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)

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

Liquid biopsy and next-generation sequencing of cell-free DNA (cfDNA) in cancer patients’ plasma offer a minimally-invasive solution to detect tumor cell genomic information to aid real-time clinical decision-making. Reliability and sensitivity in the detection of genomic alterations is crucial for patient management and it is particularly relevant in the context of targeted therapies. However, biological and technical factors, such as low cfDNA tumor fraction and sequencing errors, affect the correct interpretation of genomic data limiting the utility of non-invasive cfDNA-based tumor profiling. To address these issues, we designed a prostate cancer bespoke assay, PCF_SELECT, that includes an innovative sequencing panel covering ∼25 000 high minor allele frequency SNPs and tailored analytical solutions to enable allele-informed evaluation of patients’ tumor. The framework also implements ABEMUS, an ad-hoc computational procedure we specifically designed for cfDNA samples to accurately detect somatic point mutations that could emerge under treatment pressure and as drug resistance mechanism. When applied on serial plasma samples from three patients receiving PARP inhibition harboring DNA repair gene aberrations, PCF_SELECT demonstrated high sensitivity in detecting BRCA2 allelic imbalance with decreasing tumor fractions resultant from treatment and identified complex ATM genomic states that may be incongruent with protein losses. As a step towards a more sensitive detection of tumor features in circulation of cancer patients, we next hypothesized that recent WGS-based approaches exploiting cfDNA fragments characteristics could be extrapolated for targeted sequencing data and that gene-region specific measures could improve detection metrics. Preliminary results suggest an increased sensitivity compared to copy-number-based methods supporting the integration at no extra cost in our targeted assay. Overall, this work is relevant to the context of precision oncology. It provides an innovative platform for the management of cancer patients, delivering detailed patient-specific molecular information which could be applied to guide treatment and improve clinical outcomes.

Infertility is an issue for approximately 12% of couples attempting to have a child. Of this group, 50% of the cases are due to male factor infertility. There are many reasons for decreased fecundity in men, but there remains 10% to 15% of infertile men that are diagnosed with the most severe form of infertility, non-obstructive azoospermia (NOA). A diagnosis of NOA implies the lack of sperm cells in the ejaculate with no physiological reason. The current diagnostic test and treatment consist of microscopic examination of seminal fluid and a biopsy to extract any viable sperm from the testis. This treatment is known to be problematic because of the destructive nature of surgery as well as expense. A non-invasive diagnostic test that could identify the presence of sperm in the testis at the beginning of fertility treatment would inform the patient and the physician about the functionality of the testis and thus lead to more informed decisions about treatment and potentially a decrease in cost. The ability to identify the tissue source of DNA present in the reproductive tract could facilitate a fertility diagnostic tool. Tissue specific epigenetic mechanisms are known to play a role in an organism's development. The identification of an epigenetic signature unique to sperm DNA would allow for the identification of sperm DNA in a heterologous mixture. Our lab has been able to identify a methylation signature that can consistently differentiate between sperm DNA and somatic DNA. We compared the sperm DNA signature with that of blood and testicular tissue and found that there was no overlap in epigenetic markers. To create an assay that could evaluate the presence of sperm DNA we used an Oxford Nanopore next-generation sequencing platform. Sequencing bisulfite converted DNA; we were able to retrieve the methylation status at locations of interest. A bioinformatic tool was created to analyze the thousands of reads obtained and analyze the individual methylation points within single molecules of DNA. To create a more accessible fertility test, we used the sperm DNA analysis tool to evaluate seminal cell-free DNA (cfDNA). The presence of sperm cfDNA in a patient's seminal fluid may indicate that there is sperm somewhere in the male reproductive tract even if the cells are not intact. A clinician could use this information to better advise the patient about treatment and potentially decrease cost of care.

