Biochemistry Commons^™

Quantitation Without Calibration: Response Profile As An Indicator Of Target Amount, Mrittika Debnath

Graduate Research Theses & Dissertations

Quantitative assessment of biomarkers is essential in numerous contexts from decision-making in clinical situations to food quality monitoring to the interpretation of life-science research findings. However, appropriate quantitation techniques are not as widely addressed as detection methods. One of the major challenges in biomarker’s quantitation is the need to have a calibration for correlating a measured signal to a target amount. The step complicates the methodologies and makes them less sustainable. In this work, we addressed the issue via a new strategy: relying on the position of response profile rather than absolute signal for assessment of a target nucleic acid’s …

Quantifying The Differences In Binding Affinity Of Reduced Glutathione For Glutathione S-Transferase At Ph 6.5 And 8.5 Using Isothermal Titration Calorimetry, Clay P. Renshaw Jr., Ronald K. Gary

McNair Poster Presentations

The binding affinity between an enzyme and its substrate is often dependent on the pH of the local environment. Knowing the pH at which reduced glutathione (GSH) binds with the highest affinity to the enzyme glutathione S-transferase (GST) is useful for determining the optimal pH for purification of GST-fusion proteins during GST-affinity chromatography. In this study, GST of the species Schistosoma japonicum was purified, quantified, and utilized to study its binding interaction with GSH at pH 6.5 and 8.5 via isothermal titration calorimetry (ITC). After protein expression, extraction, and purification, the GST concentration was quantified using Qubit^TM fluorometry. …

Synthesis, Characterisation And Dna Intercalation Studies Of Regioisomers Of Ruthenium (Ii) Polypyridyl Complexes, Laura Perdisatt, Samar Moqadasi, Luke O'Neill, Gary Hessman, Alessandra Ghion, Muhammad Qasim Mushtaq Warraich, Alan Casey, Christine O'Connor

Articles

Regioisomers of the functional group of the main ligand (L) on a series of [Ru(phen)2L]2+and [Ru(bpy)2L]2+ complexes, where phen is 1,10 phenanthroline and bpy is 2,2′-bipyridine, were synthesised to investigate the interaction with deoxyribonucleic acid (DNA) as potential therapeutics. UV–Vis binding titrations, thermal denaturation and circular dichroism were used to evaluate their interaction with DNA. The conclusions indicated the significance of the auxiliary ligand; especially 1,10-phenanthroline has on the binding constants (Kb). The systematic variation of auxiliary ligand(phen or bpy), and polypyridyl ligand (4-(1H-Imidazo[4,5-f][1,10]phenanthrolin-2-yl)benzonitrile (CPIP), 2-(4-formylphenyl)imidazo[4,5-f] [1,10] phenanthroline (FPIP), 2-(4-bromophenyl)imidazo[4,5-f][1,10]phenanthroline (BPIP) and 2-(4-nitrophenyl)imidazo[4,5-f] [1,10] phenanthroline (NPIP), split in terms of …

Morphological, Genetic And Biochemical Characterization Of The Anti-Malarial Herb, Artemisia Annua Germplasm Collection At West Virginia University, Delini K. Samarasinghe

Graduate Theses, Dissertations, and Problem Reports (ETD)

Malaria is one of the deadliest diseases in human history. Nearly half of the world’s population, is at the risk in 106 countries. Only in 2016, this disease killed about 445,000 people, 72% of them being children under age five. It also accounts for US $12 billion dollars of direct costs in Africa alone. Five different species of Plasmodium cause malaria but P. falciparum is the most detrimental one, causing 50% of all malaria cases and is considered as the deadliest parasite in humans. Artemisinin (ART), a 15 C sesquiterpenoid is currently the only precursor to the most effective anti-malarial …

Quantitative Genetics Of Populus, Roba Bdeir

Dissertations, Master's Theses and Master's Reports

Evidence for bark, stem and stomatal density adaptation to different climates in the model species Populus is seen in both the natural population as well as in the greenhouse, but the genetic basis of these adaptation remains poorly understood. The present thesis investigates bark texture, bark thickness, diameter and stomatal density variations among Populus population using two quantitative genetics methods to attempt understand the genetic system controlling inheritance of these traits and to associate them with respective genes. The first approach aimed at detecting quantitative trait loci (QTL) associated with all phenotypic traits in an interspecific hybrid pedigree (Populus …

