Molecular Biology Commons^™

Prophylactic Cranial Irradiation Reduces The Incidence Of Brain Metastasis In A Mouse Model Of Metastatic Breast Cancer, Daniel L. Smith

Dissertations and Theses (Open Access)

Prophylactic cranial irradiation (PCI) is a preventative whole-brain irradiation technique used to reduce the incidence of brain metastasis and improve overall survival in select patients with small cell lung cancer and acute lymphoblastic leukemia. A population of breast cancer patients – stage IV, HER2+ or triple-negative – has emerged as having a high risk of developing brain metastases. Because only 10-20% of breast cancer patients diagnosed with brain metastases survive longer than one year, in this high-risk population the benefit of PCI – potential for reduced incidence of brain metastasis and improved overall survival – may outweigh the risks – …

Direct Regulation Of Apoptosis By Linear Ubiqutin Chain Assembly Complex (Lubac) And Feedback Regulation Of Lubac Function By Caspases, Donghyun Joo

Dissertations and Theses (Open Access)

Tumor Necrosis Factor-alpha (TNF-α) is a cytokine that plays a role in various cellular processes such as proliferation, differentiation (mainly through NF-κB signaling) and death (via apoptosis signaling). Recently, linear ubiquitination by LUBAC (linear ubiquitin chain assembly complex) was reported to have a regulatory function in TNF-α mediated NF-κB activation. Although LUBAC is suggested to control not only NF-kB signaling but also the apoptosis pathway, the precise mechanism of apoptosis regulation remains unknown. Moreover, NF-κB and apoptosis pathways have opposed but fundamental functions for various cellular processes. Although these two pathways actively interplay to balance the death and survival, the …

Histone H3 K4 Methylation Regulates The Spindle Assembly Checkpoint Through Direct Binding Of Multiple Checkpoint Components And Cdc20, Andria C. Schibler

Dissertations and Theses (Open Access)

Histone H3K4 methylation is conserved across species and is associated with active transcription. By using Saccharomyces cerevisiae, we found histone H3K4 methylation has a previously unknown role in regulating mitosis through the Spindle Assembly Checkpoint. The Spindle Assembly Checkpoint ensures duplicated chromosomes are segregated correctly and each daughter cell receives one full copy of the genome. Our data show SET1 mutants and histone H3K4 mutants display a resistance to the mitotic poison, benomyl. Moreover methylated histone H3 directly binds to Spindle Assembly Checkpoint proteins Bub3 and Mad2 as well as the activator of the Anaphase Promoting Complex (APC) protein …

The Effect Of Activation Induced Cytidine Deaminase Phosphorylation And Herpes Virus Uracil Dna Glycosylase On Antibody Diversification, Marc Macaluso

Dissertations and Theses (Open Access)

Activation-induced cytidine deaminase (AID) is a mutagenic enzyme that is expressed in mammalian B-cells and initiates the antibody diversification processes of somatic hypermuntation (SHM) and isotype class switch recombination (CSR). AID is targeted to the immunoglobulin gene locus where it deaminates cytosines to generate uracil residues in DNA. This generates guanine-uracil (U:G) mismatch lesion which are recognized by uracil DNA glycosylase (UNG), a DNA repair enzyme that removes uracil from DNA and triggers downstream repair of the lesion. While UNG is a ubiquitously expressed DNA repair enzyme, its recognition and removal of AID introduced uracils is essential in both SHM …

Dna Polymerase Θ (Polq) And The Cellular Defense Against Dna Damage, Matthew J. Yousefzadeh

Dissertations and Theses (Open Access)

In mammalian cells, DNA polymerase θ (POLQ) is an unusual specialized DNA polymerase whose in vivo function is under active investigation. The protein is comprised of an N-terminal helicase-like domain, a C-terminal DNA polymerase domain, and a large central domain that spans between the two. This arrangement is also found in the Drosophila Mus308 protein, which helps confer resistance to DNA interstrand crosslinking agents. Homologs of POLQ and Mus308 are found in eukaryotes, including plants, but a comparison of phenotypes suggests that not all of these genes are functional orthologs. Flies with defective Mus308 are sensitive to DNA interstrand crosslinking …

Investigating The Roles Of P63 And P73 Isoforms To Therapeutically Treat P53-Altered Cancers, Avinashnarayan Venkatanarayan

Dissertations and Theses (Open Access)

Investigating the roles of p63 & p73 isoforms to therapeutically treat

p53-altered cancers

Avinashnarayan Venkatanarayan, M.S.