Lymphoma is a common hematopoietic malignancy in canines. Current diagnostic techniques to diagnose lymphoma are often invasive and expensive. Additionally, tumor heterogeneity complicates the accurate classification and diagnosis of specific subtypes, hindering the development of targeted therapy and prognostic assessments. We propose a minimally invasive liquid biopsy technique involving blood collection to detect cell-free DNA from tumors using Next-generation sequencing. We hypothesize that identical tumor aberrations can be found in matching plasma and tumor DNA. Five dogs diagnosed with B-cell lymphoma through flow cytometry or PAAR were enrolled in the study. Samples collected included: (1) blood for plasma (cfDNA), (2) tumor tissue fine-needle aspirates (tumor DNA), and (3) buccal swabs (genomic DNA, germline control). Whole Genome Sequencing was performed using Illumina NovaSeq 6000, and the sequenced output was analyzed with bioinformatics tools to detect somatic variants in plasma and tumor samples. Our results revealed many shared somatic variants between matched cfDNA and tumor DNA samples, with 1.7-49% of tumor variants also found in corresponding plasma samples. Shared variants constituted only 0.5-9% of all plasma somatic variants. Specific B-cell lymphoma mutations were identified in cfDNA, including MYC, POT1, and TRAF3, alongside other cancer-related genes. Tumor samples showed mutations in genes associated with canine and human B-cell lymphoma. This study suggests that tumor-specific genomic mutations can be detected in plasma, supporting the potential of liquid biopsy as a less invasive diagnostic tool. However, cfDNA may not capture the full genetic heterogeneity of tumors due to low tumor-derived DNA content in limited plasma volumes. / Master of Science / Lymphoma is a type of blood cancer affecting white blood cells. Canine lymphoma is a common neoplasia, with an incidence rate of 20 to 100 cases per 100,000 dogs, making it a significant research focus. Current diagnostic methods are invasive and costly. Additionally, the wide variety of tumor types in lymphoma makes it challenging to determine the exact subtypes, which is crucial for selecting the best treatment approach. To overcome these challenges, we proposed a less invasive method known as "liquid biopsy". This technique involves taking a blood sample of a dog to find cell-free DNA from tumor cells using Next-Generation Sequencing technologies. We aimed to see if blood DNA could provide the same information as tumor DNA. In our study, we worked with five dogs diagnosed with B-cell lymphoma through traditional methods. We collected blood, tissue from needle biopsies, and buccal swabs from each dog. We then performed DNA extraction and sequencing on these samples. Our findings showed that 1.7-50% of the mutations in tumor DNA were also detected in matched blood DNA, though these represented only a small fraction of all changes found in blood samples. Additionally, the blood samples also revealed mutations related to canine B-cell lymphoma in genes like MYC, POT1, and TRAF3. In conclusion, our study supports the use of liquid biopsy as a feasible and less invasive method to diagnose lymphoma in dogs. However, they might not show all genetic variations of the tumor due to limited tumor DNA content.

The purpose of this investigation was to elucidate the changes in cf-DNA as it relates to fluctuations in resistance training workloads and intensities. The relationship between cell free DNA (cf-DNA), C-reactive protein (CRP), creatine kinase (CK), testosterone (T), cortisol (C), testosterone-cortisol ratio (T:C), body mass and body composition were also examined. Eight weightlifters (5 males and 3 females, age = 25 ± 3.5 yr, body mass = 88.3 ± 22.7 kg, height = 173.8 ±8.4 cm) volunteered to participate in this study. Venous blood samples, body mass and body composition were taken six times, each corresponding to the end of a training phase. CK (p = 0.018, η² = 0.409) and CK %Δ (p < 0.001, η² = 0.594) were the only biochemical variables to reach statistical significance at any point. A number of statistically significant correlations were found among variables. VLD4wk was related to CK %Δ (r = 0.86), VLD4wk %Δ was related CK %Δ (r = 0.86) and TID1wk was related to CRP (r = 0.83). cf-DNA %Δ was correlated with CRP and CRP %Δ (r = 0.83 and 0.86, respectively). CRP and CRP %Δ were correlated with BF % (r = 0.94 and 0.92, respectively). CK and CK %Δ were both related to T:C (r = 0.94 and 0.89, respectively) and T:C %Δ (r = 0.87 and 0.86, respectively). The correlation between cf-DNA and CRP suggests that cf-DNA may be a valuable indicator of inflammation in weightlifters.