Molecular Biology Commons^™

Functional Analysis Of Rpl22: Coordinating Differentiation Signals And Developmental Gene Translation To Promote Cell Fate Decisions, Stephen Kim

Graduate School of Biomedical Sciences Theses and Dissertations

The execution of meiosis and spore formation in S. cerevisiae requires a transient transcription program that is initiated by Ime I-dependent destruction of the repressor Ume6. IMEJ transcription is tightly contro1led by both cell type and nutritional cues but how its translation is regulated has been unknown. This study demonstrates that Rpl22A and Rpl22B, eukaryotic-specific ribosomal protein paralogs of the 60S large subunit, are required for efficient Ime I translation. While IME1 mRNA is induced normally in the absence of both para logs following transfer to sporulation medium, Ume6 remains stabilized, downstream early meiotic gene remain repressed, and development is …

Conformational Dynamics Of Dnaa Protein Of Bacillus Anthracis In The Initiation Of Dna Replication: A Fluorescence Resonance Energy Transfer Study, Meera J. Patel

Graduate School of Biomedical Sciences Theses and Dissertations

A precursor of the origin recognition complex proteins in eukaryotes, the DnaA protein, a single-subunit protein found in all prokaryotes, p1ays a significant role in the initiation of DNA replication. The DnaA protein in Gram-negative Escherichia coli (DnaA_EC) identifies, binds and activates specific DNA sequences at the origin of replication, oriC, initiating the duplication of the prokaryotic genome. Using DnaA_EC as a guide, we selected the full-length form of DnaA from Gram-positive Bacillus anthracis (DnaA_BA) as our model for studying. We have cloned, mutated, expressed and purified large quantities of DnaA_BA protein due to …

Structural And Functional Interactions Between Bro1 Domain Of Human Alix Protein And Nucleocapsid Packaging Rna Complex From Hiv, Scott Gross

Graduate School of Biomedical Sciences Theses and Dissertations

A virus is only as powerful as its ability to spread. Enveloped retroviruses, namely HIV-1, use exocytosis pathways that normal host cells use to release particles from the plasma membrane. The main pathways of interest in this study are the Endosomal Sorting Complex Required for Transport (ESCRT) and adjacent ALIX pathways. The ESCRT pathway is especially important for degradation of receptor/cargo complexes that form Multi-Vesicular Bodies (MVBs). Currently, there is no known therapy that targets this endosomal pathway, which would prevent the spread of the virus to other cells. The virus has adapted to jump from pathway to pathway when …

Age-Dependent Breakdown Of The Blood Brain Barrier And Associated Changes In S100b Ko Mice, Eric V. Brown

Graduate School of Biomedical Sciences Theses and Dissertations

Autoantibodies play an important role in many autoimmune diseases. Recent research has shown that breakdown of the blood brain barrier (BBB) occurs concomitant to generation of brain reactive autoantibodies in many neurodegenerative diseases, which serve as biomarkers and drivers of pathology. SI00B, a calcium binding protein found most highly expressed in astrocytes which ensheathe the BBB, has many functions in neural development and signaling. Currently literature indicates that S100B KO mice develop normally, with no phenotypic abnormalities. Here, it is demonstrated that S100B KO mice seem to develop a chronic BBB breakdown similar to that seen in human neurodegenerative diseases. …

Evolution Of Sperm Activation In Nematodes, Qing Wei

Graduate School of Biomedical Sciences Theses and Dissertations

Three hermaphroditic species in the nematode genus Caenorhabditis - C. elegans, C. briggsae and C. tropicalis evolved independently from a male/female ancestor. Thus, the hermaphroditic reproductive system provides an ideal subject for studying the origin of new complex traits. Previous studies implied that this transition required two steps: a mutation that caused XY animals to make sperm, and a mutation that allowed XX spermatids to activate and fertilize oocytes. In this dissertation, I show how sperm activation is regulated in different hermaphroditic species.

First, I used TALENs to create genetic tools for C. briggsae and C. tropicalis. These tools were …

Meiotic Checkpoint Activation Promotes Anaphase Promoting Complex/Cyclosome Substrate Specificity And Ume6/Sin3/Rpd3-Independent Reduction In Emg Expression, Rebecca Lewandowski

Graduate School of Biomedical Sciences Theses and Dissertations

Complex differentiation pathways, such as yeast meiosis and sporulation, are initiated in response to cell type and external stimuli and are driven by changes in tightly regulated and temporally expressed transcriptional programs. These programs are monitored by checkpoint mechanisms that couple meiotic progression to transcriptional activity to ensure each stage is successfully completed prior to progression into the next phase. This work investigated transcriptional regulation controlling meiotic progression when cells sense an insult to genetic integrity. These studies revealed that the early meiotic gene (EMG) repressor Ume6p is degraded during meiotic checkpoint activation triggered by the deoxyribonucleotide reductase inhibitor and …

Novel Biomarkers Can Be Used As Targets To Combat Cancer, Harini Krishnan

Graduate School of Biomedical Sciences Theses and Dissertations

Cancer kills almost 8 million people per year worldwide. Therefore, about 13 people die from cancer every minute. Clearly, current cancer treatments are not completely effective. Moreover, many chemotherapeutic reagents are not very specific for cancer cells.

