Our previous studies have demonstrated that RNA can serve as a template for double-strand break (DSB) repair in the yeast Saccharomyces cerevisiae using synthetic RNA-containing oligonucleotides (oligos). Following this initial work, we show that the RNA tract of RNA-containing oligos can be copied into DNA to transfer a genetic change at the chromosomal level also in the bacterium Escherichia coli and in human cells. Exploiting the use of oligos containing ribonucleoside monophosphates (rNMPs), we developed a molecular approach to generate RNA/DNA hybrids of chosen sequence and structure at the chromosomal level in both yeast and E. coli cells. Such technique allows us to study how rNMPs present in the DNA genome of cells are tolerated by cells, what factors recognize and target rNMPs in DNA and to what extent the embedded rNMPs may alter genome integrity. Here we proved that mispaired rNMPs embedded into genomic DNA, if not removed, serve as templates for DNA synthesis during chromosomal replication and produce a genetic change. We discovered that mispaired rNMPs that are embedded in genomic DNA are not only targeted by ribonucleases H (RNases H) but also by the mismatch repair (MMR) system both in yeast and in E. coli. Our data reveal novel substrates for the MMR system, and also uncover an unpredicted competition between RNase H and MMR for the RNA/DNA mispairs.

In particle induced desorption-ionization mass spectrometry the strength of an analyte's signal under a given set of bombardment conditions is usually considered to be representative of the analytes relative surface activity. This rationale is generally used to explain differences in the technique's sensitivity between and within various classes of compound. In liquid matrix assisted secondary ion mass spectrometry (SIMS) sensitivity enhancement of ionic analytes by pairing with surface active counterions has been demonstrated by several groups. This technique has been utilized in this work to achieve a 10,000 fold enhancement in the signal for ATP on a double focusing magnetic sector instrument and to detect femtomole quantities of nucleoside monophosphates on a time-of-flight instrument. The analyte's signal, however, is dependent on both the analyte bulk concentration and that of the surfactant. Additionally, the surfactant concentration that produces the maximum analyte signal changes with the analyte concentration. In this study, this phenomenon has been modeled in terms of conventional solution equilibria and surface chemical principles. It is assumed that the initial surface composition and the bulk concentration are the boundary conditions of a steady state established by the competing processes of surface sputtering and surface replenishment from the bulk during analysis. Calculated surface excesses correlate well with observed relative ion intensities, suggesting that equilibrium conditions are approached in the sample matrices despite the outwardly dynamic nature of the sputtering processes. / Graduation date: 1994

Dietary effects of a nucleotide mixture that contained AMP¡BGMP¡BCMP¡BUMP and IMP in equivalent amount were evaluated in 4 supplemental levels of 0, 0.05, 0.2, and 0.4 g/100g diet. Weight gain (519-616%) of juvenile groupers fed for 14 weeks with the test diets was not significantly affected by the supplements. Serum concentrations of total protein, albumin, and cholesterol, A/G ratio and activities of AST were also not significantly affected. Serum total bilirubin concentration in the 0.4 g/100g group was significantly higher than the 0 or 0.05 g/100g group. Head-kidney macrophage phagocytosis and intracellular superoxide anion production as well as serum lysozyme activity were also not different among treatments. Histological sections of fish gut indicated that villous heights of intestinal fold were significantly greater in the 0.2g/100g group than the 0.4 g/100g group, muscle layer thickness, in contrast, was not affected. When challenged with Photobacterium damsela subsp. damselae after fed the test for 3 weeks, relative percent survivals of the groupers were 87.8, 91.1, 90.0 and 94.4% (0, 0.05, 0.2, and 0.4 g/100g diet, respectively). The survivals were 74.5, 82.4, 86.3, and 85.3%, respectively when the feeding trial was extended to 18 weeks. The results show that a diet containg 38% protein and asupplemented with nucleotides at the tested levels was not effective in promoting growth, but was moderately effective in enhancing immune responses and pathogen resistance of juvenile grouper.

