Thesis (Ph. D.)--University of Washington, 1996. / Includes bibliographical references (leaves ).

Lam Yuk Lan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 147-167). / Abstracts in English and Chinese. / ABSTRACT --- p.i / 摘要 --- p.iii / ACKNOWLEDGEMENTS --- p.v / PUBLICATIONS --- p.vii / TABLE OF CONTENTS --- p.ix / LIST OF FIGURES --- p.xiii / LIST OF TABLES --- p.xiv / LIST OF ABBREVIATIONS --- p.xvi / Chapter Section 1: --- Background --- p.1 / Chapter Chapter 1: --- Cell-free circulating DNA --- p.1 / DNA and Man --- p.2 / Cell-free Circulating DNA in Plasma and Serum --- p.4 / The Discovery and Early Development --- p.4 / Clinical Implications --- p.5 / Cancers --- p.5 / Prenatal diagnosis --- p.11 / Pregnancy abnormalities --- p.14 / Organ transplantation --- p.15 / Trauma and post-traumatic complications --- p.15 / "Origin, Mechanisms and Characteristics" --- p.16 / Methods of Analysis --- p.22 / Chapter Chapter 2: --- Trauma and Organ Failure --- p.25 / Trauma and Society --- p.25 / The Problem of Organ Failure --- p.26 / Definitions --- p.27 / Pathogenesis --- p.228 / Inflammation --- p.29 / Predictions --- p.30 / Trauma Scoring Systems --- p.31 / Abbreviated Injury Scale --- p.32 / Injury Severity Score --- p.32 / Other scoring systems --- p.33 / Definition of Trauma --- p.33 / Chapter Chapter 3: --- Stroke --- p.35 / The Burden of Stroke --- p.35 / What is a Stroke ？ --- p.36 / The Causes --- p.40 / Pathophysiology --- p.41 / Diagnosis and Tests --- p.42 / Assessments and prognosis --- p.44 / Biochemical Markers --- p.47 / Chapter Chapter 4: --- Aims of the study --- p.48 / Chapter Section 2: --- Materials and Methods --- p.50 / Chapter Chapter 5: --- Methods of analysis on cell-free circulating DNA --- p.51 / Materials --- p.51 / DNA Extraction from the Plasma Samples --- p.51 / Real-time Quantitative PCR --- p.52 / Methods --- p.54 / DNA Extraction from the Plasma Samples --- p.54 / Real-time Quantitative PCR --- p.56 / Principle --- p.56 / The β-globin TaqMan Assay --- p.59 / Calibration of the β-globin TaqMan System --- p.62 / Contamination Control --- p.64 / Chapter Section 3: --- Cell-free circulating DNA after trauma --- p.65 / Chapter Chapter 6: --- Cell-free circulating DNA concentration as a prognostic marker in patients after trauma --- p.66 / Introduction --- p.66 / Methods --- p.68 / Results --- p.71 / Discussion --- p.84 / Chapter Chapter 7: --- Temporal changes of cell-free circulating DNA after trauma --- p.89 / Introduction --- p.89 / Methods --- p.90 / Results --- p.92 / Discussion --- p.106 / Chapter Section 4: --- Cell-free circulating DNA concentration after stroke --- p.109 / Chapter Chapter 8: --- Cell-free circulating DNA concentration in patients with stroke --- p.110 / Introduction --- p.110 / Methods --- p.111 / Results --- p.115 / Discussion --- p.129 / Chapter Chapter 9: --- Daily changes of cell-free circulating DNA concentration after stroke --- p.132 / Introduction --- p.132 / Methods --- p.132 / Results --- p.133 / Discussion --- p.137 / Chapter Section 5: --- Conclusion and future perspectives --- p.139 / Chapter Chapter 10: --- Conclusion and Future Perspectives --- p.140 / Conclusion --- p.140 / Future perspectives --- p.145 / BIBLIOGRAPHY --- p.147 / APPENDIX 1: Goriśةmultiple organ failure score --- p.168 / "APPENDIX 2: Definitions and criteria for ARDS, ALI and MODS" --- p.170 / APPENDIX 3: Computed axial tomography and magnetic resonance imaging --- p.172 / APPENDIX 4: Glasgow Coma Scale --- p.173 / APPENDIX 5: Post-Stroke Modified Rankin Scale --- p.174

