Molecular Biology Commons^™

Analysis Of Chromatin Accessiblity Of The Human C-Myc Replication Origin, Tu Thien Danh

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The best characterized eukaryote replication model is of the budding yeast Saccharomyces cerevisiae. Replication origins of S.cerevisiae are 100 to 200 bp in size and contain an essential 11-bp autonomous replicating sequence (ARS) consensus sequence (ACS). The origin recognition complex (ORC) binds to the ACS in order to recruit additional replication factors (Cdt1, Cdc6, MCM, Cdc45) and together they form the pre-replication complex (pre-RC).

Unlike budding yeast, the mammalian cells contain dispersed replication origins in which multiple elements distributed over large distances act as replication start sites. Mammalian DNA replication origins, such as the c-myc origin, contain a DNA unwinding …

Differences In Fecal Metabolite Profiles From Geographically Distinct Populations Of Adolescents, Jessica Moncivaiz

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Microbiota of the gastrointestinal tract have a variety of functions within the human body. They participate in protection of the host from pathogens, aid in immune system development and regulation, and carry out a variety of metabolic functions. This study focuses on the ability of gut microbiota to create metabolites through the degradation of food products. Using 1H NMR on fecal water extracts, I compared the metabolite profiles of two geographically distinct cohorts: healthy adolescents from Egypt (n=28) and healthy adolescents from the United States (n=14). Multivariate statistical analyses of binned NMR data confirmed that samples separated into groups corresponding …

Distinguishing Melanocytic Nevi From Melanoma By Dna Copy Number Changes: Array-Comparative Genomic Hybridization As A Research Tool, Ahmed Ibrahim Mahas

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Skin melanocytes can give rise to different benign and malignant neoplasms. Discrimination of an early melanoma from an unusual/atypical benign nevus can represent a significant challenge. However, previous studies have shown that in contrast to benign nevi, melanoma demonstrates pervasive chromosomal aberrations. This substantial difference between melanoma and benign nevi formed the idea of exploiting this difference to discriminate between melanoma and benign nevi. Array-comparative genomic hybridization (aCGH) is an approach that can be used on DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissues to assess the entire genome for the presence of changes in DNA copy number. In this study, …

Profiling Fatty Acid Composition Of Brown Adipose Tissue, White Adipose Tissue And Bone Marrow Adipose Tissue Of Healthy And Diet-Induced Obese Mice, Urszula Osinska Warncke

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Bone marrow adipose tissue (MAT) contains a fat depot of unknown function. Characterization of this tissue in healthy and pathophysiological conditions may be an important step in order to understand MAT contribution to development of osteoporosis and perhaps to develop means to improve bone health by manipulation with a phenotype of MAT. Here we demonstrate that MAT, brown adipose tissue (BAT), and white adipose tissue (WAT) derived from healthy mice differ in fatty acid and phospholipid composition. The profile of MAT changed to resemble a less-metabolically active tissue upon metabolic impairment as seen in diet-induced obesity. Additionally, PPARγ activation alters …

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

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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 …

Investigating The Undefined Role Of Subunit Iiiin Cytochrome C Oxidase Functioning Using Dicyclohexylcarbodiimide Chemical Modification; Insight Into Enzyme Structure And Molecular Mechanism, Kelli Nicole Fisher

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In the cell metabolic cycle, cytochrome c oxidase (COX) is the final electron acceptor of the respiratory chain which reduces molecular oxygen into water. It is bound in the inner mitochondrial membrane and on the plasma membrane of bacterial species. Energy produced through electron transfer is coupled to the pumping of protons against the electrochemical gradient in order to fuel the proton motive force for the synthesis of most of the ATP in the cell. The three mitochondrial encoded subunits of COX, I, II, and III, are conserved across species and the complete function of subunit III remains unknown. Dicyclohexylcarbodiimide …

Fabrication Of Responsive Polymer Brushes For Patterned Cell Growth And Detachment, Ashley B. Sutherland

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Patterned polymer brushes are used in biological applications to enhance cell function and to achieve selective cell growth. These patterned surfaces have a variety of applications, including cell sheet harvesting and tissue engineering. This work describes creation of chemical, topological, and responsive patterns on glass by sequential surface-initiated polymerization of poly(N-isopropylacrylamide)-co-(2-hydroxyethyl methacrylate) (pNIPAM-co-HEMA) and subsequent use of UV lithography to pattern the surface with pNIPAM. The cell adhesion peptide, RGD, is then immobilized on the surface of the copolymer pHEMA via DSC-coupling to spatially enhance cell adhesion properties. By culturing cells at 37 degrees celsius, cells selectively grow on DSC-activated …

