Synthesizing proteins for use in therapeutics is restrained by, in part, contaminants in in vivo expression systems and limited production capacity of in vitro systems. Cell free expression (CFE) systems have emerged as a potential alternative for protein expression because of the inherently lower contents of contaminants, and their flexible modular design that allows the addition of factors that aid in synthesis of complex products. Here, we investigate and establish an in-house Escherichia coli-based cell free protein synthesis (CFPS) system, explore different CFPS commercial kits, develop assays to test performance of these systems and identify potential rules that dictate expression levels. Using CFE, we were able to test different vectors and conditions of system, as well as scale-up protein synthesis reactions. In conclusion, this work shows that CFPS is a functional and easy-to-use platform and can potentially meet the requirements for the synthesis of therapeutics.

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

The recent developments in Internet-of-Things (IoT) and the next-generation wireless communication systems (5G and beyond) are posing unprecedented demands for massive connectivity, enhanced spectrum efficiency, and strengthened reliability. Moreover, the conventional orthogonal multiple access (OMA) techniques have approached their fundamental limits or the improvements in performance are marginal. To this end, a paradigm-shift from OMA to massive multiple-input multiple-output (MIMO) non-orthogonal multiple access (NOMA) technology is proposed. The proposed techniques are capable of serving multiple spatially-distributed user nodes/IoTs in the same frequency-time resource block by reaping out the benefits of power-domain NOMA, and favorable propagation and channel hardening brought by very large antenna arrays.First, a comprehensively literature survey has been conducted. Next, system, channel and signal models were developed by considering practical transmission impairments of the proposed massive MIMO NOMA. Then, novel NOMA relaying strategies via massive MIMO with pilot designs, per-hop and cascaded channel estimation, statistical-parameter based power allocation policy, and reliable precoding scheme are designed. Then, a complete analytical framework to derive the fundamental performance metrics is developed. A MATLAB-based simulation framework is developed to verify the proposed system designs.Then, the detrimental effects of residual interference caused by intra-cluster pilot sharing and error propagation caused by imperfect successive interference cancellation are quantified. The results acquired can provide insights for refining the proposed techniques in terms of signal model and pilot design.Trade-offs among massive connectivity and spectral efficiency will be established and refined for the proposed relay aided/cell-free massive MIMO NOMA via carefully designing per-hop and cascaded channel estimation, low-complexity statistical-parameter-based power allocation, and conjugate precoding schemes. The proposed technique is expected to significantly outperform the conventional OMA scheme in all overloaded system scenarios by virtue of the proposed aggressive spatial multiplexing and power-domain NOMA techniques. Hence, the proposed technique can simultaneously serve many users with fast data rates than that of the existing OMA techniques. The proposed NOMA techniques are expected to provide higher spectral and energy efficiencies with ultra-low end-to-end latency than those of existing OMA. Thus, the proposed relay-aided/cell-free massive MIMO NOMA can significantly contribute as a novel candidate technology for the next-generation wireless standards.