Supervisory Professor: Elsa R. Flores, Ph.D.

The TP53 tumor suppressor is mutated in approximately 50% of human cancers rendering cancer therapies ineffective. p53 reactivation suppresses tumor formation in mice. However, this strategy has proven difficult to implement therapeutically. An alternate approach to overcome p53 loss is to manipulate the p53-family members, p63 and p73, which interact and share structural similarities to p53. p63 and p73, unlike p53 are less frequently mutated and have two major isoforms with distinct functions …

Measuring Single Cell Responses To Lapatinib In A Heterogeneous Population, Preety Priya

Dissertations and Theses (Open Access)

Cancer is notonedisease butasaga of diseases and is the outcome of disturbed homeostasis in the normal cells due to the deregulation of its genetic makeup. With advent of technologies thatallowdetailed molecular characterizationoftumors, targeted therapies have emerged as a more promising and specific mode of treatment. However, a major challenge with targeted therapy is the acquired resistance in the cancer cells to these therapies, quite often very rapidly in the course of a few months. One of the major targets in cancer has been the EGFR/ErbB2 network in breast and other cancer types. Prior work from our lab and others have …

Regulation Of Cell Adhesion By The Ferm Proteins, Ptpn14 And Merlin, Patty Dimarco Hewitt

Dissertations and Theses (Open Access)

Cell-cell adhesion is critical for the control of tissue organization and homeostasis. A family of proteins that regulate cell-cell adhesions is the FERM (4.1 protein, Ezrin, Radixin, Moesin) domain-containing proteins.One FERM domain protein, the non-receptor tyrosine phosphatase PTPN14, is mutated or deleted in several human cancers suggesting that it may be involved in tumor development and/or progression. Additionally, the loss of the FERM domain protein Merlin is associated with tumor development and metastasis.Both PTPN14 and Merlin have been shown to localize and possibly regulate adherens junction (AJ) functions. This work sought to determine if …

Novel Posttranslational Modification In Lkb1 Activation And Function, Szu-Wei Lee

Dissertations and Theses (Open Access)

Cancer cells display dramatic alterations in cellular metabolism to meet their needs of increased growth and proliferation. In the last decade, cancer research has brought these pathways into focus, and one emerging issue that has come to attention is that many oncogenes and tumor-suppressors are intimately linked to metabolic regulation (Jones and Thompson, 2009). One of the key tumor-suppressors involved in metabolism is Liver Kinase B1 (LKB1). LKB1 is the major upstream kinase of the evolutionarily conserved metabolic sensor—AMP-activated protein kinase (AMPK). Activation of the LKB1/AMPK pathway provides a survival advantage for cells under energy stress. LKB1 forms a heterotrimeric …

Swarna Ramaswamy_Thesis, Swarna S. Ramaswamy

Dissertations and Theses (Open Access)

STRUCTURAL INVESTIGATIONS OF LIGAND GATED ION CHANNELS

Swarna Ramaswamy, B.S

Advisor: Vasanthi Jayaraman, Ph.D.

Ion channels form an integral part of membrane proteins. In the nervous system including the central and the peripheral nervous system, ligand gated ion channels form a very important part of intercellular communications. They receive chemical signals and convert them to electrical signal, mainly by allowing ion passage across the cell membrane. Ion passage also translates into downstream signaling events. Faithful translation of these signals and transmittance is crucial for several physiological functions, implying that irregular ion channel function could lead to serious consequences.