Assembly And Trafficking Of The Cystic Fibrosis Transmembrane Conductance Regulator And Associated Proteins, Zhihui Zhang

Theses and Dissertations--Chemistry

Cystic Fibrosis (CF) is an autosomal recessive genetic disease that leads to severe malfunction in many organs, but particularly the lungs. The primary cause of this malfunction is the decrease of the airway surface liquid layer on the lung epithelium. The lack of hydration leads to mucus build up on the epithelial lining, leading to blockage of airways. The underlying cause of CF is the dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR), which results from mutations in the protein. Almost 90% of CF patients are caused by the deletion of the phenylalanine at position 508 of CFTR, which …

Evaluation Of Ispe For Inhibitor Design In The Non-Mevalonate Pathway, Kataryzna Barbara Hoerchler

Graduate Research Theses & Dissertations

There is an urgent need to discover new antibiotics to combat the emergence of drug resistant Novel antibiotics that target unique pathways different from existing therapies are promising targets. The non-mevalonate pathway (MEP), which was discovered in the 1990’s, is essential for most bacteria, plants and apicomplexan parasites. Inhibition of the non-mevalonate is an attractive pharmaceutical target, since it is not present in mammals. Many compounds have surfaced as potential inhibitors, but none have made it to market. This thesis investigates the fourth step in the MEP pathway involving the enzyme 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE) from Escherichia coli (Ec), Burkholderia thailandensis …

Understanding Carbohydrate Recognition Mechanisms In Non-Catalytic Proteins Through Molecular Simulations, Abhishek A. Kognole

Theses and Dissertations--Chemical and Materials Engineering

Non-catalytic protein-carbohydrate interactions are an essential element of various biological events. This dissertation presents the work on understanding carbohydrate recognition mechanisms and their physical significance in two groups of non-catalytic proteins, also called lectins, which play key roles in major applications such as cellulosic biofuel production and drug delivery pathways. A computational approach using molecular modeling, molecular dynamic simulations and free energy calculations was used to study molecular-level protein-carbohydrate and protein-protein interactions. Various microorganisms like bacteria and fungi secret multi-modular enzymes to deconstruct cellulosic biomass into fermentable sugars. The carbohydrate binding modules (CBM) are non-catalytic domains of such enzymes that …

Protein Engineering In The Study Of Protein Labeling And Degradation, Xinyi Zhang

Theses and Dissertations--Chemistry

Proteins are large macromolecules that play important roles in nature. With the development of modern molecular biology techniques, protein engineering has emerged as a useful tool and found many applications in areas ranging from food industry, environmental protection, to medical and life science. Biomimetic membrane incorporates biological elements, such as proteins, to form membranes that mimic the high specificity and conductance of natural biological membranes. For any application involving the usage of proteins, the first barrier is always the production of proteins with sufficient stability, and the incorporation of proteins into the artificial matrix. This thesis contains two major parts, …

Protein Suppression Of Flavin Semiquinone As A Mechanistically Important Control Of Reactivity: A Study Comparing Flavoenzymes Which Differ In Redox Properties, Substrates, And Ability To Bifurcate Electrons, John Patrick Hoben

Theses and Dissertations--Chemistry

A growing number of flavoprotein systems have been observed to bifurcate pairs of electrons. Flavin-based electron bifurcation (FBEB) results in products with greater reducing power than that of the reactants with less reducing power. Highly reducing electrons at low reduction midpoint potential are required for life processes of both aerobic and anaerobic metabolic processes. For electron bifurcation to function, the semiquinone (SQ) redox intermediate needs to be destabilized in the protein to suppress its ability to trap electrons. This dissertation examines SQ suppression across a number of flavin systems for the purpose of better understanding the nature of SQ suppression …

Study Of The Mechanism Of Action For Ru(Ii) Polypyridyl Complexes As Potential Anticancer Agents, Yang Sun

Theses and Dissertations--Chemistry

Application of chemotherapeutic agents in current cancer treatment has been limited by adverse effects as poor selectivity results in systemic toxicity; most chemotherapy approaches also experience inherited or acquired drug resistance which lead to reduced treatment outcome. Research efforts have focused on the discovery of novel chemotherapies that overcome the limitations mentioned above. Ru(II) polypyridyl complexes with anti-cancer properties have been extensively studied as traditional cytotoxic agents and photodynamic therapy agents due to their photophysical and photochemical characteristics.