Histone Deacetylation As A Mechanism Of Ypel3 Down-Regulation In Er-A Positive Breast Cancer Cell Lines, Remah Ali

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YPEL3 is a growth inhibitory gene that was established by our laboratory to trigger senescence in both primary and tumor cells. We recently reported YPEL3 to be directly repressed by estrogen in estrogen receptor-a positive (ER-a +ve) breast cancer cell lines. Here, we set out to determine whether the repression of YPEL3 involves histone deacetylation and, if so, elucidate the molecular mechanism of this activity. MCF-7, T-47D, and ZR-75.1 (all ER-a +ve) breast cancer cell lines were treated with varying doses of the global histone deacetylase inhibitors (HDACIs), trichostatin A (TSA) or suberoyl anilide hydroxamic acid (SAHA), for 24, 48 …

Exploration Of Ypel3 Response To Hormones And Ability To Induce Senescence, Joseph E. Rotsinger

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p53 activation through different cellular senescence pathways can trigger cell cycle arrest via regulation of p53 target genes. One such target gene is YPEL3 which is expressed upon binding of tumor suppressor protein p53 at its p53 binding sites (Kelley, 2010). The ability of p53 to induce YPEL3 gene expression led to the discovery that YPEL3 is one of several p53 target genes which induce cellular senescence (Kelley, 2010). Additionally YPEL3 can be regulated independently of p53 by estrogen signaling through estrogen receptor α (Tuttle, 2011). The loss of estrogen receptor α or removal of estrogen induces YPEL3 gene expression …

Study Of Microrna-34a Mediated Post Transcriptional Regulation Of Mdm4, Pooja P. Mandke

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MDM4 is an important negative regulator of the tumor suppressor p53. In normal unstressed cells the activity of p53 is kept under control by MDM4 and its homologue MDM2. MDM4 is said to possess oncogenic potential based on the evidence of its overexpression in many cancers. Until recently it was believed MDM4 is constitutively transcribed; however a decrease in full length MDM4 in response to genotoxic stress was observed paving way for exploring the mechanism responsible for this.

It was observed miR-34a a member of the miR34 family which is a direct transcriptional targets of p53 could have a potential …

Lipase-Kinase Associations Involving Pld2, Jak3 And Fes That Underlie Cancer Cell Proliferation And Invasion, Qing Ye

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Phospholipase D (PLD) is an enzyme that breaks down phospholipids in the cell membrane. It has been suggested that PLD may play a role during cell proliferation and cell invasion of cancer cells. The objective of this thesis was to define new molecular signaling pathways in which PLD2 might be involved in terms of cell proliferation (first part) and cell invasion (second part). To this, I compared molecular and biochemical aspects between untransformed cell lines with highly invasive, transformed breast cancer cells. In the first part, I investigated the interaction of two tyrosine kinases with PLD2 and the effect of …

Bio-Functionalized Clay Nanoparticles For Wound Healing Applications, Christopher Anthony Vaiana

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Wound healing is a complex, multi-step process that can be summarized into three stages, namely hemostasis and inflammation, proliferation, and finally tissue remodeling. Battlefield wound healing demands rapid hemostasis using clotting or cauterizing agents to immediately limit blood loss, but this occurs at the expense of proper tissue repair beyond hemostasis. Layered silicate clays such as kaolin and montmorillonite (MMT) have been previously shown to induce blood clotting due to their ability to form charged interactions with clotting factors. The charge characteristics of sodium MMT (Na-MMT) also enable functionalization with active biomolecules. Herein we first functionalize three types of alumoinosilicate …

Interrogation Of The Distal Gut Microbiota Of Healthy Adolescents And Those With Irritable Bowel Syndrome, Laura J. Rigsbee

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The human-associated microbiota has been the focus of much current research, with the microbiota inhabiting the gastrointestinal tract of particular interest. These organisms play many roles in human health and well-being. However, shifts in the composition of the intestinal microbiota have been associated with diseases such as irritable bowel syndrome, inflammatory bowel disease, and colon cancer. Several recent studies have reported on the distal gut microbiota composition of healthy adults and those with IBS, while there is a lack of studies devoted to adolescents. This study utilized a custom-designed Affymetrix Microbiota Array capable of detecting 775 phylo-species of intestinal bacteria …

Functional Metabolomics' Enhances Assessment Of Tissue Dysfunction As Demonstrated In A Rat Model Of Sub-Acute D-Serine Exposure, Isaie Sibomana