This thesis …

Jab1 Negatively Regulates Pten And Promotes Resistance To Trastuzumab In Her2-Positive Breast Cancer, Thuy T. Vu

Dissertations and Theses (Open Access)

HER2-positive breast cancer, which is characterized by the over-expression of the HER2 onco-protein, accounts for approximately 20% of all breast cancer cases. Trastuzumab (Herceptin), the first targeted therapy approved for HER2-positive disease, potently prevents the activation of signaling pathways downstream of HER2 and significantly improves patients’ outcomes. However, resistance to trastuzumab is inevitable; such resistance can occur through reduced expression of PTEN protein.

Jab1 is over-expressed in 50% of primary cancers and 90% of metastatic tumors. Our lab previously showed that depletion of Jab1 in combination with trastuzumab treatment up-regulated PTEN in mouse xenografts refractory to trastuzumab. PTEN was not …

Pi3k- And Mtor-Dependent Mechanisms Of Lapatinib Resistance And Resulting Therapeutic Opportunities, Samuel Brady

Dissertations and Theses (Open Access)

Breast cancers with HER2 amplification represent 20-25% of breast cancer cases and are frequently responsive to the HER2 kinase inhibitor lapatinib, but generally for only short duration. We aimed to understand how breast cancers with HER2 amplification become resistant to lapatinib, in order to identify potential therapies that can overcome lapatinib resistance. To establish lapatinib resistance models we treated three HER2+ breast cancer cell lines with lapatinib for several months until they became lapatinib-resistant. We then compared lapatinib-sensitive (parental) cells with their lapatinib-resistant (LapR) counterparts to identify changes conferring lapatinib resistance. We found that activation of PI3K, specifically the p110α …

Roles For B-Raf Kinase In The Specific Regulation Of Α4Β1 Integrin In T Cells, Wells S. Brown

Dissertations and Theses (Open Access)

The regulation of integrin-mediated adhesion is of vital importance to adaptive and innate immunity. Integrins are versatile proteins and mediate T cell migration and trafficking by binding to ECM or other cells, as well as initiating intracellular signaling cascades promoting survival or activation. The mitogen activated-protein kinase (MAPK) pathway is known to be downstream from integrins and regulate survival, differentiation, and motility. However, secondary roles for canonical MAPK pathway members are being discovered. We show chemical inhibition of RAF by Sorafenib or shRNA-mediated knockdown of B-Raf reduces T cell resistance to shear stress to α4β1 integrin ligands vascular cell adhesion …

Egfr Modulates Microrna Maturation In Response To Hypoxia Through Phosphorylation Of Argonaute2, Jia Shen

Dissertations and Theses (Open Access)

MicroRNAs (miRNAs) are generated by two-step processing to yield small RNAs that negatively regulate target gene expression at posttranscriptional level. Deregulation of miRNAs has been linked to diverse pathological processes, including cancer. Recent studies have also implicated miRNAs in regulatory roles to cope with a spectrum of stresses, such as hypoxia, which is frequently encountered in the poorly angiogenic core of a solid tumor. However, the upstream regulators of miRNA biogenesis machineries remain obscure, raising the question of how tumor cells efficiently coordinate and impose specificity on miRNA expression and function in response to stresses. Here, we show that EGFR, …

Energy Stress Causes Chaperones To Assemble Into Cytoplasmic Complexes, Kimberly J. Cope

Dissertations and Theses (Open Access)

The majority of proteins require molecular chaperones to assist their folding into tertiary and quaternary structures. Certain stresses can compromise the weak hydrophobic forces responsible for these structures and lead to protein unfolding, misfolding, and aggregation. Aggregates of proteins are hallmarks of devastating diseases such as Alzheimer’s, Parkinson’s, and Huntington’s diseases. Fortunately, bacteria, plants, and fungi have a potent disaggregase, named Hsp104 in Saccharomyces cerevisiae. Recently, heat-induced aggregates, termed Q-bodies, were found to contain three molecular chaperones: Hsp70, Hsp104, and Hsp42. Their coalescence from small puncta into larger inclusions required Hsp104. During glucose deprivation, a stress that isn’t known to …