Thesis (Ph. D.)--University of Chicago, Department of Biochemistry and Molecular Biology, 2001. / Includes bibliographical references. Also available on the Internet.

Šilumos taikymas įvairioms ligoms gydyti jau buvo naudojamas senovės Egipto, Graikijos, Romos civilizacijose daugiau kaip prieš 2000 m. pr. Šiuo metu hipertermija plačiai taikoma visame pasaulyje gydant vėžį, nes tai pigus ir patogus metodas turintis mažą šalutinį poveikį. Mokslininkai nustatė, kad vėžinės ląstelės greičiau žūsta esant aukštesnei už fiziologinę (41-45°C) temperatūrai, todėl hipertermija, derinama su kitais vėžio gydymo metodais (radioterapija, chemoterapija, imunoterapija ir chirurgija), tampa efektyvesniu metodu. Kadangi yra mažai žinoma apie hipertermijos poveikio mechanizmą sveiko audinio ląstelėms karščiavimo, hiperterminio vėžio gydymo ar gydymo termoabliacija metu, todėl yra svarbu nustatyti hipertermijos paveiktų ląstelių gyvybingumą bei hipertermijos poveikį adenino ir piridino nukleotidų koncentracijoms. Šio darbo tikslas buvo įvertinti hipertermijos, būdingos nutolusioms nuo termozondo audinio sritims poveikį, adenino ir piridino nukleotidų koncentracijoms žiurkės kepenų ląstelėse bei audinyje. Buvo naudojamas jonų porų efektyviosios skysčių chromatografijos metodas, leidžiantis vienoje chromatografinėje analizėje išskirstyti labai skirtingo hidrofobiškumo junginius. Taip pat buvo vertintas gyvų ir negyvų ląstelių skaičius gautoje hepatocitų suspensijoje panaudojant tripano mėlio metodą bei NAD(P)H fluorescencijos pokyčiai kepenų audinyje termoabliacijos metu. Gauti rezultatai parodė, kad išskirti hepatocitai pasižymėjo dideliu gyvybingumu (8... [toliau žr. visą tekstą] / The application of heat in the treatment of disease was first recorded in the ancient civilizations of Egypt, Greece, and Rome from as early as 2000 BC. Nowadays hiperthermia is widely using in cancer diseases in all the world. It was determined by many scientists that cancer cells are more sensitive for supraphysiological temperature (41-45°C) killing compared to normal cells. There are numerous evidences that hyperthermia can increase the effectiveness of other cancer therapies: radiotherapy, chemotherapy, immunotherapy and surgery. There is little known about the mechanisms of hyperthermia effects on healthy tissue, which are important in fever, in hyperthermic treatment of neighboring tumour and in thermoablation. Therefore it is very important to determinate the vability of cells during different hyperthermic treatment and hyperthermic effects of adenine ir pyridine nucleotides concentrations.The aim of study was to value the effect of hyperthermia,which is typical remote from thermoprobe tissue areas, on the concentration of adenine and pyridine nucleotides in hepatocytes and liver tissue. It was used ion-pair high-performance liquid chromatography method, which allows to disperse different combinations of hydrophobicity. Also were evaluated live and dead cells quantity in the suspension through tripan blue method and NAD(P)H fluorescence changes in liver tissue during the ablation. The results showed that isolated hepatocytes exhibited with high viability (80%)... [to full text]

DNA is the genetic material of a cell and is copied in the form of pre-mRNA through transcription in eukaryotes. RNA polymerase II is responsible for the transcription of all genes that express proteins. Transcription is a significant source of the stochasticity in gene expression. In this thesis, I discuss the development of a biochemically detailed model of eukaryotic transcription, which includes pre-initiation complex (PIC) assembly, abortive initiation, promoter-proximal pausing and termination as the points that can be slow steps for transcription. The stochastic properties of this model are studied in detail by stochastic simulations with some preliminary mathematical analysis. The results of this model suggest that PIC assembly can play the most significant role in affecting the transcription dynamics. In addition, promoter-proximal pausing has been identified as a potential noise regulatory step in eukaryotic transcription. These results show excellent agreement with many experimental studies. / x, 107 leaves : ill. ; 29 cm

