Michael Albosta

Wayne State University Dissertations

Cyclin J (CycJ) is a highly conserved cyclin that is uniquely expressed specifically in ovaries in Drosophila. Deletion of the genomic region containing CycJ and adjacent genes resulted in a genetic interaction with neighboring piRNA pathway gene, armitage (armi). Here I assessed oogenesis in CycJ null in the presence or absence of mutations in armi or other piRNA pathway genes. Although CycJ null flies had decreased egg laying and hatching rates, ovaries appeared normal indicating that CycJ is dispensable for oogenesis under normal conditions. Further double mutant analysis of CycJ and neighbor armi, as well as two other piRNA pathway …

Analyzing The Interactions Of Kdm5/Lid And Sin3 In Drosophila Melanogaster, Ambikai Gajan

Wayne State University Dissertations

SIN3, the scaffold protein of a histone modifying complex is conserved from yeast to mammals. Drosophila SIN3 associates with both a histone deactylase RPD3 and a histone demethylase dKDM5/LID. Immunopurification of dKDM5/LID verifies a previously observed interaction with SIN3 and RPD3. Furthermore, deficiency of dKDM5/LID phenocopies deficiency of SIN3 in many cellular and developmental processes. Knockdown of both Sin3A and lid hinder cell proliferation in Drosophila cultured cells and developing flies. Knockdown of these genes also results in a curved wing phenotype implicating a role in wing development. Analysis of underlying gene expression changes upon decreased expression of SIN3, dKDM5/LID …

The Role Of Crebh In Hepatic Energy Regulation Under Metabolic Stress, Roberto Mendez

Wayne State University Dissertations

Lipid metabolism is tightly regulated by nuclear receptors, transcription factors, and cellular enzymes in response to nutritional, hormonal, and stress signals. Hepatocyte specific, cyclic AMP responsive element-binding protein (CREBH) is a transcription factor that is preferentially expressed in the liver and localized in the endoplasmic reticulum (ER) membrane. CREBH is known to be activated by ER stress, inflammatory stimuli, and metabolic signals to regulate hepatic acute-phase response, lipid metabolism, and glucose metabolism. In my thesis research, I have characterized the roles and mechanisms of CREBH in these functions, as well as the overall phenotype of CrebH-null mice. I demonstrated that …

Crebh, A Novel Liver Clock Keeper For Energy Metabolism, Ze Zheng

Wayne State University Dissertations

Circadian rhythms play crucial roles in orchestrating diverse physiological processes that are critical for health and disease. Cyclic AMP responsive element binding protein 3-like 3 (CREB3L3, also known as CREBH) is a liver-enriched, endoplasmic reticulum (ER)-tethered transcription factor known to regulate hepatic acute-phase response and energy homeostasis under stress conditions. Here, we demonstrate that CREBH is regulated by the circadian clock and functions as a diurnal regulator of hepatic lipid and glucose metabolism. CREBH is required to maintain circadian profiles of blood triglycerides, fatty acids, and glucose as well as hepatic glycogen storage. CREBH rhythmically regulates expression levels and amplitudes …

Hrd1 Partners In Endoplasmic Reticulum-Associated Degradation, Aaron Alexander Burr

Wayne State University Dissertations

Protein Quality Control (PQC) comprises cellular pathways that regulate the turnover of short-lived, misfolded proteins. A main component of PQC is Endoplasmic Reticulum (ER)-Associated Degradation (ERAD), which controls the degradation of proteins synthesized in the ER. Aberrations in ERAD have been linked to malignancies such as sarcomas, breast, and pancreatic carcinomas, as well as neurodegenerative disease. The machinery in this system is complex and while significant progress has been made to understand ERAD, it is not clear how the different components come together, or how they are regulated. HRD1 is a resident ubiquitin ligase that has been proposed as a …

Soy Isoflavones Mediate Radioprotection Of Normal Lung Tissue By Modulating The Radiation-Induced Inflammatory Response, Lisa Marie Abernathy

Wayne State University Dissertations

Radiation-induced lung injury (RILI) is caused by an early inflammatory process triggered by damage to lung parenchyma, epithelial cells, vascular endothelial cells and stroma. Initially, oxidative injuries after radiation induce altered expression of pro-inflammatory cytokines. Infiltrating inflammatory cells are stimulated and activated, producing additional mediators, resulting in a cytokine cascade. The expansion and perpetual activation of inflammatory cells, as well as lung parenchyma, lead to clinical pneumonitis. Activated cells produce molecular mediators and growth factors that affect the proliferation and gene expression of lung fibroblasts. This process leads to increased collagen synthesis and deposition, eventually leading to the development of …