Functional Analysis Of Cytosolic Hsp70 Nucleotide Exchange Factor Networks In Yeast, Jennifer Lynn Abrams

Dissertations and Theses (Open Access)

The Hsp70 class of molecular chaperones play critical roles in protein homeostasis via an ATP-dependent folding cycle. Cytosolic Hsp70s in the budding yeast Saccharomyces cerevisiae, Ssa and Ssb, interact with up to three distinct nucleotide exchange factors (NEFs) homologous to human counterparts; Sse1/Sse2/HSP110, Fes1/HspBP1, and Snl1/Bag1. In an effort to understand the differential functional contributions of the cytosolic NEFs to protein homeostasis (“proteostasis”), I carried out comparative genetic, biochemical and cell biological analyses. For these studies, I developed protocols to monitor protein disaggregation and reactivation in a near real-time coupled assay that revealed the importance of aggregate dynamics in the …

Characterization Of Ftsa-Ftsn Interaction During Escherichia Coli Cell Division, [email protected] K. Busiek

Dissertations and Theses (Open Access)

Division of a bacterial cell into two equal daughter cells requires precise assembly and constriction of the division machinery, or divisome. The Escherichia coli divisome includes nearly a dozen essential cell division proteins that assemble at midcell between segregating sister chromosomes. FtsZ, a homolog of eukaryotic tubulin, is the first essential cell division protein to localize at midcell where it polymerizes into a ring-shaped scaffold (Z ring). Establishment of the Z ring is required for recruitment of downstream cell division proteins including FtsA, a cytoplasmic protein that tethers the Z ring to the inner membrane. Following localization of FtsA and …

Diabetes And Obesity Induce Transcriptomic And Metabolomic Changes Enhancing Pancreatic Cancer Aggressiveness, Guermarie Velázquez Torres

Dissertations and Theses (Open Access)

Pancreatic cancer is one of the most aggressive types of cancer, with poor prognosis that lacks effective diagnostic markers and therapies. It is expected that in 2014 the incidence and the mortality of pancreatic cancer in the United States will be 46,420 and 39,590 respectively. Diabetes and obesity are modifiable risk factors associated with accelerated pancreatic carcinogenesis and tumor progression, but the biological mechanisms are not completely understood. The purpose of this study is to demonstrate direct evidence for the mechanisms mediating these epidemiologic phenomena. Our hypothesis is that obesity and diabetes mellitus type 2 (DM2) accelerate pancreatic cancer and …

The Regulation Of Microrna Biogenesis By Ribosome-Interacting Proteins, Brian Pickering

Dissertations and Theses (Open Access)

MicroRNA (miRNA) are small, non-coding RNAs that affect gene expression through degradation of complementary mRNA targets or inhibition of translation. As they affect approximately 50% of all cellular processes, miRNA are tightly regulated by the cell through transcriptional and post-transcriptional mechanisms. Transcribed miRNA are capped and polyadenylated (referred to as pri-miRNA) which are cleaved by Drosha and DGCR8 to generate 60-90 nucleotide precursor miRNA. The precursors are cleaved again by Dicer and loaded into the RNA-induced silencing complex (RISC) of which Argonaute 2 is the functional component. Many of the proteins involved in miRNA biogenesis share a common role in …

Characterization Of The Rna Binding And Rna Degrading Subunits Of The Eukaryotic Exosome, Borislava Tsanova

Dissertations and Theses (Open Access)

The exosome is an essential complex of ten proteins involved in the processing and degradation of many RNAs in the cell. These include various stable RNAs, mRNAs, and aberrant transcripts both in the nucleus and in the cytoplasm.

