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

>Magister Scientiae - MSc / The discovery of cell free foetal DNA (cffDNA) in 1997 allows for the combination of accuracy as well as non-invasiveness for prenatal diagnosis. This non-invasive genetic test requires only a maternal blood sample from which the cffDNA can be isolated and analysed. In this work cffDNA was isolated from a maternal blood sample using a micro-fluidic device which was fabricated using hot embossing and laser ablation techniques. The DNA sample was first pre-concentration by electrokinetic trapping (EKT) and then isotachophoresis (ITP). The concentrated sample was then separated by size using capillary electrophoresis (CE), all in a single device. All parameters and processes concerned with the micro-fluidic device were optimised sequentially. These parameters include both the chemical components as well as the physical processes which occur. The DNA used for the optimisation protocol was analysed using fluorescence spectroscopy, agarose gel electrophoresis as well as an Agilent Bioanalyser. The optimised protocol included a 9% acrylamide/pDMA matrix, 3 M N,N-dimethylurea as a denaturing agent, with tris based buffers for pre-concentration steps and 1X TBE (tris/borate/EDTA) buffer for capillary electrophoresis. The applied voltage of ITP was 300 V and CE was carried out at 180 V. The timing at which DNA was extracted from the device was kept at time = 60 s intervals. The optimised protocol was then used for real sample analysis and these samples were obtained from mothers pregnant with male foetuses. The DNA extracted from the micro-fluidic device was then analysed using real time PCR (RT-PCR) in order to distinguish which was maternal and which was foetal. This was carried out by amplification of male and general (present in male and female) genes respectively. RT-PCR results confirmed that only the male specific gene was amplified in initial samples exiting the device and it was thus successful in isolating cffDNA from a maternal plasma sample.

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第13792号 / 工博第2896号 / 新制||工||1427(附属図書館) / 26008 / UT51-2008-C708 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 今中 忠行, 教授 青山 安宏, 教授 濵地 格 / 学位規則第4条第1項該当

Cell-free transcription and translation systems promise to accelerate and simplify the engineering of synthetic proteins, biological circuits or metabolic pathways. Microfluidic droplet platforms can generate millions of reactions in parallel. This allows cell-free reactions to be miniaturized down to picoliter volumes. Nevertheless, the true potential of microfluidics have not been reached for cell-free bioengineering. Better approaches are needed for reaching sufficient in-drop expression levels while efficiently creating DNA diversity among droplets. This work develops a droplet microfluidic platform for single or multiple protein expression from a single DNA coated bead per droplet. This opens up the possibility to diversify a million droplets for synthetic biology applications.

Platelet-rich plasma (PRP) is the cell free plasma, which has an enriched concentration of platelets and clotting factors with the ability to enhance the natural healing process. PRP is often used by physicians in an office setting to accelerate the healing of a variety of sports related injuries, chronic wounds and enhance skin rejuvenation. PRP mimics the wound healing cascade by enhancing the recruitment, proliferation and differentiation of cells involved in tissue regeneration. Although PRP is used to enhance healing, the efficacy thereof is debated as no clear-cut set of parameters is available that device manufacturers and protocols should follow. The lack of uniformity in the PRP preparation methods results in differing PRP volume, platelet contents and unavoidably platelet-derived growth factors. Therefore, the aim of this study was to develop a simple and rapid method for preparing autologous PRP in an office setting using a tabletop centrifuge for point-of-care use. The simplified preparation procedure involved a single centrifugation step of 18 ml of whole blood, which sufficiently enriched the platelet content in the PRP fraction. As activated platelets express and release growth factors and cytokines that mediate the different phases of the wound healing cascade, the extracted PRP fraction was activated with an ethanol, calcium chloride (CaCl2) and platelet poor plasma (PPP) preparation in glass containers, without the collection of additional blood as required in some protocols. The activated PRP formed a fibrin clot, trapping the degranulating platelets and its released growth factors. The concentration of TGF- 1 obtained from the fibrin clot was 45.49 ± 3.80 ng/ml, in range with the available literature. During the in vitro studies, the extracted PRP by the developed method was able to significantly induce cell proliferation in a dose dependent manner. Cells enumerated with the crystal violet assay indicated that the cells treaded with 5% or 10% PRP significantly increased the percentage of viable cells to 165-176% and 156-158%, when compared to the positive controls. Cells enumerated with the MTT-assay indicated that the cells treaded with 5% or 10% PRP increased the percentage of viable cells to 79-91% and 87-105% which is comparable to that of the positive control. Data from the cellular proliferation assays indicate that sufficient plateletderived growth factors had been obtained with the preparation procedure. Furthermore, data from the in vivo studies indicated that the extracted PRP was able to augment soft tissue regeneration and bone formation. Treatment with the activated PRP resulted in symptom reduction and accelerated healing of various injuries. The simplified preparation and the use of the provided study product packaged in a kit developed during this study will enable physicians to easily obtain autologous PRP, in an office setting for point-of-care use, with the ability to induce tissue regeneration. / Dissertation (MSc)--University of Pretoria, 2013 / gm2014 / Pharmacology / unrestricted

