Biochemistry, Biophysics, and Structural Biology Commons^™

Protein-Protein Interactions In Cell Cycle Proteins: An In Silico Investigation Of Two Important Players, Andriele Eichner

Dissertations, Theses, and Capstone Projects

The examination of the cell cycle carries significant implications for the biology, health, and overall existence of all living things. These implications span from the development and growth of these organisms to the aging process and cancer, as well as the potential of stem cell therapies to repair diseases and injuries. Numerous proteins of the cell cycle are essential for cellular division and proliferation and are widely conserved over the course of evolution. In this work, we aimed to investigate the molecular processes of protein-protein interactions in cell cycle proteins, centering on two key players: Cdc6 in budding yeast and …

Determining The Roles Of The Oligomerization And C-Terminal Domains In Mutant P53 Gain-Of-Function Activities, George K. Annor

Dissertations, Theses, and Capstone Projects

The tumor suppressor p53 (TP53) gene is often mutated in cancer, with missense mutations found in the central DNA binding domain, and less often in the oligomerization domain (OD) and C-terminal domain (CTD). The OD and CTD have been found to be critical for the tumor suppressor functionality of wild-type p53 (wtp53). Specific missense mutations in the DNA binding domain have been found to confer new gain-of-function (GOF) activities. Mutations that destabilize tetramer formation, or deletion of key lysine residues within the CTD, downregulate the ability of wtp53 to transactivate (increase the rate of transcription of) its target …

Cdc6 Is Sequentially Regulated By Pp2a-Cdc55, Cdc14, And Sic1 For Origin Licensing In S. Cerevisiae, Jasmin Philip

Dissertations, Theses, and Capstone Projects

Control of DNA replication is critical for progression of the cell cycle and genomic stability. Cyclin-dependent kinases (CDKs) coordinate numerous phosphorylation events to accomplish two biological tasks for all living organisms: DNA replication and cell division. One CDK, Cyclin-Cdc28, is responsible for cell cycle progression in budding yeast. DNA replication requires a stepwise assembly of the pre-replicative complex on DNA, including Orc1-6, Cdc6, Cdt1 and Mcm2-7, during M-G1 phase. Cdc6 contains eight Cdc28 consensus sites, SP or TP motifs. Clb5-Cdc28 phosphorylates Cdc6-T7 to recruit Cks1, the Cdc28 phospho-adaptor, for subsequent multisite phosphorylation during S phase. There are two phospho-degrons at …

Study Of The Gain-Of-Function Mutant P53 And Parp1 In Triple-Negative Breast Cancer, Devon Lundine

Dissertations, Theses, and Capstone Projects

Cancer cells often lose expression of the p53 protein or express mutant forms of p53. Some of these mutant p53 proteins, called gain-of-function mutant p53, have gained oncogenic functions. Previously, our group observed mutant p53 R273H interacts with replicating DNA and upregulates the chromatin localization of several DNA replication factors including PCNA, MCM2-7, and PARP1 (termed the mtp53-PARP-MCM axis). In this thesis, we explore the contribution of mutant p53 and PARP1 in castration-resistant prostate cancer (mutant p53 P223L and V274F) and triple-negative breast cancer (mutant p53 R273H). In the castration-resistant prostate cancer cell line DU145, we examine two mutant p53 …

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 …

Development Of Ligand Guided Selection (Ligs) To Identify Specific Dna Aptamers Against Cell Surface Proteins, Hasan Ekrem Zumrut

Dissertations, Theses, and Capstone Projects

Oligonucleotide aptamers (nucleic acid-based affinity reagents) are an emerging class of synthetic molecules that display high affinity and specificity towards their targets. Aptamer molecules for a target of interest are obtained using a combinatorial chemistry-based method termed systematic evolution of ligands by exponential enrichment (SELEX). SELEX is an in vitro selection process in which a random oligonucleotide library is subjected to repeated cycles of target incubation, separation, and amplification until target-specific evolved sequences become prevalent in the library. Typically, SELEX is used against target molecules such as small molecules and proteins, in their purified state. However, aptamers selected against purified …

Probing The Limits Of Singular Gene Expression Through The Activity Of High Representation Odorant Receptor Transgenes, Eugene Lempert

Dissertations, Theses, and Capstone Projects

Singular gene expression is a common phenomenon in biology, making its appearance in immunoglobulin selection, protocadherin expression, X chromosome-inactivation, random monoallelic expression, and olfactory receptor choice. Singularity involves an activation and a feedback step. The mechanisms of singular gene choice have some capacity to integrate additional member genes while still maintaining singularity, but will activate an additional member if an earlier choice was incapable of triggering the feedback step. Odorant Receptor (OR) genes are substantially divergent from each other in terms of coding sequence, promoter structure, and genomic locus, all of which plays a role in how many Olfactory Sensory …