In conclusion, the studies in this thesis have provided new information on the molecular nature of circulating DNA. Alteration of the size of circulating DNA is demonstrated in different physiological and pathological conditions. The non-bisulfite-based approach described in this thesis has provided a more sensitive and precise way for the detection and quantification of aberrantly methylated DNA sequences in the circulation. This method has tremendous potential for being applied for noninvasive cancer detection and prenatal diagnosis. / In the second chapter of this thesis, the molecular nature of circulating Epstein-Barr virus (EBV) DNA is studied. Circulating EBV DNA has previously been shown to be a valuable marker for the detection, monitoring and prognostication of nasopharyngeal carcinoma and several cancers associated with EBV infection. Using DNase digestion and ultracentrifugation analysis, circulating EBV DNA was shown to be free DNA fragments instead of being associated with viral particles. Furthermore, a quantitative system was developed for measuring the size of these EBV DNA molecules and showed that over 80% of the circulating EBV DNA molecules are shorter than 180 bp. / In the subsequent chapters, this DNA size measurement technique has been applied for analyzing the integrity of plasma genomic DNA in cancer patients and pregnant women. Increased plasma DNA integrity was observed in both of these groups of individuals. Moreover, the size of plasma DNA in cancer patients was shown to normalize after successful treatment and the failure of such normalization was shown to be associated with poor prognosis. In pregnant women, in addition to the overall increase in plasma DNA size, the maternal-derived DNA molecules were further shown to be longer than the fetal-derived ones. This observation opens up the possibility for fetal DNA enrichment through size fractionation of maternal plasma DNA. / The discovery of circulating nucleic acids in plasma and serum has led to the development of numerous promising noninvasive diagnostic tests. To date, circulating nucleic acid analysis has been applied to many different areas, including cancer detection, prenatal diagnosis, monitoring of organ transplant recipients and acute medicine. However, despite the extensive investigations on their clinical applications, the information on the molecular characteristics of the circulating nucleic acids is lacking. / The latter part of the thesis describes the principle of a non-bisulfite-based method for the detection of aberrant DNA methylation in plasma/serum. Using this technique, a universal fetal DNA marker has been developed based on an epigenetic approach. The placentally derived hypermethylated RASSF1A sequence has been developed as a gender and polymorphism-independent marker for fetal DNA in maternal plasma. In pregnant women undergoing noninvasive prenatal rhesus D genotyping, false negative cases were successfully identified through the analysis of this new fetal DNA marker. The quantitative analysis of circulating methylated RASSF1A sequences has further been shown to be useful for the detection and prognostication of hepatocellular carcinoma. / Chan, Kwan Chee. / Adviser: Y.M. Dennis. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3307. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 143-162). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 146-160).

In recent years, a number of nuclear transcription factors have been shown to be present in the mitochondria where they have distinct roles in regulating mitochondrial function. Signal Transducer and Activator of Transcription 3 (STAT3), classically activated by the JAK family of receptor associated tyrosine kinases to drive nuclear gene expression, is one such transcription factor with a unique mitochondrial role. There, it has been shown to support oxidative phosphorylation, regulate mitochondrial-encoded transcripts, and be key for the transformation and growth of a number of different cancers. Despite its well-characterized functional importance at the level of the mitochondria, the mechanism through which mitochondrial STAT3 acts and how it is regulated has not been as well studied. Using various cell culture models, we now show that mitochondrial STAT3 is dynamically regulated by oxidative stress and cytokine treatment in the acute setting. Under these conditions we have observed a rapid loss of mitochondrial STAT3 that recovers to baseline conditions with time. During this recovery phase we have noted that mitochondrial STAT3 becomes competent to bind to Cyclophilin D (CypD), the key regulator and activator of the mitochondrial permeability transition pore (MPTP). This is particularly the case with oxidative insults, which we believe may represent an important homeostatic mechanism for the cell. Intriguingly, chronic stimulation with certain stressors seems to increase mitochondrial STAT3 levels suggesting differential regulation in the acute versus chronic setting. The regulation of mitochondrial STAT3 levels by various stimuli points to a novel signaling pathway potentially linking mitochondrial responses with those of the cell. Unification of responses throughout the cell would seem to serve a clear adaptive advantage, particularly in coupling nuclear regulation with metabolic demands as dictated by the mitochondria. Extramitochondrial signaling, also known as the mitochondrial retrograde response, has emerged as an important homeostatic mechanism in lower organisms, but its signaling components have not been well characterized at the mammalian level. Our results point to a role for mitochondrial STAT3 in sensing cellular inputs, whereby its regulation and subsequent association with CypD may have implications in overall mitochondrial quality control. Though the inner workings of this signaling cascade are just beginning to be elucidated, they suggest the existence of a previously unappreciated pathway at the mitochondrial level.