These agents attack rapidly dividing cells, including a wide array of normal cells, in addition to cancer cells, in the body. This lack of specificity can cause collateral damage and significant side effects in patients. More targeted therapies are needed to successfully combat cancer. Specific cancer biomarkers need to identified and characterized in order to develop better targeted anti-cancer drugs.

Tumor cells can …

Nack Is An Integral Component Of The Notch Transcriptional Activation Complex And Is Critical For Development And Tumorigenesis, Kelly L Weaver, Marie-Clotilde Alves-Guerra, Ke Jin, Zhiqiang Wang, Xiaoqing Han, Prathibha Ranganathan, Xiaoxia Zhu, Thiago Dasilva, Wei Liu, Francesca Ratti, Renee M Demarest, Cristos Tzimas, Meghan Rice, Rodrigo Vasquez-Del Carpio, Nadia Dahmane, David J Robbins, Anthony J Capobianco

Rowan-Virtua School of Osteopathic Medicine Departmental Research

The Notch signaling pathway governs many distinct cellular processes by regulating transcriptional programs. The transcriptional response initiated by Notch is highly cell context dependent, indicating that multiple factors influence Notch target gene selection and activity. However, the mechanism by which Notch drives target gene transcription is not well understood. Herein, we identify and characterize a novel Notch-interacting protein, Notch activation complex kinase (NACK), which acts as a Notch transcriptional coactivator. We show that NACK associates with the Notch transcriptional activation complex on DNA, mediates Notch transcriptional activity, and is required for Notch-mediated tumorigenesis. We demonstrate that Notch1 and NACK are …

The Development Of A Comprehensive Antifungal Susceptibility Testing Assay For Vulvovaginal Candidiasis Therapy, David Hardaker

Graduate School of Biomedical Sciences Theses and Dissertations

Vulvovaginal candidiasis is the most common fungal infection of the female urogenital tract, commonly caused by Candida albicans and C. glabrata. However, treatment can be difficult when caused by non-albicans strains due to resistance to the oral antifungal drug fluconazole. The mechanism through which strains of Candida, particularly C. glabrata, develop resistance to different antifungal classes has not been completely characterized. The ergosterol biosynthesis pathway, an important component of the cell membrane, is the major target of antifungals such as fluconazole, an inhibitor of the ERG 11 gene. Current research shows that an upregulation of specific genes in C. glabrata …

Structural And Functional Analysis Of Harv1 Lipid Binding, Jamie Francisco

Graduate School of Biomedical Sciences Theses and Dissertations

Cardiovascular disease (CVD) has the highest mortality rate worldwide and is considered epidemic in the United States. Statins as the primary and almost sole method of treatment for cholesterol-related CVD, but they have limitations. Proper sterol transport is needed to maintain normal blood lipid levels. Studies have shown that misregulation of lipid trafficking aids in the progression of CVD and in the activation of the unfolded protein response (UPR). Misregulation of lipid and sterol homeostasis also results in the loss of lipid rafts, known cellular signaling hubs and major components of ER associated membrane contact sites. Arv1 is a highly …

Molecular Pap Biomarkers For Cervical Cancer Risk Assessment In Thinprep® Clinical Specimens, Jennifer Taylor

Graduate School of Biomedical Sciences Theses and Dissertations

Approximately 500,000 patients are diagnosed with cervical cancer worldwide each year, thus making it the second most common cause of cancer-related deaths in women. Persistent high-risk human papillomavirus (HR-HPV) infections induce cervical dysplasia by increasing the expression of two viral oncogenes, E6 and E7. Utilization of the Papanicolaous smear test (Pap test) has contributed to a decrease in morbidity and mortality of cervical cancer; however, subjective analysis of cell morphology with a limited amount of cells has led to a substantial rate of false positive and false negative diagnoses. A more objective and sensitive diagnostic tool for the detection of …

The Effects Of Lipoxin A4 (Lxa4) On Neutrophil Biology In Sepsis, Benedict Wu

Graduate School of Biomedical Sciences Theses and Dissertations

During sepsis, neutrophils are inappropriately activated and have prolonged lifespans, thus becoming dysfunctional. Excessive neutrophil activation can lead to tissue injury while neutrophil dysfunction can lead to decreased free radical production and reduced phagocytosis, preventing the host from clearing preexisting infections. Lipoxin A4 (LXA4) is a specialized pro-resolution mediator which reduces neutrophil migration and expression of proinflammatory mediators. Intact neutrophil functions are critical for the host to efficiently clear invading pathogens. The effects of LXA4 on neutrophil function in sepsis have not been established. Using the cecal ligation and puncture (CLP) model of sepsis, LXA4 administered 1 h after sepsis …