Sepsis is an uncontrolled response to infection. Severe sepsis is associated with organ dysfunction, and has mortality rate of 30-50%. Identification of severity of sepsis and prediction on mortality is crucial in making clinical decisions. Recently, cell-free DNA (cfDNA) in blood was found to have high discriminative power in predicting ICU mortality in patients with severe sepsis. In an analysis of 80 severely septic patients, the mean cfDNA level in survivors (1.16±0.13μg/ml) was similar to that of healthy volunteers (0.93±0.76μg/ml), while that of non-survivors (4.65±0.48μg/ml) was notably higher. Therefore, rapid quantification of cfDNA concentration in blood will enable physicians to quickly predict mortality of sepsis and decide on treatment. Current methods for quantification of cfDNA involve multiple steps including centrifugation, DNA-extraction from plasma, and its quantification either through spectroscopic methods or quantitative PCR. The whole process is time consuming, thus is not suitable for immediate bedside assessment. To solve the problems, a microfluidic device is designed and fabricated in this thesis, which is potential for cfDNA quantification directly using blood in 5 minutes. The goal is to use this device for distinguishing survivors or healthy donors from non-survivors in patients with severe sepsis. The two-layer device consists of a sample channel (top) and an accumulation channel (bottom) that intersect each other. The accumulation channel is preloaded with 1% agarose gel, and the blood containing cfDNA and intercalating fluorescent dye is loaded in the sample channel. Fluorescently labeled DNA is able to be trapped and concentrated at the intersection using a DC electric field, and fluorescent intensity of the accumulated DNA is representative of its concentration in the blood. The simulated electric field in the sample channel reveals that both the magnitude and the gradient of electric field reach their maximum values at the intersection. Force analysis shows that DNA was driven into the gel by the dominate electrophoretic force, while red blood cells moved away from the gel due to a strong dielectrophoretic force. In this thesis, 4 types of samples have been used to characterize the performance of the device. It showed that DNA was efficiently accumulated at the intersection, and the fluorescent intensity could be measured using a fluorescent microscope. Samples from healthy donors were able to be distinguished from that of severely septic patients in 5 minutes. However, better resolution was needed for differentiating various cfDNA concentrations in patient samples. The discussion on the effect of applied voltage showed that 9V is an optimized setting compared with 3V and 15V. In addition, it has been proved that the fluorescent reagent could be immobilized in the device and the sample preparation could be absolutely eliminated. In summary, the device proposed in this thesis is capable of distinguishing severely septic patients from healthy donors using clinical plasma in 5 minutes, and is potential to be applied in clinical blood samples. It has low cost, and is ready to be developed into a fully functioned system. This tool can be a valuable addition to the ICU to rapidly assess the severity of sepsis for informed decision making. / Thesis / Master of Applied Science (MASc)

Microrobotics is an emerging discipline with the potential to radically affect fields ranging from medicine to environmental stewardship. Already, there have been remarkable breakthroughs; small scale robots have been made that can selectively traverse the gastrointestinal tract, and others have been built that can fly in a manner inspired from bees. However, there are still significant challenges in microrobotics, and it remains difficult to engineer reliable power sources, actuators, and sensors to create robust, modular designs at the microscale. The miniaturization of the robotic system makes design and efficiency of these components particularly difficult. However, biological systems demonstrate the key features of robotics " sensing, actuation, processing" and are remarkably complex at the microscale. As such, many researchers have turned to biology for inspiration and living robotic components. In our laboratory we have engineered an Escherichia coli (E. coli) capable of producing surface display proteins to either anchor the cells, bind to functionalized nanoparticles, or capture small molecules from the environment, all complex actuation features. Additionally, we have created a processing unit that can create signals based on biological components, yet is non-living. This thesis focuses on the characterization of the surface display E. Coli system and the creation of programmable microparticle scaffolds that may be controlled by biological circuitry. In particular, by leveraging the strong interaction between biotin and streptavidin, I have created programmable microparticle scaffolds capable of attenuating the intensity of a fluorescent response in response to perturbations in the local environmental conditions. We believe this is an excellent enabling technology to facilitate the creation of complex behaviors at the microscale and can be used as a processing unit for simple decision making on microrobots. We foresee this technology impacting disciplines from medical microrobotics to environmental sensing and remediation. / Master of Science