In this work I characterize the three members of the exosome “cap”, the RNA binding proteins Rrp4, Rrp40, and Csl4. I determine that in spite of their structural similarity, they each have a unique essential role. Second, I determine that two of the cap proteins Rrp4 and Rrp40 have a role in bridging subunits of the PH ring of the exosome. The …

Studying Aggregate Formation By Amyotrophic Lateral Sclerosis-Associated Mutant Sod1 Protein In Drosophila Model, Michael Mccarthy

Dissertations and Theses (Open Access)

A common pathological hallmark of most neurodegenerative disorders is the presence of protein aggregates in the brain. Understanding the regulation of aggregate formation is thus important for elucidating disease pathogenic mechanisms and finding effective preventive avenues and cures. Amyotrophic Lateral Sclerosis (ALS), also known as Lou Gehrig’s disease, is a selective neurodegenerative disorder predominantly affecting motor neurons. The majority of ALS cases are sporadic, however, mutations in superoxide dismutase 1 (SOD1) are responsible for about 20% of familial ALS (fALS). Mutated SOD1 proteins are prone to misfold and form protein aggregates, thus representing a good candidate for studying aggregate formation. …

Characterization Of Jak, Stat, And Src Interactions In Head And Neck Squamous Cell Carcinoma, Reshma Jaseja, Reshma Jaseja

Dissertations and Theses (Open Access)

Recurrence of Head and Neck Squamous Cell Carcinoma (HNSCC) is common; thus, it is essential to improve the effectiveness and reduce toxicity of current treatments. Proteins in the Src/Jak/STAT pathway represent potential therapeutic targets, as this pathway is hyperactive in HNSCC and it has roles in cell migration, metastasis, proliferation, survival, and angiogenesis. During short-term Src inhibition, Janus kinase (Jak) 2, and signal transducer and activator of transcription (STAT) 3 and STAT5 are dephosphorylated and inactivated. Following sustained Src inhibition, STAT5 remains inactive, but Jak2 and STAT3 are reactivated following their early inhibition. To further characterize the mechanism of this …

Mechanisms Underlying The Heterogeneous Sensitivities Of Cancer Cells To Proteasome Inhibitors, Matthew C. White

Dissertations and Theses (Open Access)

The mechanisms underlying cellular response to proteasome inhibitors have not been clearly elucidated in solid tumor models. Evidence suggests that the ability of a cell to manage the amount of proteotoxic stress following proteasome inhibition dictates survival. In this study using the FDA-approved proteasome inhibitor bortezomib (Velcade®) in solid tumor cells, we demonstrated that perhaps the most critical response to proteasome inhibition is repression of global protein synthesis by phosphorylation of the eukaryotic initiation factor 2-α subunit (eIF2α). In a panel of 10 distinct human pancreatic cancer cells, we showed marked heterogeneity in the ability of cancer cells to induce …

Genome-Wide Profiling Unveils Criticial Functions Of P53 In Human Embryonic Stem Cells, Kadir C. Akdemir

Dissertations and Theses (Open Access)

Embryonic stem cells (ESCs) possess two unique characteristics: infinite self-renewal and the potential to differentiate into almost every cell type (pluripotency). Recently, global expression analyses of metastatic breast and lung cancers revealed an ESC-like expression program or signature, specifically for cancers that are mutant for p53 function. Surprisingly, although p53 is widely recognized as the guardian of the genome, due to its roles in cell cycle checkpoints, programmed cell death or senescence, relatively little is known about p53 functions in normal cells, especially in ESCs. My hypothesis is that p53 has specific transcription regulatory functions in human ESCs (hESCs) that …

Interaction Between Brk And Her2 In Breast Cancer, Midan Ai

Dissertations and Theses (Open Access)

INTERACTION BETWEEN BRK AND HER2 IN BREAST CANCER

Midan Ai, Ph.D.

Supervisory Professor: Zhen Fan, M.D.