Dictyostelium Discoideum Protein Kinase C-Orthologue Pkca Regulates The Actin Cytoskeleton Through Interaction With Phospholipase D And P21-Activated Kinase, Sean Singh

Dissertations, Theses, and Capstone Projects

Proper regulation of the actin cytoskeleton is crucial to many cellular processes. Many of these processes are regulated by extracellular signaling cues, which direct changes in the actin cytoskeleton, resulting in changes to cellular morphology, and directed motility. The social amoeba, Dictyostelium discoideum, is used as a simple model system to study the translation of extracellular signals to the actin cytoskeleton. When starved, these unicellular amoebae undergo a multicellular developmental process characterized by a tightly regulated sequence of signaling events. This results in chemotaxis and formation of a multicellular aggregate, and ultimately cell differentiation and the formation of a fruiting …

Through The Back Door: Proteins Escape Cells Without Conventional Permission, Michael J. Cohen

Dissertations, Theses, and Capstone Projects

Proteins secreted to the extracellular environment play a fundamental role as signals, in metabolism, and a variety of other processes. The process of secretion through the endoplasmic reticulum and Golgi to the plasma membrane is well documented, and all cargo in this pathway contains a signal peptide. However, a variety of proteins secreted from eukaryotes lack a signal peptide and are called unconventionally secreted proteins. Here we discuss known mechanisms of unconventional protein secretion, as well as model proteins which follow characterized pathways. Additionally, we summarize the roles various unconventionally secreted proteins play outside of cells and suggest criteria for …

Hyaluronan At The Brain-Environment Interface, Donald M. Thevalingam

Dissertations, Theses, and Capstone Projects

Hyaluronan (HA; Hyaluronic Acid), a primary scaffolding component of the brain extracellular matrix, serves as an integral structural component to the brain extracellular space (ECS). The fossorial African naked mole-rat (Heterocephalus glaber; NM-R), a mammal which lives in a low-oxygen environment and is capable of tolerating hypoxia and hypercapnia, has been shown to synthesize and sustain a unique high-molecular-mass variant of hyaluronan macromolecule (HMM-HA). This body of work highlights HA’s role in mediating the interplay between brain ECM composition, ECS structure, and cell viability.

Here we employ the NM-R as a unique animal model to observe the role of the …

Towards A Mathematical Model Of Motility Using Dictyostelium Discoideum: Proteins And Geometric Features That Regulate Bleb-Based Motility, Zully Santiago

Dissertations, Theses, and Capstone Projects

A variety of biological functions depend on actin organization. The organization of actin is tightly regulated by a plethora of extracellular and intracellular signaling, scaffolding, and actin-binding proteins. Dysfunctions in this regulation lead to immune diseases, increased susceptibility to pathogens, neurodegenerative diseases, developmental disorders, and cancer metastasis. A variety of actin-dependent processes, including cell motility, are regulated by several proteins of interest: Paxillin, a scaffolding protein; WASP, an actin nucleating protein; SCAR/WAVE, another WASP family actin nucleating protein; Talin, a cortex-to-membrane binding protein; Myosin II, an F-actin contracting motor protein; and Protein Kinase C, a protein kinase. D. discoideum cells …

The Role And Regulation Of Alternative Polyadenylation In The Dna Damage Response, Michael R. Murphy

Dissertations, Theses, and Capstone Projects

Cellular homeostasis is achieved by the dynamic flux in gene expression. Post-transcriptional regulation of coding and non-coding RNA offers a fast method of adapting to a changing cellular environment, including deadenylation, microRNA (miRNA) pathway, and alternative polyadenylation (APA). In this dissertation, I explored some of the mechanisms involved in the post-transcriptional regulation of gene expression. The main hypothesis in these studies is that a single APA event after DNA damage is governed by specific conditions and factors outside of current known regulators of APA, and that the resultant transcript has a role in the DNA damage response (DDR). My aims …

Renal Risk Variants Of Apolipoprotein L-1 Form Channels At The Plasma Membrane That Lead To A Cytotoxic Influx Of Calcium, Joseph A. Giovinazzo

Dissertations, Theses, and Capstone Projects

Apolipoprotein L-1 (APOL1) is a secreted protein that provides protection against several protozoan parasites due to its channel forming properties. Recently evolved variants, G1 and G2, increase kidney disease risk when present in two copies. In mammalian cells, overexpression of G1 and G2, but not wild-type G0, leads to swelling and eventual lysis. However, the mechanism of cell death remains elusive with multiple pathways being invoked, such as autophagic cell death mediated by a BH3 domain in APOL1, which we evaluated in this study. We hypothesized that the common trigger for these pathways is the APOL1 cation channel, which is …