abstract: The fundamental building blocks for constructing complex synthetic gene networks are effective biological parts with wide dynamic range, low crosstalk, and modularity. RNA-based components are promising sources of such parts since they can provide regulation at the level of transcription and translation and their predictable base pairing properties enable large libraries to be generated through in silico design. This dissertation studies two different approaches for initiating interactions between RNA molecules to implement RNA-based components that achieve translational regulation. First, single-stranded domains known as toeholds were employed for detection of the highly prevalent foodborne pathogen norovirus. Toehold switch riboregulators activated by trigger RNAs from the norovirus RNA genome are designed, validated, and coupled with paper-based cell-free transcription-translation systems. Integration of paper-based reactions with synbody enrichment and isothermal RNA amplification enables as few as 160 copies/mL of norovirus from clinical samples to be detected in reactions that do not require sophisticated equipment and can be read directly by eye. Second, a new type of riboregulator that initiates RNA-RNA interactions through the loop portions of RNA stem-loop structures was developed. These loop-initiated RNA activators (LIRAs) provide multiple advantages compared to toehold-based riboregulators, exhibiting ultralow signal leakage in vivo, lacking any trigger RNA sequence constraints, and appending no additional residues to the output protein. Harnessing LIRAs as modular parts, logic gates that exploit loop-mediated control of mRNA folding state to implement AND and OR operations with up to three sequence-independent input RNAs were constructed. LIRA circuits can also be ported to paper-based cell-free reactions to implement portable systems with molecular computing and sensing capabilities. LIRAs can detect RNAs from a variety of different pathogens, such as HIV, Zika, dengue, yellow fever, and norovirus, and after coupling to isothermal amplification reactions, provide visible test results down to concentrations of 20 aM (12 RNA copies/µL). And the logic functionality of LIRA circuits can be used to specifically identify different HIV strains and influenza A subtypes. These findings demonstrate that toehold- and loop-mediated RNA-RNA interactions are both powerful strategies for implementing RNA-based computing systems for intracellular and diagnostic applications. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2019

Metabolic engineering has enabled studying microorganisms by the modification of their genetic material and analysis of their metabolism for the isolation of microbial strains capable of producing high yields of high value chemicals and biofuels. In this research, novel tools were developed to improve genetic engineering of microbial cells. In this matter, λ-PCR (lambda-PCR) was developed enabling the construction of plasmid DNA. This technique allows DNA assembly and manipulation (insertion, substitution and/or deletion) at any location of a vector. λ-PCR addresses the need for an easy, highly-efficient, rapid and inexpensive tool for genetic engineering and overcoming limitations encountered with traditional techniques. Then, novel synthetic small RNA (sRNA) regulators were designed in a cell-free-system (in vitro) in order to modulate protein expression in biosynthetic pathways. The ability of the sRNAs to regulate mRNA expression with statistical significance was demonstrated. Up to 70% decrease in protein expression level was achieved by targeting specific secondary structures of the mRNA with antisense binding regions of the sRNA. Most importantly, a sRNA was identified capable of protein overexpression by up to 65%. An understanding of its mechanism showed that its mRNA target region(s) likely lead to occlusion of RNase E binding. This mechanism was translated for expression of a diaphorase enzyme, which has relevance to synthetic biology and metabolic engineering in in vitro systems. Results were successful, showing a greater than 75% increase in diaphorase expression in a cell-free protein synthesis reaction. Next, Raman spectroscopy was employed as a near real-time method for microbial phenotyping. Here, Raman spectroscopy was used in combination with chemometric analysis methods through RametrixTM Toolboxes to study the effects of environmental conditions (i.e. illumination, glucose, nitrate deprivation, acetate, sodium chloride and magnesium sulfate) on the phenotypic response of the cyanobacterium Synechocystis sp. PCC6803. The RametrixTM LITE Toolbox for MATLAB® enabled processing of Raman spectra and application of principal component analysis (PCA) and discriminant analysis of principal components (DAPC). Two studies were performed. PCA and DAPC produces distinct clustering of Raman spectra, representing multiple Synechocystis phenotypes, based on the (i) presence of glucose in the growth medium, (ii) illumination, (iii) nitrate limitation, and (iv) throughout a circadian rhythm growth cycle, in the first study. The second study focused on the phenotypic response based on (i) growth in presence of acetate, (ii) presence of high concentrations of sodium chloride and (iii) magnesium sulfate starvation. RametrixTM PRO was applied for the validation of the DAPC models through leave-one-out method that allowed calculation of prediction accuracy, sensitivity and selectivity for an unkown Raman spectrum. Statistical tests (ANOVA and pairwise comparison) were performed on Raman spectra to identify statistically relevant changes in Synechocystis phenotypes. Next, comparison between Raman data and standardized analytical methods (GF-FID, UPLC, spectrometric assays) was established. Overall, good correlation were obtained (R > 0.7). Finally, genomic DNA libraries were enriched to isolate a deoxynivalenol detoxifying enzyme. To do this, library fragments from microorganisms was generated through oligonucleotide primed polymerase chain reaction (DOP-PCR) and transformed in a DON-sensitive yeast strain. Rounds of subculture were performed in the presence of DON and ferulic acid in order to isolate a strain capable of enzymatic degradation of DON. / Doctor of Philosophy / Metabolic engineering is the use of genetic engineering to modify microorganisms in order to produce high yields of valuable commodity chemicals. The goal of this research is to develop new methods to improve genetic modification and selection of microbial cells. The specific objectives were to: (i) develop new tools for DNA assembly and manipulation, (ii) utilize small synthetic RNA to control protein expression level, (iii) use Raman spectroscopy to study phenotypic responses to environmental changes and (iv) enrich for microorganisms that detoxify dangerous toxins. First, a new technique for DNA assembly, named λ-PCR (lambda-PCR), was developed. This method allows the easy manipulation of plasmid DNA with high-efficiency and low-cost compared to traditional techniques. Second, novel synthetic small RNA (sRNA) regulators were designed in a cell-free-system in order to modulate (downregulate or overexpress) fluorescent protein expression. Next, Raman spectroscopy was used to assess phenotypic response of cyanobacterial cells to different environmental modifications (light settings, salts, sugar, etc…). Finally, genomic library was used to discover and characterize enzymes capable of degrading a mycotoxin.