Bird schistosomes of the genus Trichobilharzia are known as causative agents of hyper-immune skin reaction called cercarial dermatitis (swimmer's itch). They use pulmonary water snails from family Lymnaeidae as the intermediate host and mostly anatid birds as the definitive host. The first larva, miracidium, actively moves in water environment, penetrates the snail and develops to the mother sporocyst. Then the daughter sporocysts are formed and migrate to the hepatopancreas of the snail where the high number of cercariae is assexually produced. Cercariae leave the intermediate host, actively move in a water and penetrate the skin of definitive host. Within a host body they mature and lay eggs. Cercariae can penetrate also the mammalian skin, including human, where they are immediately eliminated by the immune system of the host, which is followed by inflammatory reaction. Until now, for humans, there is no effective method enabling to differ cercarial dermatitis from other hyper-immune skin reactions and for birds the reliable diagnostic method of trichobilharziasis is missing. The main aim of this thesis was to use the molecular methods for diagnostic of bird schistosomes infection in natural (ducks) and accidental hosts (mice, human). For optimization, the conventional PCR was used for detection...

No description available.

The objectives of this dissertation were to investigate the utility of cf-DNA as a marker of systemic inflammation, fatigue, and training status in a long-term athlete monitoring program (LTAMP). In study one, cf-DNA, other biochemical markers, volume load, and training intensity were measured in weightlifters over 20 weeks. The changes and relationships between these variables were investigated in order to determine which variables may be indicative of an athlete’s training status. In study two, cf-DNA, other biochemical markers, and session rating of perceived exertion (sRPE) were measured over the course of a 15-week soccer season in order investigate the utility of cf-DNA as an indicator of systemic inflammation and fatigue. In study one, CK was statistically greater T2 than T4, T5, and T6 at p = 0.015, 0.025, and 0.030 respectively. cf-DNA %Δ was correlated with CRP percent change and BF% (r = 0.86 and r = 0.91 respectively). The correlation between cf-DNA and CRP suggests that cf-DNA may be a valuable indicator of inflammation. Upon further visual inspection, cf-DNA and CRP also appeared to rise and fall with changes in volume load with displacement (VLwD). In study 2, G1, cf-DNA (P = 0.001), CRP (P = 0.000), CK (P = 0.003), cf-DNA %Δ (P = 0.002), CRP %Δ (P = 0.002), and CK %Δ (P = 0.002) were all significantly higher than T1 at T2 and T3. In G2, CRP (P = 0.057) and CRP %Δ (P = 0.039) were significantly higher at T2 than T1. Despite the lack of statistically significant differences across all 3 testing times, cf-DNA %Δ, CRP %Δ, and CK %Δ increased throughout the season in G1. In G2, cf-DNA %Δ, CRP %Δ, and CK %Δ were all higher at T2 and T3 than T1 but fewer significant differences were present, potentially a result of the lower sRPE values in G2 versus G1.These results suggest that cf-DNA may a useful marker to reflect accumulated training and competitive stressors. The correlation between cf-DNA and CRP in study 1 suggests that cf-DNA may be a valuable indicator of inflammation.

