Shing, Ka Fai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 158-188). / Abstracts in English and Chinese. / ABSTRACT --- p.i / 摘要 --- p.iv / ACKNOWLEDGEMENTS --- p.vi / TABLE OF CONTENTS --- p.vii / LIST OF TABLES --- p.x / LIST OF FIGURES --- p.xii / Chapter SECTION I: --- BACKGROUND --- p.1 / Chapter CHAPTER 1: --- CELL-FREE NUCLEIC ACIDS IN HUMAN BODILY FLUIDS --- p.2 / Chapter 1.1 --- Early studies on the presence of cell-free nucleic acids in human bodily fluids --- p.2 / Chapter 1.2 --- Circulating nucleic acids in plasma and serum --- p.2 / Chapter 1.2.1 --- Cancer Detection --- p.3 / Chapter 1.2.1.1 --- Circulating tumor-derived DNA --- p.3 / Chapter 1.2.1.2 --- Circulating tumor-derived RNA --- p.5 / Chapter 1.2.2 --- Prenatal diagnosis --- p.7 / Chapter 1.2.2.1 --- Circulating fetal DNA --- p.7 / Chapter 1.2.2.2 --- Circulating fetal messenger RNA --- p.11 / Chapter 1.2.2.3 --- Circulating placental microRNA --- p.13 / Chapter 1.3 --- Cell-free nucleic acids in urine --- p.14 / Chapter 1.3.1 --- Transrenal DNA (Tr-DNA) --- p.15 / Chapter 1.3.1.1 --- Biology of Tr-DNA --- p.15 / Chapter 1.3.1.2 --- Detection of fetal-derived Tr-DNA --- p.15 / Chapter 1.3.1.3 --- Potential problems associated with the detection of Tr-DNA --- p.16 / Chapter 1.3.2 --- Cell-free DNA in urine as released from the urinary tract --- p.17 / Chapter 1.4 --- Other bodily fluids with cell-free nucleic acids --- p.18 / Chapter 1.4.1 --- Amniotic fluid --- p.19 / Chapter 1.4.2 --- Cerebrospinal fluid (CSF) --- p.20 / Chapter 1.4.3 --- Peritoneal fluid --- p.20 / Chapter CHAPTER 2: --- MICRORNA IN HUMANS --- p.21 / Chapter 2.1 --- Introduction --- p.21 / Chapter 2.2 --- Biogenesis --- p.21 / Chapter 2.2.1 --- Transcription of microRNA genes --- p.21 / Chapter 2.2.2 --- Processing and maturation of microRNA precursors --- p.23 / Chapter 2.3 --- Mechanisms of gene regulation --- p.24 / Chapter 2.3.1 --- Cleavage of target mRNA --- p.24 / Chapter 2.3.2 --- Translational repression of mRNA --- p.25 / Chapter 2.4 --- Functional roles of microRNAs --- p.25 / Chapter 2.4.1 --- Oncogenesis --- p.25 / Chapter 2.4.2 --- Programmed cell death --- p.26 / Chapter 2.4.3 --- Cellular differentiation and development --- p.27 / Chapter 2.4.4 --- Regulation of physiological and cellular processes --- p.28 / Chapter 2.5 --- Aim of this thesis --- p.28 / Chapter SECTION II: --- MATERIALS AND METHODS --- p.30 / Chapter CHAPTER 3: --- QUANTITATIVE ANALYSIS OF CIRCULATING AND URINARY NUCLEIC ACIDS --- p.31 / Chapter 3.1 --- Preparation of samples --- p.31 / Chapter 3.1.1 --- Preparation of plasma --- p.31 / Chapter 3.1.2 --- Preparation of blood cells --- p.32 / Chapter 3.1.3 --- Preparation of placental tissue --- p.32 / Chapter 3.1.4 --- Preparation of urine and urine cell pellet --- p.32 / Chapter 3.2 --- Nucleic acid extraction --- p.33 / Chapter 3.2.1 --- "Extraction of small RNA-containing total RNA from plasma, blood cells and placental tissue" --- p.33 / Chapter 3.2.2 --- Extraction of DNA from urine --- p.37 / Chapter 3.3 --- Quantitative measurements of nucleic acids --- p.38 / Chapter 3.3.1 --- Principle of real-time quantitative PCR --- p.38 / Chapter 3.3.2 --- One-step QRT-PCR assays for mRNA quantification --- p.40 / Chapter 3.3.2.1 --- Principle --- p.40 / Chapter 3.3.2.2 --- Quantification of human placental lactogen （hPL) mRNA --- p.40 / Chapter 3.3.3 --- Two-step QRT-PCR assays for microRNA quantification --- p.45 / Chapter 3.3.3.1 --- Principle --- p.45 / Chapter 3.3.3.2 --- Advantages --- p.46 / Chapter 3.3.3.3 --- TaqMan® MicroRNA Assays --- p.47 / Chapter 3.3.4 --- QPCR assays for DNA quantification --- p.53 / Chapter 3.3.4.1 --- Principle --- p.53 / Chapter 3.3.4.2 --- Quantification of the leptin gene and the sex-determining region on Ychromosome gene --- p.53 / Chapter 3.4 --- Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) --- p.57 / Chapter 3.4.1 --- Principle --- p.57 / Chapter 3.4.2 --- Zinc finger protein gene assay for determining the fractional concentration of male DNA --- p.58 / Chapter 3.5 --- Statistical analyses --- p.65 / Chapter SECTION III: --- CIRCULATING PLACENTAL MICRORNAS IN MATERNAL PLASMA AS MARKERS FOR PRENATAL DIAGNOSIS　 --- p.66 / Chapter CHAPTER 4: --- THE EXISTENCE AND QUANTITATIVE DETECTION OF CELL-FREE MICRORNAS IN PLASMA --- p.67 / Chapter 4.1 --- Introduction --- p.67 / Chapter 4.2 --- Materials and methods --- p.69 / Chapter 4.2.1 --- Sample collection --- p.69 / Chapter 4.2.2 --- Experimental design --- p.69 / Chapter 4.2.3 --- RNA extraction and quantification --- p.72 / Chapter 4.3 --- Results --- p.75 / Chapter 4.3.1 --- Validation of two-step QRT-PCR system for miRNA quantification --- p.75 / Chapter 4.3.2 --- Detection of cell-free miRNA in maternal plasma --- p.82 / Chapter 4.4 --- Discussion --- p.82 / Chapter CHAPTER 5: --- SYSTEMATIC IDENTIFICATION AND CHARACTERIZATION OF PLACENTAL MICRORNAS IN MATERNAL PLASMA --- p.86 / Chapter 5.1 --- Introduction --- p.86 / Chapter 5.2 --- Materials and methods --- p.88 / Chapter 5.2.1 --- Sample collection --- p.88 / Chapter 5.2.2 --- Experimental design --- p.88 / Chapter 5.2.3 --- RNA extraction and miRNA quantification --- p.91 / Chapter 5.3 --- Results --- p.93 / Chapter 5.3.1 --- A systematic search for placental miRNAs in maternal plasma using two-step QRT-PCR assays --- p.93 / Chapter 5.3.2 --- Detection rate and clearance kinetics of placental miRNAs in maternal plasma --- p.97 / Chapter 5.3.3 --- Effects of filtering maternal plasma on the concentration of placental miRNA and mRNA --- p.99 / Chapter 5.3.5 --- Temporal profile of placental miRNA concentrations in maternal plasma across different trimesters of pregnancies --- p.103 / Chapter 5.4 --- Discussion --- p.115 / Chapter SECTION IV: --- DETECTION OF CELL-FREE DNA IN URINE --- p.119 / Chapter CHAPTER 6: --- HEMATOPOIETIC STEM CELL TRANSPLANTATION RECIPIENTS AS A MODEL TO STUDY CELL-FREE DNA IN URINE --- p.120 / Chapter 6.1 --- Introduction --- p.120 / Chapter 6.2 --- Materials and methods --- p.123 / Chapter 6.2.1 --- Sample collection --- p.123 / Chapter 6.2.2 --- Experimental design --- p.124 / Chapter 6.2.3 --- DNA extraction and quantification --- p.125 / Chapter 6.3 --- Results --- p.128 / Chapter 6.3.1 --- Validation of the zinc finger protein gene assay --- p.128 / Chapter 6.3.2 --- Fractional concentration of male DNA in blood cells and plasma of sex-mismatched HSCT patients --- p.129 / Chapter 6.3.3 --- Fractional concentration of male DNA in the urine and the urine cell pellets of sex-mismatched HSCT patients --- p.131 / Chapter 6.3.4 --- Size distribution of cell-free DNA in peripheral blood and urine samples of sex-mismatched HSCT patients --- p.132 / Amplicon size --- p.138 / Chapter 6.4 --- Discussion --- p.143 / Chapter SECTION V: --- CONCLUDING REMARKS --- p.147 / Chapter CHAPTER 7: --- CONCLUSION AND FUTURE PERSPECTIVES --- p.148 / Chapter 7.1 --- Circulating miRNA is a valuable resource for molecular analysis --- p.148 / Chapter 7.2 --- The presence of donor-derived DNA in the urine of HSCT recipients --- p.150 / Chapter 7.3 --- Prospects for future work --- p.152 / APPENDIX 1 --- p.154 / REFERENCES --- p.158

Quantifying human posture and range of motion remains challenging due to the need for specific technologies, time for data collection and analysis, and space requirements. The demand for affordable and accessible human body position measurement requires alternative methods that cost less, are portable, and provide similar accuracy to expensive multi-camera systems. This thesis developed and evaluated a novel augmented reality mobile app for human posture measurement to bring marker-based body segment measurement to the point of patient contact. The augmented reality app provides live video of the person being measured, AprilTag2 fiducial markers locations in the video, processes marker data, and calculates angles and distances between markers. Results demonstrated that the mobile app can identify, track, and measure angles and distances between AprilTag2 markers attached to a human body in real-time with millimetre accuracy, thereby allowing researchers and clinicians to quantify posture measurements anywhere, at anytime.