Most research has focused on the design of Ru(II) polypyridyl complexes that have affinities to nucleic acids as inspired by the classic …

Crystal Structure Of (2-{[(8-Aminona­Phthalen-1-Yl)Imino]­Meth­Yl}-4,6-Di-Tert-Butyl­Phenolato-Κ3n,N′,O)Bromido­Nickel(Ii), Patrick O’Brien, Matthias Zeller, Wei-Tsung Less

Chemistry: Faculty Publications and Other Works

The title compound, [NiBr(C25H29N2O)], contains an NiII atom with a slightly distorted square-planar coordination environment defined by one O and two N atoms from the 2-{[(8-aminonaphthalen-1-yl)imino]methyl}-4,6-di-tert-butylphenolate ligand and a bromide anion. The Ni—O and Ni—N bond lengths are slightly longer than those observed in the phenyl backbone counterpart, which can be attributed to the larger steric hindrance of the naphthyl group in the structure of the title compound. The molecule as a whole is substantially distorted, with both the planar naphthalene-1,8-diamine and imino–methyl– phenolate substitutents rotated against the NiN2OBr plane by 38.92 (7) and 37.22 (8), respectively, giving the molecule …

Identification Of Genes And Metabolites Involved In Durian Durio Zibethinus Fruit Ripening, Pinnapat Pinsorn

Chulalongkorn University Theses and Dissertations (Chula ETD)

Durian (Durio zibethinus M.), a major economic fruit, actively produces infamous sulfur-containing compounds during ripening. However, sulfur metabolism which is important in this fruit is rarely investigated. In this study, the integration of metabolomics using CE-TOF/MS and transcriptomics using de novo RNA-seq assembly was performed to reveal metabolic changes involved in sulfur metabolism and chemical composition related to fruit quality. During ripening, the expression level of genes involved in sulfur metabolism is concurred with the reduced sulfate ion content in placenta and the up-accumulation of sulfur metabolism-related metabolites in pulps. It is noteworthy that durian pulps highly accumulate γ-glutamylcysteine (γ-EC) …

Development Of Simple Devices For Point-Of-Care Screening Of Bacterial Infection And Inflammatory Markers, Julaluk Noiphung

Chulalongkorn University Theses and Dissertations (Chula ETD)

A paper-based analytical devices (PADs) provides a great benefit in medical diagnostics, especially in the developing world. The first part of this dissertation describes the effect of sample viscosity on PADs for the measurement of pH and nitrite concentration in human saliva. Preliminary study found that viscosity had a significant effect on the calibration curve of nitrite and a consistent curve could be generated after added washing buffer. Nitrite measurement in artificial saliva with viscosity 1.87-5.10 mPa-s was tested on the proposed device and also showed a good correlation with the standard spectrophotometry method (p=0.8484, paired sample t-test, n=20). Moreover, …

Polyamine Accumulation And Responses Of Mango Fruit To Postharvest Polyamine Application, Bussarin Wonnabussapawich

Chulalongkorn University Theses and Dissertations (Chula ETD)

Mango (Mangifera indicia L.) is a popular tropical fruit which has a great value in both domestic and international markets. Mango is a climacteric fruit which ethylene production increases dramatically, thus leading to a quick ripening process and a short postharvest life. Polyamines (PAs) have been reported to be involved in fruit ripening. Changes in endogenous free polyamines accumulation were measured in two mango cultivars; ‘Nam Dok Mai No.4’ and ‘Nam Dok Mai Si Tong’. Mangoes were allowed to ripen at 25±1 ºC after harvest. It was found that ‘Nam Dok Mai No.4’ mango exhibited higher ethylene production, respiration rate, …

Functional Characterization Of Heat Shock Proteins During Stress Response In Pacific White Shrimp Penaeus Vannamei And Brine Shrimp Artemia Franciscana, Wisarut Junprung

Chulalongkorn University Theses and Dissertations (Chula ETD)

