Molecular Biology Commons^™

Urinary Metabolomics To Detect Polycystic Kidney Disease At Early Stage, Amnah Mahmoud Obidan

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Urinary metabolomics abilities in detection of biological effects (i.e., toxicity, disease) is compounded by high background variability. Previously, we showed mild kidney dysfunction was detectable under the stress imposed by furosemide (diuretic). Here we tested whether furosemide (FUR) can enhance the sensitivity to detect autosomal dominant polycystic kidney disease (ADPKD) at an early stage. ADPKD is one of the most common inherited renal disorders and is characterized by the growth of numerous cysts in the kidneys. Urinary metabolomics analyses, using nuclear magnetic resonance (NMR) spectroscopy, were conducted in control (WT) and diseased mice (RC) at 7 and 24 weeks of …

Role Of Erk3 In Regulating Rhogdi1-Paks Signaling Axis, Hitham Abdulrahman Aldharee

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Extracellular signal-regulated kinase 3 (ERK3) is an atypical protein kinase of the mitogen-activated protein kinase (MAPK) family. In comparison to well-investigated ERK1/2 (classical) MAPKs, much less has been discovered about ERK3 signaling and its cellular functions. Recent studies have shown that ERK3 is overexpressed in various types of cancers such as lung cancer and breast cancer and that ERK3 promotes cancer cell migration and invasion. How ERK3 regulates cancer cell motility and invasiveness, however, is still largely unknown. RhoGTPases, including Rho, Cdc42 and Rac1, play critical roles in regulating cell motility and invasiveness through activating downstream effectors such as p21-activated …

Dnp63a Suppresses Cell Invasion By Targeting Rac1 Through Mir-320a, Amjad Ahmed Aljagthmi

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DNp63a, a member of the p53 family of transcription factors, is overexpressed in a number of cancers and known to play a role in proliferation, differentiation, migration and invasion. DNp63a has been shown to regulate several microRNAs that play a role in both development and cancer, but to date there has not been a global analysis of p63- regulated miRNA. Using next-generation sequencing of small RNA from wild type and sip63 transfected HaCaT cells, our laboratory recently identified a number of DNp63a- regulated miRNAs by RNA-Seq studies which may serve as biomarkers of cancer progression. We identified a novel miRNA, …

Exploring The Role Of Lipin1 In Mitophagy Process Using Lipin1 Deficient-Egfp Tagged Lc3 Transgenic Mice, Abdullah Ali Alshudukhi

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Lipin1 (phosphatidic acid phosphatase) is a key molecule in the cells with two functions: first, it converts phosphatidic acid into diacylglycerol in the cytosol which in turn makes triglycerides. Second, in nucleus lipin1 acts as a transcriptional factor which regulates the expression of genes involved in the fatty acid oxidation and lipid metabolism. Clinically, Lpin1 gene mutations have been detected in patients with severe rhabdomyolysis accompanied with aggregated and dysfunctional mitochondria in their type II muscle fiber. Previously, we have observed that mice with lipin1 deficiency had aggregated mitochondria and abnormal autophagosomes formations by electron microscopy. The mechanism underlying the …

Novel Insight Into The Role Of Lxrα In Metabolic Regulation Via Dna Binding As A Heterodimer With Pparα And As A Homodimer, Andrea M. Klingler

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Liver X receptor a (LXRa) plays a critical role in the maintenance of energy homeostasis within a cell through tight transcriptional regulation of genes involved in metabolism of lipids, glucose, and cholesterol. Although LXRa has been established to function as a heterodimer with the retinoid X receptor a (RXRa), recent studies have determined that LXRa also interacts directly with peroxisome proliferator-activated receptor a (PPARa). However, little is known regarding the functionality of this heterodimer, if any exists at all. This study determined that a heterodimer of PPARa and LXRa is capable of binding to candidate response elements in vitro with …

Effects Of Synthetic Ligands On Heterodimer Pairs Regarding Full-Length Human Pparα, Rxrα And Lxrα, Emily Delman