Phospholipase D-Dependent Mtorc1 Activation By Glutamine, Elyssa Bernfeld

Dissertations, Theses, and Capstone Projects

Glutamine, the conditionally essential amino acid and most abundant amino acid in human sera, is a key nutrient required for sustaining cell proliferation. Glutamine is essential for nucleotide, protein, and lipid synthesis, all of which are essential for cell proliferation. The mammalian target of rapamycin complex 1 (mTORC1) is a highly conserved protein complex that acts as a sensor of nutrients, relaying signals for the shift from catabolic to anabolic metabolism. While glutamine plays an important role in activating mTORC1, the mechanism is not completely clear. Here we describe a Rag-independent mechanism of mTORC1 activation by glutamine that is dependent …

Mechanisms Adopted By Dengue-2 Viruses To Induce Autophagy In Mammalian Cells, Sounak Ghosh Roy

Dissertations, Theses, and Capstone Projects

Dengue, the most rapidly spreading flavivirus, threatens to affect almost half of the human global population. We previously showed that dengue-2 protects canine kidney cells (MDCK) from cytotoxic chemicals. We showed, independently, that cell protection, as well as viral replication and maturation, are positively regulated by PI3K-dependent autophagy. However, we had not identified the specific pathway that induces autophagy in infected cells. The current study explores the role of a specific branch of the endoplasmic reticulum (ER) stress-mediated Unfolded Protein Response (UPR), the PERK/eIF2α/ATF4 pathway in the induction of autophagy by …

Regulation Of The Tubulin Homolog Ftsz In Escherichia Coli, Monika S. Buczek

Dissertations, Theses, and Capstone Projects

Escherichia coli is a well-known pathogen, and importantly, a widely used model organism in all fields of biological sciences for cloning, protein purification, and as a model for Gram-negative bacterial species. And yet, researchers do not fully understand how this bacterium replicates and divides. Every year additional division proteins are discovered, which adds complexity to how we understand E. coli undergoes cell division. Due to their specific roles in cytokinesis, some of these proteins may be potential targets for development of antibacterials or bacteriostatics, which are much needed for fighting the current global antibacterial deficit. My thesis work focuses on …

Mechanisms For Survival And Drug Resistance In Cancer Cells, Matthew B. Utter

Dissertations, Theses, and Capstone Projects

PART I

Prostate cells are hormonally driven to grow and divide. Typical treatments for prostate cancer involve blocking the hormone androgen from activating the androgen receptor (AR) and thus inhibit growth and proliferation of the cancer. Androgen deprivation therapy (ADT) can lead to the selection of cancer cells that grow and divide independently of androgen receptor activation. Prostate cancer cells that are insensitive to androgens commonly display metastatic phenotypes and reduced long-term survival of patients. In this study, we provide evidence that androgen-insensitive prostate cancer cells have elevated phospholipase D (PLD) activity relative to the androgen-sensitive prostate cancer cells. PLD …

Lipid Sensing By Mammalian Target Of Rapamycin, Deepak Menon

Dissertations, Theses, and Capstone Projects

Mammalian target of Rapamycin (mTOR) is a protein kinase that integrates nutrient and growth factor signals to promote cellular growth and proliferation. mTOR exists in two complexes - mTORC1 and mTORC2 that are distinguished by their binding partners and signaling inputs. mTORC1 is responsive to growth factors, amino acids and glucose and is associated with Raptor; whereas, mTORC2 is responsive primarily to growth factors and is associated with Rictor. Raptor and Rictor confer substrate specificity to mTORC1 and mTORC2 respectively. Phosphatidic acid (PA), a lipid second messenger and a central metabolite for membrane phospholipid biosynthesis, is required for the stability …

Transcriptional And Post-Transcriptional Regulation Of Histone Variant H2a.Z During Sea Urchin Development, Mihai Hajdu

Dissertations, Theses, and Capstone Projects

Histone variant H2A.Z promotes chromatin accessibility at transcriptional regulatory elements and is developmentally regulated in metazoans. We characterize the transcriptional and post-transcriptional regulation of H2A.Z in the purple sea urchin Strongylocentrotus purpuratus. H2A.Z depletion by antisense translation-blocking morpholino oligonucleotides during early development causes developmental collapse, in agreement with its previously demonstrated general role in transcriptional multipotency. During H2A.Z peak expression in 24-h embryos, endogenous H2A.Z 3’ UTR sequences stabilize GFP mRNAs relative to those with SV40 3’ UTR sequences, although the 3’UTR of H2A.Z does not determine the spatial distribution of H2A.Z transcripts during embryonic and postembryonic development. We …

Molecular Analysis Of Ftsz-Ring Assembly In E. Coli Cytokinesis, Kuo-Hsiang Huang

