Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2007.

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)

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...

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.

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)

Circulating DNA is fragments of DNA which can be found in the blood of healthy as well as diseased individuals. Higher levels of these nucleic acid molecules can be found in diseased and pregnant individuals in contrast to healthy controls. The origin of circulating DNA has not been elucidated, but release of DNA after apoptosis or necrosis or active release by living cells has been hypothesized. It was concluded in this study that apoptosis or necrosis may only be a minor source of circulating DNA and that release of DNA by living cells might play a major role in the origin, while disturbance of the equilibrium between release by living cells and clearance mechanisms may cause the rise in the levels of circulating DNA observed in different conditions. Before circulating DNA can be analyzed, it has to be isolated from the blood. A number of different preanalytical conditions can have an impact on the quantity and quality of circulating DNA that can be isolated. Furthermore, the choice of isolation and quantification method may also influence the results obtained. Quantitative analysis of circulating DNA was done by real-time PCR analysis of the &Globin gene and the DNA levels obtained for healthy controls and cancer patients correlated with levels reported in the literature. Characterization of total circulating DNA may be beneficial in diagnosis and prognosis and may also contribute to determining the source and function of circulating DNA. In order for characterization to take place a method to clone total circulating DNA was developed and standardized and thirty-five clones were obtained and analyzed. It was found that the sequences contain a large amount of Alu repeats and the significance of this has not been determined yet. This is a first step towards future studies. / Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2007

Circulating DNA is fragments of DNA which can be found in the blood of healthy as well as diseased individuals. Higher levels of these nucleic acid molecules can be found in diseased and pregnant individuals in contrast to healthy controls. The origin of circulating DNA has not been elucidated, but release of DNA after apoptosis or necrosis or active release by living cells has been hypothesized. It was concluded in this study that apoptosis or necrosis may only be a minor source of circulating DNA and that release of DNA by living cells might play a major role in the origin, while disturbance of the equilibrium between release by living cells and clearance mechanisms may cause the rise in the levels of circulating DNA observed in different conditions. Before circulating DNA can be analyzed, it has to be isolated from the blood. A number of different preanalytical conditions can have an impact on the quantity and quality of circulating DNA that can be isolated. Furthermore, the choice of isolation and quantification method may also influence the results obtained. Quantitative analysis of circulating DNA was done by real-time PCR analysis of the &Globin gene and the DNA levels obtained for healthy controls and cancer patients correlated with levels reported in the literature. Characterization of total circulating DNA may be beneficial in diagnosis and prognosis and may also contribute to determining the source and function of circulating DNA. In order for characterization to take place a method to clone total circulating DNA was developed and standardized and thirty-five clones were obtained and analyzed. It was found that the sequences contain a large amount of Alu repeats and the significance of this has not been determined yet. This is a first step towards future studies. / Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2007

The colorectal cancer (CRC) is the third most common malignancy in the world and in Brazil, the fifth most diagnosed and the third cause of cancer deaths. The detection of molecular markers in peripheral blood applies to the early diagnosis of cancer, before and after surgery, reducing the time of identification of CCR and improving the treatment, it is less invasive and reducing costs. This work is quantified by Real Time PCR in absolute and direct, free of DNA fragments found in the serum of patients with colorectal cancer, thereby determining values that characterize the condition of carrier of tumor. For this, a method was developed in which the measurement used as reference samples of the actual fragments ALU115 and ALU247 purified and quantified. We studied 3 major groups: Control, with healthy volunteers; surgery, patients who have already been submitted to surgical removal of colorectal tumor and non-surgery, patients who have not removed the tumor through surgery. Observing the results we note that the groups differ mainly by the values of quantification ALU247. For the control group, the limits were between 91 fentogramas and 1.55 picograms. The non operated group had amounts ranging from 8.02pg to 23.54pg and the group operators, 80fg to 5.95pg. In contrary, the results presented by quantification ALU115 did not set limits defined capable of differentiating at least one of the groups. The control group presented as limits 2pg and 69.45pg and the non operated group, 9.71pg and and 381.56pg, the group operated, 10.69pg and 196.85pg. The non operated group showed a mean significant when compared to the other two groups, but showed minor differences in the quantification ALU247 (14.62 ± 4.73 pg - p <0.05). It could be concluded that direct measurement, using reference concentrations of the fragments themselves ALU115 and ALU247 is a simple methodology, low cost and with reliable results. Moreover, the quantification ALU247 is able to identify the condition of carrier of tumor. / Fundação de Amparo a Pesquisa do Estado de Alagoas / O câncer colorretal (CCR) é a terceira neoplasia maligna mais frequente no mundo, sendo no Brasil a quinta mais diagnosticada e a terceira causa de morte por câncer. A detecção de marcadores moleculares no sangue periférico aplica-se ao diagnóstico precoce de neoplasias, antes e após procedimento cirúrgico, diminuindo assim o tempo de identificação do CCR e melhorando o tratamento, que se torna menos invasivo e diminuindo os custos. O objetivo deste trabalho é quantificar, através da PCR em Tempo Real de forma absoluta e direta, os fragmentos de DNA Livre encontrados no soro de pacientes com câncer colorretal, determinando assim valores que caracterizam a condição de portador de tumor. Para isso, foi desenvolvido um método em que a quantificação utiliza como amostras de referências os próprios fragmentos ALU115 e ALU247 purificados e quantificados. Foram estudados 3 grandes grupos: Controle, com voluntários saudáveis; Operados, pacientes que já se submeteram à cirurgia para retirada de tumor colorretal; e Não-Operados, pacientes que ainda não retiraram o tumor através de cirurgia. Observando os resultados podemos notar que os grupos se diferenciam principalmente através dos valores da quantificação ALU247. Para o grupo Controle, os limites ficaram entre 91 fentogramas e 1,55 picogramas. O grupo Não-Operados apresentou quantidades que vão de 8,02pg a 23,54pg e, o grupo Operados, de 80fg a 5,95 pg. De forma contrária, os resultados apresentados pela quantificação ALU115 não permitiram estabelecer limites definidos capazes de diferenciar pelo menos um dos grupos. O grupo Controle apresentou como limites 2pg e 69,45pg; o grupo Não-Operados, 9,71pg e 381,56pg e; o grupo Operados, 10,69pg e 196,85pg. O grupo Não-Operados apresentou uma média significativa quando comparado aos outros dois grupos, porém apresentou menor heterogeneidade na quantificação ALU247 (14,62pg ± 4,73 - p<0,05). Foi possível concluir que a quantificação direta, utilizando como concentrações de referência os próprios fragmentos ALU115 e ALU247, mostrou-se uma metodologia simples, de baixo custo e com resultados confiáveis. Além disso, a quantificação ALU247 é capaz de identificar a condição de portador de tumor.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21995号 / 医博第4509号 / 新制||医||1037(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 戸井 雅和, 教授 万代 昌紀, 教授 武藤 学 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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