Breast tumor kinase (Brk) is a nonreceptor protein-tyrosine kinase that is highly expressed in approximately two thirds of breast cancers but is not detectable or is expressed at very low levels in normal mammary epithelium. Brk plays important roles in promoting proliferation, survival, invasion, and metastasis of breast cancer cells, but the mechanism(s) of which remain largely unknown. Recent studies showed that Brk is frequently co-overexpressed with human epidermal growth factor receptor-2 (HER2) and is physically associated with HER2 in breast cancer. The mechanism …

Structure-Function Analysis Of Human Integrator Subunit-4, Anupama Sataluri

Dissertations and Theses (Open Access)

Structure-function analysis of human Integrator subunit 4

Anupama Sataluri

Advisor: Eric. J. Wagner, Ph.D.

Uridine-rich small nuclear RNAs (U snRNA) are RNA Polymerase-II (RNAPII) transcripts that are ubiquitously expressed and are known to be essential for gene expression. snRNAs play a key role in mRNA splicing and in histone mRNA expression. Inaccurate snRNA biosynthesis can lead to diseases related to defective splicing and histone mRNA expression. Although the 3′ end formation mechanism and processing machinery of other RNAPII transcripts such as mRNA has been well studied, the mechanism of snRNA 3′ end processing has remained a mystery until the recent …

Functional Analysis Of Drosophila Integrator Complex In Snrna 3' End Processing, Jiandong Chen

Dissertations and Theses (Open Access)

Uridine-rich small nuclear RNAs (U snRNAs) play essential roles in eukaryotic gene expression by facilitating the removal of introns from mRNA precursors and the processing of the replication-dependent histone pre-mRNAs. Formation of the 3’ end of these snRNAs is carried out by a poorly characterized, twelve-membered protein complex named Integrator Complex.

In the effort to understand Integrator Complex function in the formation of the snRNA 3’ end, we performed a functional RNAi screen in Drosophila S2 cells to identify protein factors required for snRNA 3’ end formation. This screen was conducted by using a fluorescence-based reporter that elicits GFP expression …

A Study On The Function Of 14-3-3sigma In Regulating Cancer Energy Metabolism, Liem M. Phan, Liem M. Phan

Dissertations and Theses (Open Access)

Metabolic reprogramming has been shown to be a major cancer hallmark providing tumor cells with significant advantages for survival, proliferation, growth, metastasis and resistance against anti-cancer therapies. Glycolysis, glutaminolysis and mitochondrial biogenesis are among the most essential cancer metabolic alterations because these pathways provide cancer cells with not only energy but also crucial metabolites to support large-scale biosynthesis, rapid proliferation and tumorigenesis. In this study, we find that 14-3-3σ suppresses all these three metabolic processes by promoting the degradation of their main driver, c-Myc. In fact, 14-3-3s significantly enhances c-Myc poly-ubiquitination and subsequent degradation, reduces c-Myc transcriptional activity, and down-regulates …

Fancm And Faap24 Maintain Genomic Stability Through Cooperative And Unique Functions, Yucai Wang

Dissertations and Theses (Open Access)

Fanconi anemia (FA) is a rare recessive genetic disease with an array of clinical manifestations including multiple congenital abnormalities, progressive bone marrow failure and profound cancer susceptibility. A hallmark of cells derived from FA patients is hypersensitivity to DNA interstrand crosslinking agents such as mitomycin C (MMC) and cisplatin, suggesting that FA- and FA-associated proteins play important roles in protecting cells from DNA interstrand crosslink (ICL) damage. Two genes involved in the FA pathway, FANCM and FAAP24, are of particular interest because they contain DNA interacting domains. However, there are no definitive patient mutations for these two genes, and the …

Differential Activity Of The Kras Oncogene By Method Of Activation: Implications For Signaling And Therapeutic Intervention, Nathan Ihle

Dissertations and Theses (Open Access)

Despite having been identified over thirty years ago and definitively established as having a critical role in driving tumor growth and predicting for resistance to therapy, the KRAS oncogene remains a target in cancer for which there is no effective treatment. KRas is activated b y mutations at a few sites, primarily amino acid substitutions at codon 12 which promote a constitutively active state. I have found that different amino acid substitutions at codon 12 can activate different KRas downstream signaling pathways, determine clonogenic growth potential and determine patient response to molecularly targeted therapies. Computer modeling of the KRas structure …