The Role Of Mammalian Cyclin C In Stress Response And Thyroid Disease Progression, Kun Wang

Graduate School of Biomedical Sciences Theses and Dissertations

The cellular response to stress involves changes in gene expression programs as well as the remodeling of subcellular organelles including the mitochondria. Important for this study, the mitochondria undergo extensive fragmentation when the cell is exposed to a variety of stressors. The cyclin C-Cdk8 kinase is a highly conserved transcription factor that associates with the RNA polymerase II holoenzyme. Using cyclin C knockout (CCNC^-/-) mouse embryonic fibroblast (MEF) cells, I revealed that cyclin C is required for the extensive mitochondrial fragmentation following treatment with pro-oxidants or the anti-cancer drug cisplatin. However, Cdk8 is not required for this process …

Bacterial Transcription Factors Grea, Greb And Dksa: Characterization Of Their Interaction With Rna Polymerase And Molecular Mechanism Of Action, Andrey Parshin

Graduate School of Biomedical Sciences Theses and Dissertations

The activity of RNA polymerase (RNAP), the key enzyme of transcription process, is regulated by a large number of transcription factors acting at each step of the transcription cycle. Our research project is focused on studying the structure and function of bacterial transcript cleavage factors GreA, GreB and the stringent response regulator DksA. Gre factors bind in the secondary channel of RNAP in backtracked (inactive) ternary elongation complexes and stimulate the enzyme's intrinsic RNase activity, which is required for suppression of transcription pause and arrest, enhancement of transcription fidelity, and efficient promoter escape. DksA also acts through the RNAP secondary …

Modulation Of Bax/Bak Dependent Apoptosis By Sirtuin 3 And Mitochondrial Permeability Transition By Sirtuin 4, Manish Verma

Graduate School of Biomedical Sciences Theses and Dissertations

Mitochondria are dynamic organelles that regulate a myriad of cellular functions, including energy production and metabolic regulation. Mitochondria are also a critical regulator of cell death signaling cascades modulating both apoptotic and necrotic cell death. However, what determines which cell death pathway is activated is still unclear. The mitochondrial/intrinsic pathway of apoptosis is dependent on the activation of pro-apoptotic proteins, Bax and Bak, which induce mitochondrial outer membrane permeabilization (MOMP). Once the integrity of outer mitochondrial membrane (OMM) is compromised, pro-apoptotic intermembrane space proteins like cytochrome c, Smac/Diablo, Omi/HtrA2 and AIF are released into the cytoplasm, which activates the post-mitochondrial …

Med13p Prevents Stress-Independent Mitochondrial Hyperfragmentation And Aberrant Apoptosis Activation In Saccharomyces Cerevisiae By Controlling Cyclin C Nuclear Localization, Svetlana Khakhina

Graduate School of Biomedical Sciences Theses and Dissertations

During aging, and as a result of environmental changes, cells are exposed to elevated levels of reactive oxygen species (ROS). High ROS levels induce lipid oxidation, protein aggregation, mitochondrial hyperfragmentation, DNA damage and programmed cell death (PCD), also called apoptosis. PCD is a highly regulated process and its misregulation has been linked to neurodegenerative diseases and cancer development.

Our hypothesis is that cyclin C plays a role in the initiation of apoptosis. During normal conditions, cyclin C represses the transcription of stress response genes (SRG). In response to stress, cyclin C translocates to the cytoplasm where it facilitates mitochondrial hyperfragmentation …

Diabetes Mellitus And Hypercholesterolemia Are Risk Factors For Alzheimer’S Disease And Appear To Affect The Integrity Of The Blood Brain Barrier, Jacqueline Dash

Graduate School of Biomedical Sciences Theses and Dissertations

Studies have shown that the vascular risk factors common to diabetes mellitus and hypercholesterolemia are also risk factors for Alzheimer’s disease (AD). It is currently unknown how these diseases are associated with AD, but they may cause a leak in the blood brain barrier (BBB), which is one of the hallmarks of AD. In this preliminary study, over 150 pig brain slides were tested for the expression levels of tight junction proteins occludin and claudin V in the BBB microvasculature. There were three groups of pig brains used in this study namely, control pigs, pigs with diabetes mellitus and hypercholesterolemia …