A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, in fulfilment of the requirements for the degree of Master of Science, Johannesburg 2017 / The high mortality rates of pancreatic ductal adenocarcinoma (PDAC) are largely attributed to a delayed diagnosis, of which in advanced disease, patients are unable to receive surgical resection with curative intent. Clinical presentations and genetic features shared between PDAC and other pancreatic conditions such as chronic pancreatitis (CP) are insufficient to facilitate the disease and often lead to diagnostic uncertainty at an early stage. The purpose of this study was to develop sensitive and specific non-invasive markers to aid in the detection and disease monitoring of PDAC. Here, circulating cell-free DNA (cfDNA) isolated from plasma samples of patients with PDAC (n= 155) and two control groups consisting of patients with either CP (n= 46) or critical limb ischemia (CLI) (n= 88) revealed significant differences in measured concentrations between the three patient groups (p= 0.006-Kruskal-Wallis test).When two groups were compared with each other using the Wilcoxon rank-sum test, observable differences were seen between the two pancreatic diseases: PDAC and CP (p= 0.002), and between the two controls: CP and the CLI groups (p= 0.007). A strong association was also observed in elevated cfDNA levels of CLI patients with HIV (p= 0.03), indicating a poor prognosis for patients. Results from methylationspecific PCR (MSP) in age-matched patient samples showed promoter methylation to account for the loss of Smad4 in late-stage PDAC; with an observed association with overall increasing cfDNA levels (p= 0.03).This study indicates the potential clinical utility of cfDNA as a non-invasive tool to predict disease progression both quantitatively and qualitatively, as well as to trace epigenetic changes in tumour markers associated with PDAC. Further investigation to identify hypermethylated genes in cfDNA for the early detection of PDAC is warranted. / XL2018

Serving as key players in cell signaling, nearly all cells in the human body contain GPCRs. As the largest and most diverse superfamily of proteins in the human body, GPCRs are linked to some of the most prevalent current disease states including cardiovascular disease, type II diabetes, and various types of cancers. The development of new biosensors capable of simple, specific, sensitive, high-throughput screenings of the ligand-binding events of GPCRs are crucial to the diagnosis and maintenance of such diseases. To this end, this research is focused on the development of a novel biosensor platform incorporating ICCRs reconstituted into BLMs. Although ICCRs have been expressed previously in oocytes and HEK293 cells, no occurrence of cell-free expression has yet been performed. The advantages of such a platform include the specificity and real-time measurement capabilities of GPCRs, the innate sensitivity of electrophysiological ion channel flux measurements, and the simplified cellular mimicking of the BLM and cell-free expression. The majority of the presented research was based in the molecular cloning of M2Kir6.2, an ICCR incorporating a muscarinic acetylcholine receptor (M2), from Xenopus oocyte vector pGH2 into cell-free expression vector pT7CFE1-CGST-HA-His. Much optimization of the cloning procedure (PCR, digestion, and ligation) was necessary involving studies into polymerase fidelity, inclusion of DpnI for degradation of methylated DNA, and ligation parameter alterations in time, temperature, and insert:vector ratios. It was discovered that Deep VentR polymerase was beneficial to preventing mutations within the sequence of M2Kir6.2 during PCR, DpnI was capable of degrading unwanted residual M2Kir6.2 pGH2, and ligation performance was optimal using a 1:1 (insert:vector) ratio and reaction time and temperature of 18 h and 4 °C, respectively. With the successful ligation of M2Kir6.2 into cell-free expression vector pT7, expression of the ICCR via cell-free expression lysate kit was performed with direct reconstitution into a micropipette suspended BLM attempted. Five reconstitution trials were performed with electrophysiological single-channel recording results suggesting ICCR insertion based on ion channel currents of ~ 3 pA and mean open-times of ~ 3 ms observed corresponding to literature values for native Kir6.2 channels. Additionally, a Western blot analysis of the cell-free expression mixture contained products with molecular weights corresponding to monomer (~ 100 kDa) and tetramer (~ 400 kDa) constructs of M2Kir6.2. With the successful optimization of the M2Kir6.2 pT7 cloning procedure, this procedure can be used in future cloning attempts with similar ICCR constructs, such as D2Kir6.2. Although preliminary electrophysiological results suggest ICCR expression and BLM reconstitution, further work needs to be done in controlling the amount of ICCR insertion and optimizing BLM stability. Additionally, in order to confirm the functionality of both M2 and Kir6.2 ligand dose response curves must be performed. The evidence supporting ICCR expression and direct reconstitution into suspended BLMs via cell-free protein expression is both exciting and promising. Not only has this research involved the first cell-free expression of M2Kir6.2 but also has great benefits to the further development of such novel ligand-binding biosensor platforms.

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.