Functional Analysis Of A Highly Conserved Cyclin, Cyclin Y, In Drosophila Melanogaster, Nermin Gerges

Wayne State University Dissertations

Cyclin Y is a highly conserved member of the Cyclin superfamily of proteins. In Drosophila the Cyclin Y gene (CycY) is required for progression through several stages of development but the specific pathways that Cyclin Y belongs to and that account for its requirement are not known. Studies in human and Drosophila cell lines have shown that membrane-localized Cyclin Y is required for phosphorylation of the wingless/Wnt co-receptor, arrow/LRP6, and for full activation of the canonical wingless/Wnt pathway. CycY null Drosophila, however, do not phenocopy loss-of-function mutations in canonical wingless pathway genes, suggesting that Cyclin Y may have additional roles …

Towards A Unified Model Of Sperm Chromatin Structure, Graham Johnson

Wayne State University Dissertations

Sperm possess several layers of information that are delivered to the oocyte alongside the paternal DNA. Examples of potential sperm borne molecular cues of probable use to the embryo include RNAs and local and global chromatin structure. To identify candidate sperm RNAs that likely reach the oocyte cytoplasm following fertilization patterns of transcript compartmentalization in the mature gamete were identified. Though all sperm RNAs exhibited a preferential peripheral enrichment, a subset of RNAs were identified in which this trend was reduced. These RNAs are thought to be embedded with perinuclear theca and are correlated with late spermatogenic transcription. Malat1, a …

Identification, Description, And Activity Of Proteins In The Tergal Glands Of The German Cockroach, Blattella Germanica (L.), Aaron Myers

Open Access Theses

German cockroaches are important urban pests that have been linked to asthma and serious allergic reactions in sensitized individuals. In this research project I, (i) identified different proteins expressed in the tergal glands of male German cockroaches, (ii) determined the expression levels of these proteins in different cockroach life stages and tissues, and (iii) investigated the role of the tergal gland alpha-amylase (BGTG-1) protein. ^ Four major tergal gland proteins were separated on denaturing polyacrylamide gels. With peptide sequencing two of these proteins were identified as alpha-amylase (BGTG-1) and Blattella germanica allergen 2 (Bla g 2). Both of these proteins …

Transcriptional Stress Response In Foodborne Pathogens, Aaron M. Pleitner

Open Access Dissertations

Novel molecular method use is leading to better identification and understanding of microbial presence in food and food processing environments. The application of molecular methods targeting foodborne pathogens provides genetic profiling and targeted intervention methods. Whole-genome sequencing (WGS) provides information on the genomic uniqueness of specific pathogens and is being implemented in epidemiological investigations. Transcriptomic profiling allows for tracking the differential expression of genes and elucidating stress responses. Effective methods and treatments require an understanding of intervention (e.g. sanitizers) and treatment (e.g. antibiotics) mechanisms and efficacies. Accurate novel molecular method use requires proper learning of theory and data management. ^ …

Mach: A Model For Explaining Molecular And Cellular Mechanisms, Caleb M. Trujillo

Open Access Dissertations

Biologists use mechanistic explanations to understand behaviors of the immense complexity of molecular and cellular systems. In undergraduate biology courses, students are expected to explain molecular and cellular mechanisms, but teaching this skill presents many challenges due to the highly abstract, intangible nature of the cellular world, the influence of everyday language, and the tendency of students to overestimate how much they can explain. Therefore, across three studies this dissertation addresses these obstacles to teach undergraduate biology students to explain molecular and cellular mechanisms. ^ The first step was to model how biology experts explain molecular and cellular mechanisms, and …

Liver Perilipin 5 Expression Worsens Hepatosteatosis But Not Insulin Resistance In High Fat-Fed Mice, Michelle B. Trevino, David Mazur-Hart, Yui Machida, Timothy King, Joseph Nadler, Elena V. Galkina, Arjun Poddar, Sucharita Dutta, Yumi Imai

Mathematics & Statistics Faculty Publications

Perilipin 5 (PLIN5) is a lipid droplet (LD) protein highly expressed in oxidative tissues, including the fasted liver. However, its expression also increases in nonalcoholic fatty liver. To determine whether PLIN5 regulates metabolic phenotypes of hepatosteatosis under nutritional excess, liver targeted overexpression of PLIN5 was achieved using adenoviral vector (Ad-PLIN5) in male C57BL/6J mice fed high-fat diet. Mice treated with adenovirus expressing green fluorescent protein (GFP) (Ad-GFP) served as control. Ad-PLIN5 livers increased LD in the liver section, and liquid chromatography with tandem mass spectrometry revealed increases in lipid classes associated with LD, including triacylglycerol, cholesterol ester, and phospholipid classes, …