Generation And Characterization Of Peptide Fusion Proteins, Brianna L. Probasco

Graduate School of Biomedical Sciences Theses and Dissertations

Pathogenic Th17 cells drive progression of many autoimmune diseases. Th17 cells develop from naïve T cells in the immune system after antigen-driven stimulation in a specific cytokine environment. Normally, T cells act to fight off infection, but when not properly controlled, they can cause disease. The cytokine interleukin-23 (IL-23) plays an essential role in the expansion of pathogenic Th17 cells. IL-23 is a heterodimeric protein, composed of a p19 alpha chain and a p40 beta chain. The p40 is also part of IL-12 and binds to the IL-12 receptor beta 1 (IL-12Rβ1) subunit. Thus, it follows that the IL-23 receptor …

Notch1 Functions As A Tumor Suppressor In A Model Of K-Ras–Induced Pancreatic Ductal Adenocarcinoma, Linda Hanlon, Jacqueline L Avila, Renée M Demarest, Scott Troutman, Megan Allen, Francesca Ratti, Anil K Rustgi, Ben Z Stanger, Fred Radtke, Volkan Adsay, Fenella Long, Anthony J Capobianco, Joseph L Kissil

Rowan-Virtua School of Osteopathic Medicine Departmental Research

K-ras is the most commonly mutated oncogene in pancreatic cancer and its activation in murine models is sufficient to recapitulate the spectrum of lesions seen in human pancreatic ductal adenocarcinoma (PDAC). Recent studies suggest that Notch receptor signaling becomes reactivated in a subset of PDACs, leading to the hypothesis that Notch1 functions as an oncogene in this setting. To determine whether Notch1 is required for K-ras-induced tumorigenesis, we used a mouse model in which an oncogenic allele of K-ras is activated and Notch1 is deleted simultaneously in the pancreas. Unexpectedly, the loss of Notch1 in this model resulted in increased …

Prolonged Cyclooxygenase-2 Induction In Neurons And Glia Following Traumatic Brain Injury In The Rat, Kenneth I. Strauss, Mary F. Barbe, Renee Marshall Demarest, Ramesh Raghupathi, Samir Mehta, Raj K. Narayan

Rowan-Virtua School of Osteopathic Medicine Departmental Research

Cyclooxygenase-2 (COX2) is a primary inflammatory mediator that converts arachidonic acid into precursors of vasoactive prostaglandins, producing reactive oxygen species in the process. Under normal conditions COX2 is not detectable, except at low abundance in the brain. This study demonstrates a distinctive pattern of COX2 increases in the brain over time following traumatic brain injury (TBI). Quantitative lysate ribonuclease protection assays indicate acute and sustained increases in COX2 mRNA in two rat models of TBI. In the lateral fluid percussion model, COX2 mRNA is significantly elevated (>twofold, p < 0.05, Dunnett) at 1 day postinjury in the injured cortex and bilaterally in the hippocampus, compared to sham-injured controls. In the lateral cortical impact model (LCI), COX2 mRNA peaks around 6 h postinjury in the ipsilateral cerebral cortex (fivefold induction, p < 0.05, Dunnett) and in the ipsilateral and contralateral hippocampus (two- and six-fold induction, respectively, p < 0.05, Dunnett). Increases are sustained out to 3 days postinjury in the injured cortex in both models. Further analyses use the LCI model to evaluate COX2 induction. Immunoblot analyses confirm increased levels of COX2 protein in the cortex and hippocampus. Profound increases in COX2 protein are observed in the cortex at 1-3 days, that return to sham levels by 7 days postinjury (p < 0.05, Dunnett). The cellular pattern of COX2 induction following TBI has been characterized using immunohistochemistry. COX2-immunoreactivity (-ir) rises acutely (cell numbers and intensity) and remains elevated for several days following TBI. Increases in COX2-ir colocalize with neurons (MAP2-ir) and glia (GFAP-ir). Increases in COX2-ir are observed in cerebral cortex and hippocampus, ipsilateral and contralateral to injury as early as 2 h postinjury. Neurons in the ipsilateral parietal, perirhinal and piriform cortex become intensely COX2-ir from 2 h to at least 3 days postinjury. In agreement with the mRNA and immunoblot results, COX2-ir appears greatest in the contralateral hippocampus. Hippocampal COX2-ir progresses from the pyramidal cell layer of the CA1 and CA2 region at 2 h, to the CA3 pyramidal cells and dentate polymorphic and granule cell layers by 24 h postinjury. These increases are distinct from those observed following inflammatory challenge, and correspond to brain areas previously identified with the neurological and cognitive deficits associated with TBI. While COX2 induction following TBI may result in selective beneficial responses, chronic COX2 production may contribute to free radical mediated cellular damage, vascular dysfunction, and alterations in cellular metabolism. These may cause secondary injuries to the brain that promote neuropathology and worsen behavioral outcome.