Dissertations, Theses, and Capstone Projects

An essential first step in bacterial division is the assembly of a cytokinetic ring (Z-ring) formed by the tubulin-like FtsZ at midcell. The highly conserved core domain of FtsZ has been reported to mediate assembly of FtsZ polymers in vivo and in vitro. Species-specific differences in the FtsZ C-terminal domain such as the FtsZ CTV region and interactions with several modulatory proteins such as ZapC and ZapD, restricted to certain bacterial classes, also serve as key determinants of FtsZ protofilament bundling. Here, we characterize (i) the roles of the FtsZ CTV region in mediating both longitudinal and lateral interactions …

Chamber-Specific Patterns Of Epicardium Formation In Zebrafish, Sana Khan

Dissertations, Theses, and Capstone Projects

The outer cardiac layer, the epicardium, coordinates the final steps of vertebrate heart development. This cardiac tissue arises from cells in the proepicardial organ (PEO) that forms around the base of the inflow tract. Its general location is conserved across species despite morphological differences. Cellular mechanisms of migration differ across species. Three strategies of PEO migration are described: 1) The floating cyst model - PEO cells released into the pericardial cavity are directed by fluid movements to migrate onto the myocardium; 2) Villi transfer - cardiac contractions may mediate multicellular PEO villi contact to the myocardium; and 3) Tissue bridge-mediated …

Polymerase Alpha Components Associate With Telomeres To Mediate Overhang Processing, Raffaella Diotti

Dissertations, Theses, and Capstone Projects

Telomeres consist of TTAGGG repeats, which end with a 3' G-overhang and are bound by a six-protein complex, known as Shelterin. In humans, telomeres shorten at each cell division, unless telomerase is expressed and able to add telomeric repeats to the 3' G-overhang. However, for effective telomere maintenance, the DNA strand complementary to that made by telomerase must be synthesized. In this study, I focused on the Polα/primase complex, in particular the subunits p68 (POLA2, the regulatory subunit) and p180 (Polα, the catalytic subunit), and their potential roles at telomeres. I was able to detect p180, p68 and OBFC1, a …

Metabolic Checkpoints In Cancer Cell Cycle, Mahesh Saqcena

Dissertations, Theses, and Capstone Projects

Growth factors (GFs) as well as nutrient sufficiency regulate cell division in metazoans. The vast majority of mutations that contribute to cancer are in genes that regulate progression through the G1 phase of the cell cycle. A key regulatory site in G1 is the growth factor-dependent Restriction Point (R), where cells get permissive signals to divide. In the absence of GF instructions, cells enter the quiescent G0 state. Despite fundamental differences between GF signaling and nutrient sensing, they both have been confusingly referred to as R and therefore by definition considered to be a singular event in G1. Autonomy from …

Lipid Dependence In Ras-Driven Tumors, Darin Salloum

Dissertations, Theses, and Capstone Projects

Over past decade, metabolic alterations in cancer cells have received a substantial amount of interest. It had been established that cancer cells undergo a significant amount of metabolic alterations, and some of these alterations are similar to those in normal highly proliferative cells. However, it is becoming more apparent that many of the metabolic alterations are specific to particular oncogenic signaling pathways. Although altered metabolic machinery makes cancer cells more efficient at promoting growth when nutrients are supplied at the sufficient amounts, the dependency of cancer cells on particular metabolic reprogramming deems cancer cells susceptible to disruptions within metabolic network. …

The Sex Of The Cell Dictates Its Response, Carlos Ganesh Penaloza

Dissertations, Theses, and Capstone Projects

Male and female differences in frequency of occurrence in disease have perplexed scientists for some time. This in part derives from limitations in the systems in which one can evaluate sex differences. At the organismal level, differences can be hidden by a myriad of extensive and complex factors. Additional limitations exist since most biomedical studies are performed almost exclusively on male subjects, as the female hormonal milieu is intrinsically more variable and too troublesome for routine inclusion in research protocols. Research documenting sex differences continues to grow, and while most researchers suggests that sex hormones are at the core of …

Identification And Characterization Of Protein Phopshatases Regulating The Sma/Mab Pathway In C. Elegans, Sheng Xiong

Dissertations, Theses, and Capstone Projects

TGF-beta signaling is a conserved signaling pathway among eukaryotes, which controls various normal cellular responses from cell proliferation to cell death. The mutations in its components are found in developmental disorders and cancer. Therefore, this signaling pathway is extensively investigated so that new therapeutic targets could be discovered and novel drugs could be developed. Previous studies suggested the involvement of phosphatases in regulation of TGF-beta signaling, but these studies were performed in cell culture rather than intact organisms. C. elegans is a tractable organism in which to study signaling in vivo. In C. elegans, growth is controled by a conserved …