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Nuclear receptor study is critically relevant in therapeutic medicine since the intricate details of disease states pertaining to atherosclerosis and diabetes are poorly understood. Three nuclear receptors of interest regulate target genes pertaining to cholesterol and fatty acid regulation, linking these receptors to therapeutic medicine. The first is the peroxisome proliferator-activated receptor alpha (PPARa), which resides in liver and muscle, coordinating lipoprotein and fatty acid homeostasis [1]. Cholesterol homeostasis is dictated by the liver X receptor alpha (LXRa), targeting genes pertaining to the kidney, intestine, liver and adipose tissues [2]. A common partner receptor to PPARa and LXRa is known …

Instability At Trinucleotide Repeat Dnas, Rujuta Yashodhan Gadgil

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Trinucleotide repeats (TNRs) are sequences prone to formation of non-B DNA structures and mutations; undergo expansions in vivo to cause various inherited neurodegenerative diseases. Hairpin structures formed during DNA replication or repair can cause replication fork stalling and if left unrepaired could cause single or double strand DNA breaks. To test and study this hypothesis we have devised a novel two color marker gene assay to detect DNA breaks at TNRs. By inducing replication stress our results show that TNRs are prone to DNA strand breaks and it is dependent on the repeat tract length. Double strand breaks at structured …

Functional Characterization Of Cancer-Related Mutations Of Erk3, Hadel Mohammed Alsaran

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Extracellular signal-regulated kinase 3 (ERK3) is an atypical member of the mitogen-activated protein kinase (MAPK) family. Recent studies have shown that ERK3 is highly upregulated in multiple cancers, such as lung cancer and colon cancer. Importantly, ERK3 promotes cancer cell migration and invasion by phosphorylating steroid receptor activator 3 (SRC-3), hence upregulating pro-invasive matrix metalloproteinase genes. While the link between ERK3 and cancers has been recognized, little is known about ERK3 mutations in cancer progression. In this study, we have investigated ERK3 mutations on arginine 64 (arginine 64 mutated to cysteine or histidine, R64C or R64H) and leucine 290 (leucine …

Biomimetic Production Techniques For Mechanical And Chemical Characterization Of Sucker Ring Teeth Isoform-12 From The Dosidicus Gigas Squid, Marcus T. Grant

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The unique protein-based structure of Sucker Ring Teeth (SRT) of cephalopods have spurned research into the molecular design, physical characteristics, functionality and mechanical properties to explore biomimetic engineering and biochemical potential for eventual industrial production. Previous research has elucidated the potential for scientific and industrial exploitation. However, much of the previous research focused on the most abundant protein isoform of the sucker ring teeth, suckerin-19 (also known as suckerin-39) from the Jumbo or Humboldt Squid (Dosidicus Gigas). There is little known about the characteristics of the other 37 protein isoforms of Sucker Ring Teeth. Although the other isoforms have similar …

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 …

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, …

Improving Dna Quality Using Ffpe Tissues For Array Comparative Genomic Hybridization To Find Single Nucleotide Polymorphisms (Snps) In Melanoma, Keerti Potluri

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Important classes of benign and malignant neoplasms are composed of melanocytic cells which produce a pigment called melanin. Benign nevi, which are non-malignant melanocytic lesions, can sometimes give rise to malignant melanoma. Melanoma can be a lethal melanocytic neoplasm, a deadly and aggressive form of skin cancer. Finding prognostic or diagnostic markers can be very useful to reduce the deaths caused by melanoma. Array Comparative Genomic Hybridization (aCGH), a cytogenetic technique, analyzes the whole genome or chromosome for detecting genetic aberrations/variations such as single nucleotide polymorphisms (SNPs) in a cancer. formalin fixed paraffin embedded FFPE tissues are usually taken from …

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 …

Purification Of Recombinant Δ Np63 Α And Characterization Of Peptide Binding, Amal Abdulah Albati

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δ NP63α, the primary p63 isoform of the p53 transcription factor family, is a proto-oncogene implicated in non-melanoma skin cancers. Expressed in the basal layer of the epidermis, δ NP63α promotes cell survival and proliferation. Inhibition of this protein could potentially be beneficial in non-melanoma skin cancer patients. The first goal of this project was the purification of recombinant δ NP63α in Escherichia Coli. Recombinant δ NP63α was expressed as GST-δ NP63α followed by GST cleavage using GST trap affinity column chromatography yielding pure δ NP63α The second objective of this project was to test the binding capabilities of peptides …

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 …

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 …

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 …

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 …

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 …

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 …

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 …

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 …

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 …

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. …