Acute hepatopancreatic necrosis disease (AHPND) caused by the bacterium Vibrio parahaemolyticus carrying toxin produced plasmid (VPAHPND), has led to severe mortalities in farmed penaeid shrimp throughout Asia. Our studies demonstrated that a chronic non-lethal heat shock (chronic-NLHS) could enhance resistance of the Pacific white shrimp Penaeus vannamei to VPAHPND infection. Shrimp exposed to chronic-NLHS (28 °C to 38 °C, 5 min for 7 days) had higher survival rate (>50%) than that of the control non-heated shrimp (20%) when they were challenged with VPAHPND. The qRT-PCR analysis revealed that the expression of heat shock proteins, LvHSP70 and LvHSP90, as well …

Production And Purification Of Mannan Oligosaccharide And Its Role In Tight Junction Assembly In Epithelial Cells, Chatchai Nopvichai

Chulalongkorn University Theses and Dissertations (Chula ETD)

Mannan Oligosaccharide (MOS) is well-known as prebiotic supplement that help increasing life quality of livestocks and animals. Many reports revealed that MOS supplement can help improve intestinal morphology, enhancing growth performance, raising body weight, and increasing metabolic and stress response. MOS feeding also affected several biological processes in peripheral blood mononuclear cells in pigs and modulates expression of many immune-related genes that helps to prevent and reduce the severity of viral infection. However, the mechanism of action of MOS is remained unclear and the production, purification, identification of bioactive MOS molecular type is needed. In this study, we developed a …

Drug Resistance Mechanism Of Ns3/4a Protease And Identification Of Potent Compounds Inhibiting Reverse Transcriptase Of Hepatitis Virus, Jirayu Kammarabutr

Chulalongkorn University Theses and Dissertations (Chula ETD)

Viral Hepatitis has become a public health concern which caused 1.34 million deaths in 2015 and 95% of sickness and untimely deaths come from chronic hepatitis B and C virus infections. The epidemic of viral hepatitis B and C affects around 325 million people worldwide and is 10 times more than the global HIV epidemic. Hepatitis C virus (HCV), which is the main cause of hepatitis C, has a high mutation rate due to the lack of proofreading activity of the RNA polymerase enzyme. One of an attractive target for anti-HCV drug design and development is the NS3/4A serine protease. …

Effect Of Feedback-Resistant Arog Overexpression On L-Phenylalanine Production In Escherichia Coli, Maria Ulfah

Chulalongkorn University Theses and Dissertations (Chula ETD)

L-Phenylalanine (L-Phe) is one of the most important amino acids in food and pharmaceutical industries. It is widely used as a nutritional supplement and a precursor for the synthesis of food additives. The market of L-Phe has been stimulated by increasing demand for the low-calorie sweetener, aspartame. In Escherichia coli, the key enzyme which catalyzes the first committed step of aromatic amino acid biosynthesis pathway is 3-deoxy-D-arabino-heptulosonate-7- phosphate synthase (DAHP-synthase). The enzyme has 3 isoforms, AroG, AroF and AroH, which are feedback inhibited by L-Phe, L-Tyr and L-Trp, respectively. AroG, encoded by aroG, is the major isoform contributed about 80% …

Functional Characterization Of White Spot Syndrome Virus-Responsive Mir-750 In Black Tiger Shrimp Penaeus Monodon, Nichaphat Kanoksinwuttipong

Chulalongkorn University Theses and Dissertations (Chula ETD)

In the RNA interference (RNAi) process, small RNAs called microRNAs (miRNAs) modulate gene expression post-transcriptionally by acting as guides to direct Argonaute (Ago)-containing RNA-induced silencing complexes (RISCs) to targeted mRNAs. The Penaeus monodon miR-750 was previously reported as the highly upregulated miRNA during white spot syndrome virus (WSSV) infection. In this study, the computational program and 2D gel electrophoresis technique were used to identify miR-750 target genes. Bioinformatic analysis suggested that the predicted target mRNAs of miR-750 were ubiquitin-conjugating enzyme E2 Q1 gene (UbcQ1), Actin1 (Act1) and Sarcoplasmic calcium-binding protein (Scp). Furthermore, the alteration of protein expression in the stomach …