<p>Advances in genetics, proteomics and chromatography techniques have enabled the successfully generation of a cell-free bacterial translation system composed of highly pure and active components. This system provided an ideal platform for better elucidating the mechanism of each individual step of the prokaryotic protein biosynthesis and the function of the translation factors involved in the process. </p><p>In doing so, we have discovered that the N-terminal domain or complete deletions of the ribosomal protein L11 reduced the termination efficiency of RF1 on cognate stop codons by four to six folds. The L11 deletions also conferred a two folds decrease in the missense error suggesting the increased nonsense termination accuracy of RF2 by two folds, which would clarified previous in vivo observations. </p><p>The versatility of the cell-free system has provided the additional possibility to study the effects of class II release factor RF3 mutations in mediating fast dissociation of class I release factors RF1 and RF2 from the post-termination ribosome complexes. The results show a series of mutations within RF3 conferring considerable reduction of the class I release factors recycling rate. These observations together with sequence alignment studies suggest the possible location on RF3 of the class I release factors interaction site. </p><p>In addition, the utilization of the cell-free system has made it possible to develop a new biotechnological approach for continuous production of polypeptides, based on gel filtration chromatography. The pilot trials have so far resulted in a six fold production increase of the MFTI test peptide compared to the conventional batch method.</p>

Advances in genetics, proteomics and chromatography techniques have enabled the successfully generation of a cell-free bacterial translation system composed of highly pure and active components. This system provided an ideal platform for better elucidating the mechanism of each individual step of the prokaryotic protein biosynthesis and the function of the translation factors involved in the process. In doing so, we have discovered that the N-terminal domain or complete deletions of the ribosomal protein L11 reduced the termination efficiency of RF1 on cognate stop codons by four to six folds. The L11 deletions also conferred a two folds decrease in the missense error suggesting the increased nonsense termination accuracy of RF2 by two folds, which would clarified previous in vivo observations. The versatility of the cell-free system has provided the additional possibility to study the effects of class II release factor RF3 mutations in mediating fast dissociation of class I release factors RF1 and RF2 from the post-termination ribosome complexes. The results show a series of mutations within RF3 conferring considerable reduction of the class I release factors recycling rate. These observations together with sequence alignment studies suggest the possible location on RF3 of the class I release factors interaction site. In addition, the utilization of the cell-free system has made it possible to develop a new biotechnological approach for continuous production of polypeptides, based on gel filtration chromatography. The pilot trials have so far resulted in a six fold production increase of the MFTI test peptide compared to the conventional batch method.