Our ability to sense small molecules with high specificity, over a broad range of concentrations, is limited and difficult to accomplish in a way that is inexpensive and continuous. The most commercially successful biosensor is the enzyme-based blood glucose electrochemical biosensor, yet for nearly all other biomolecules, detection and monitoring require specialized equipment, trained personnel, and long lead times, and are not amenable to continuous monitoring. Industries in need of enzyme-based small-molecule biosensors, including medical diagnostics, industrial production, environmental monitoring, food safety analysis, and international security, would benefit greatly from the development of new devices capable of measuring biomolecules of interest. Environmental microbes have been gaining attention because of the vast array of biomolecules that they are capable of sensing and degrading. These microbes do so, in part, through redox enzymes with diverse substrate specificities that represent an immense resource for developing electrochemical biosensors. However, the development of new enzyme biosensors has largely been limited by the lack of a general high-throughput method to identify these redox enzymes, making discovery slow, laborious, and ad hoc. To address this need, a high-throughput functional screening approach has been developed to isolate microbial oxidase enzymes from complex metagenomic DNA libraries based solely on the enzyme-mediated degradation of any target analyte. The approach can be applied to DNA isolated from any complex microbial sample, including unidentified or unculturable bacteria. In this research, I first describe the development of a general assay to capture the activity of oxidase enzymes expressed in E. coli cells. I then demonstrate how the assay can be used to screen for the nicotine degrading oxidase NicA2 from a genomic DNA library generated from the microbe P. putida. Lastly, I describe the use of this screen to identify a new hydrocortisone-responsive oxidase from a pooled genomic DNA library of eight microbes, representing over 43 Mb of DNA sequence space. This hydrocortisone oxidase represents the first of many new enzymes that can be discovered leveraging our screening platform, which is poised to revolutionize the electrochemical biosensing field and substantially broaden the number of molecules these electrochemical biosensors can detect continuously and quantitatively. / 2023-02-17T00:00:00Z

Sepsis is a dysregulated systemic response to infection and is one of the leading causes of in-hospital mortality in Canada. Accurate distinction between survivors and non-survivors of sepsis has recently been demonstrated through quantification of cell-free DNA (cfDNA) concentration in blood. In an analysis of 80 septic patients, non-survivors of sepsis had significantly higher cfDNA concentration levels than that of survivors or healthy patients. Real time separation of cfDNA from contaminants in blood has also been done using a cross channel microfluidic device. Current methods for DNA quantification utilize time consuming and complicated laboratory equipment and therefore are not suitable for bedside real-time testing. Thus a handheld cfDNA fluorescence device coined the Sepsis Check was designed that can perform DNA characterization in a reservoir device and DNA detection in a microfluidic cross channel device. The goal is to use this system along with the cross channel devices to set apart survivors or healthy donors from non-survivors in patients with sepsis. The design consists of a 470𝑛𝑚 light emitting diode (LED) with 170𝑚𝑊 of optical power (LED470L – ThorLabs), an aspherical uncoated lens with a focal length of 15𝑚𝑚 (LA1540-ML – ThorLabs), a 488𝑛𝑚 bandpass filter with a 3𝑛𝑚 full width at half maximum (FWHM) (FL05488-3 – ThorLabs), an aspherical uncoated lens with a focal length of 25𝑚𝑚 (LA1560-ML – ThorLabs), an aspherical uncoated lens with a focal length of 35𝑚𝑚 (LA1027-ML – ThorLabs), a 525𝑛𝑚 longpass filter with an optical density >4.0 (F84744 – Edmund Optics), and a Raspberry Pi Camera V2 (Raspberry Pi Foundation). The Sepsis Check is made to excite the dsDNA specific PicoGreen fluorophore which has a peak absorbance at 502𝑛𝑚 and a peak emission at 523𝑛𝑚. In summary, the Sepsis Check in this thesis is capable of calibrating dsDNA concentration from 1𝜇𝑔/𝑚𝐿 to 10𝜇𝑔/𝑚𝐿 and detect DNA accumulation of 5𝜇𝑔/𝑚𝐿 and 10𝜇𝑔/𝑚𝐿 in the cross channel device. 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)