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We describe a methodology that combines urinary metabolomics with a tissue-specific stressor administration to enhance assessment of tissue function. Kidney function in rats was mildly compromised with a sub-acute dose of D-serine and stressed with furosemide. NMR-based metabolomics analyses showed no detectable effects due to D-serine alone; but furosemide or D-serine + furosemide groups, classified separately from each other, and from control. Furosemide alone caused a ca. 2-fold increase in glucose, lactate, choline, and a 30% decrease in TCA intermediates (p≤0.05). D-serine suppressed these effects and produced a 1.7-fold increase in a p-phenolic acid-derivative of tyrosine (PAdY) relative to control …

Structural And Functional Alteration Of Full Length Pparα And Lxrα By Fatty Acids And Their Thioesters, Madhumitha Balanarasimha

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Peroxisome proliferator-activated receptors (PPAR) and liver X receptors (LXR) are known to play important roles in fatty acid metabolism, interact with each other, and function as heterodimeric partners. Although previous studies indicate that PPARα is activated by long chain fatty acyl-CoA thioesters (LCFA-CoA) and polyunsaturated fatty acids, little is known about the effects of these ligands on the function or interaction of PPARα and LXRα. In this study, hPPARα and hLXRα were shown to directly interact by circular dichroism, fluorescent binding assays, and co-immunoprecipitation. Further experiments suggested that although fatty acids resulted in small structural changes, they significantly altered binding …

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

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

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

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

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

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

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

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

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

Due-B, A New Human Dna Replication Protein, Is The Functional Homolog Of S. Cerevisiae Sld3, Jianhong Yao

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DNA unwinding elements (DUEs) are commonly found at DNA replication origins. The DUE binding protein (DUE-B) is crucial for the initiation of DNA replication in eukaryotes. The unique 59 amino acid C-terminal part of DUE-B shares nearly 50% similarity with yeast the C-terminus of Sld3. DUE-B plays a key role in eukaryotic DNA replication because it is required for the loading of Cdc45, the MCM helicase activator, on chromatin. Here we show that DUE-B, just like yeast Sld3, binds to Cdc45 and TopBP1 through its C-terminus in Sf9 cells and in vitro. We also show that DUE-B, Cdc45 and TopBP1 …

Dose-Dependent Effects Of Oxygen On Metabolism In Rat Cortico-Hippocampal Brain Tissue Slices, Jennifer Lynne Hollyfield

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Studies have shown that 95% oxygen increases neuronal excitability and ROS production. We wanted to investigate the dose-dependent effects of oxygen on brain slice metabolism. We exposed rat brain cortico-hippocampal tissue slices to 0.40, 0.95, and 4.50 ATA O2 for 60 minutes, made dual-phase tissue extracts, and used multi-nuclear NMR experiments to elucidate the slice metabolism. We found that low doses of oxygen may shift metabolism toward anaerobic glycolysis. Elevated lactate suggests this shift, along with elevated ratios of NAD+/NADH which may drive the reactions toward the production of lactate. The results also suggest that high doses of oxygen may …

Validation Of A Custom-Made Microarray To Study Human Intestinal Microflora, Harshavardhan Kenche

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Intestinal microflora refers to all the different species of bacteria that reside in the human gut and is an important organ of the human body because almost all the digestive reactions of the host occur in the intestine. The bacteria of the intestine play a key role in this process by supplementing the intestine with various enzymes and proteins that are required for the digestive process. At the same time, these bacteria were shown to be implicated in a variety of gastrointestinal disorders like Irritable Bowel Syndrome, Inflammatory Bowel Disorder and Gastrointestinal Cancer, but with the current knowledge about the …

Transcriptional Regulation Of Lamb3 By P53, Meghna Jani

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The p53 tumor-suppressor plays a very important role in the prevention of cancer and it is known that about 50% of all human tumors possess p53 mutations. Although mutations in p53 are most prevalent in human cancers, inactivation of wild-type p53 occurs through many different mechanisms that are independent of p53 mutation or deletion. In an effort to determine novel p53 target genes, our lab employed a microarray method in which p53 was re-activated by RNAi mediated knockdown of Hdm2 and HdmX in MCF7 human breast cancer cell line, harboring wild-type p53 and elevated levels of Hdm2 and HdmX. Gene …

In Search For New P53 Regulated Genes, Meldrick Daniel Mpagi

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The p53 tumor suppressor protein has the ability to transactivate its target genes whose gene products are involved in carrying out cell cycle arrest, apoptosis, DNA repair, and senescence. Here, I report that two genes may be p53 regulated. Utilizing a microarray method to search for novel p53 target genes, I was able to identify a possible transcriptional target of p53 being solute carrier family 1a1 (SLC1a1). Along with that finding I also identified an E2F-target gene, minichromosome maintenance 10 (MCM10), as being p53 regulated. Gene expression profiling of MCF7 breast cancer cells treated with RNAi targeting Hdm2 and HdmX …