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Molecular diagnostics is a fast growing field with new technologies being developed constantly. There is a demand for more sophisticated molecular tools able to detect a multitude of molecules on a single molecule level with high specificity, able to distinguish them from other similar molecules. This becomes very important for infectious diagnostics with the increasing antibiotic resistant viruses and bacteria, in gene based diagnostics and for early detection and more targeted treatments of cancer. For increased sensitivity, simplicity, speed and user friendliness, novel readouts are emerging, taking advantage of new technologies being discovered in the field of nanotechnology.  This thesis, based upon four papers, examines two novel electrical readouts for amplified single molecule detection. Target probing is based upon the highly specific amplification technique rolling circle amplification (RCA). RCA enables localized amplification resulting in a long single stranded DNA molecule containing tandem repeats of the probing sequence as product. Paper I demonstrates sensitive detection of bacterial genomic DNA using a magnetic nanoparticles-based substrate-free method where as few as 50 bacteria can be detected. Paper II illustrates a new sensor concept based on the formation of conducting molecular nanowires forming a low resistance circuit. The rolling circle products are stretched to bridge an electrode gap and upon metallization the resistance drops by several orders of magnitude, resulting in an extremely high signal to noise ratio. Paper III explores a novel metallization technique, demonstrating the efficient incorporation of boranephosphonate modified nucleotides during RCA.  In the presence of a silver ion solution, defined metal nanoparticles are formed along the DNA molecule with high spatial specificity. Paper IV demonstrates the ability to manipulate rolling circle products by dielectrophoresis. In the presence of a high AC electric field the rolling circle products stretch to bridge a 10 µm electrode gap.

The mammalian ADP-dependent glucokinase is the most recent mammalian glucokinase to have been discovered, and is unique in its ability to catalyse the phosphorylation of glucose to glucose-6-phosphate using ADP as the phosphoryl donor. Up until the discovery of this enzyme, the traditional biochemical view was that the first step of glycolysis was solely catalysed by ATP-dependent hexokinases, types I-IV. The particular role played by ADP-GK in the mammalian cell and the significance of this role has not yet been determined, although it is hypothesised that the ADP-dependent glucokinase could be potentially significant in contributing to the survival of cells under low energy and hypoxic or ischemic conditions. By using ADP as the energy investment in phase one of the glycolytic cycle instead of ATP, it is predicted that glycolysis could be sustained for longer during lower energy conditions (conditions of high ADP:ATP ratios). Since the phosphorylation of glucose by ADP-GK results in the production of AMP, it may also be possible that this has a direct effect on the energy charge of the cell. The AMP produced could lead to the regulation of cellular metabolism during hypoxia and/or ischemia via the activation of the cell-energy regulator AMPK. The study of mammalian ADP-dependent glucokinase is a very new area, and prior to this no investigation of the human ADP-GK enzyme had been undertaken. The main objective of this project was to clone, express and purify the recombinant ADP-GK so it could be kinetically characterised and directly compared with the recombinant mouse kinetic characteristics, the only other mammalian ADP-GK to have been studied. Unfortunately, due to complications in the expression and purification of soluble recombinant human ADP-GK, the project did not incorporate the kinetic characterisation of the enzyme. Acquiring data on the kinetic characteristics of the human ADP-GK will, in the long term, assist in the elucidation of the metabolic role of this enzyme, so the continuation of this project would be worthwhile.

Thesis (Ph. D.)--University of Hong Kong, 2008. / Includes bibliographical references (leaves 140-147) Also available in print.