Nmr Analyses Show Tcdd Elicits Differences In Hepatic Metabolism In Female C57bl/6 Mice And Sprague-Dawley Rats, Meghan Katherine Makley

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TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) elicits tissue-, sex-, and species-specific effects. This study compares the hepatic response to an oral dose of TCDD in immature ovariectomized (i.o.) C57BL/6 mice (30 µg/kg) and i.o. Sprague-Dawley rats (10 µg/kg), at 72, 120, and 168 h post-dose. Hepatic lipid extracts were analyzed by ¹³C and ³¹P NMR, and aqueous extracts by ¹H and ³¹P NMR.

Consistent with increased lipid content in mice (p≤0.05), TCDD induced increases in hepatic triacylglycerides (TAG), cholesterol, and fatty acids. Principal component analysis of ¹³C spectra show treatment groups separate in mice, but not rats. …

Mdm2 Amplification In Nih3t3l1 Preadipocytes Leads To Mdm2 Elevation In Terminal Adipogenesis, Vaughn Litteral

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The p53 protein is a tumor suppressor protein that is mutated or non-functional in nearly all cancers. The Mdm2 protein has the ability to functionally inactivate p53 and these two proteins have been studied extensively in the context of cellular proliferation. In this study, expression of the murine double minute 2 (mdm2) gene was examined in the mouse NIH3T3L1 cell line. Under the proper conditions, the immortalized NIH3T3L1 cells have the ability to differentiate from fibroblasts to adipocytes (Green et al., 1975). This well characterized cell line provides an excellent model to study mdm2 in differentiation. While evaluating the regulation …

Detection And Destruction Of Escherichia Coli Bacteria And Bacteriophage Using Biofunctionalized Nanoshells, Joseph E. Van Buren

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The ability to detect chemical and biological agents is arguably one of the highest priority technical challenges today. The capability to obtain specific information at and near single-molecule resolution is the ultimate goal in chemical and biological agent detection. Metallic nanostructures, nanoshells and nanorods in particular, are attractive substrates because of their plasmonic properties. Combining the specificity of biomolecular recognition with these nanostructures might lead to increased sensitivity and selectivity. Localization of biological recognition motifs to the surface of these nanostructures could provide a mechanism for highly specific and directed energy transfer when bound to its target. This study utilizes …

Due-B In Chromatin And Nuclear Speckles, Nadia Katrangi

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The DNA unwinding element binding protein (DUE-B) was first identified by using a yeast one hybrid screen with the DNA unwinding element (DUE) from the c-myc origin as bait. DUE-B's orthologue in the yeast Saccharomyces cerevisiae lacks the last 60 C-terminal amino acids and has been identified as a D-tyrosyl-tRNA deacylase. A substantial group of evidence suggests a role for DUE-B in the regulation of replication initiation. Here we show that DUE-B is focused in nuclear speckles and colocalizes with spliceosome associated protein 145 (SAP145), an mRNA splicing factor 3B subunit. Mass spectrometry results show that SAP145 co-purifies with the …

Differential Effects Of Mutant Tap63Γ On Transactivation Of P53 And/Or P63 Responsive Genes And Their Effects On Global Gene Expression, Shama Khan Khokhar

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p63, a member of the p53 gene family, known to play a role in development, has more recently also been implicated in cancer progression. Mice lacking p63 exhibit severe developmental defects such as limb truncations, abnormal skin, and absence of hair follicles, teeth, and mammary glands. Germline missense mutations of p63 have been shown to be responsible for several human developmental syndromes including SHFM, EEC and ADULT syndromes and are associated with anomalies in the development of organs of epithelial origin. The contrasting phenotypes associated with the different classes of p63 mutations might be in part due to the differential …

Characterization Of Photosynthetic Reaction Centers From Bradyrhizobium Strain Btai 1, Isaac P. Forquer

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Photosynthetic rhizobia have been studied for about 15 years now. They are now considered to be metabolically aligned with a relatively recently discovered group of bacteria, the anoxygenic aerobic phototrophs (AAP’s).Rhizobia form symbiotic relationships with plants from the Fabaceae family. Photosynthetic rhizobia not only nodulate the roots, as most other rhizobia do, but they also form nodules on the stems of certain leguminous plants. The plant provides carbon to the bacteria and the bacteria provides the plant with soluble nitrogen fixed from the biologically inert but abundant atmospheric N2. A key question regarding photosynthetic rhizobia and other AAP’s derives from …