Almond (Prunus dulcis) is the most important nut crop in terms of world production. Due to its health benefit and high nutritional value the consumption and world supply of almond is increasing. To remain competitive in the world market, the Australian almond breeding program was established to produce cultivars with better adaptation to Australian conditions. As part of this program an almond mapping population consisting of 93 F₁ progeny derived from a cross between the American cultivar ‘Nonpareil’ (NP) and the European self-compatible cultivar ‘Lauranne’ (LA) was produced to construct the genetic linkage maps. The first almond linkage map developed prior to the commencement of this project failed to produce the eight linkage groups similar to the basic chromosome number of almond (x = 8) and many large gaps were also observed on the linkage groups. Therefore, more markers were needed to saturate the maps. Microsatellite markers are considered one of the best choices for mapping studies. 195 microsatellite markers isolated from Prunus species were obtained from published papers or by personal communication. Polymorphism was revealed by three different methods, and in general, polyacrylamide gel electrophoresis (PAGE) compared to the fluorescent labelled marker detection using an automated DNA sequencer or agarose gel electrophoresis, showed the most efficient and cost effective method of genotyping. A subset of 54 markers which produced reliable and easily interpretable polymorphic bands was selected to screen the whole mapping population. Microsatellites originally isolated from almond species showed the highest rate of amplification and polymorphism followed by peach microsatellites and the least informative markers were isolated from cherry. It seems that the level of transportability and usefulness of microsatellite markers is related to the genetic distance of the closely related species. Almond and peach belong to the same subgenus (Amygdalus) and other Prunus species are classified in Prunophora subgenus. The nut, or kernel, is the commercial part of the almond tree, thus to improve the quality of fruit an understanding of environmental influence, heritability and correlation of traits is required. Pomological and quality characters such as: shell hardness, kernel size, shape, taste, pubescence, colour, and percentage of doubles were measured during three consecutive years (2005-2007) on the total mapping population, but data analysis (ANOVA) was performed only on trees that survived for all three years. Most of the traits showed high broad-sense heritability and kernel shape showed the highest heritability of H² = 0.92 suggesting high genetic control of this trait. Occasionally larger kernels than either parent were found in the progeny indicating potential for improvement of this trait even with smaller kernel size parent that encompass many desirable characters. High correlation was also found between the in-shell and kernel weight (r = 0.74), kernel length / kernel width (r = 0.67), kernel weight to kernel length (r = 0.78) and kernel width (r = 0.80). This correlation estimation pointed out in this study indicates that the improvement of one character may result the progress in another trait. Neither of the parents in the mapping population had bitter or obvious slightly bitter taste but slightly bitter kernels were observed among the progeny. Amygdalin was assumed to be responsible for bitter taste in almond; therefore we measured the amount of amygdalin in sweet and slightly bitter kernel progeny by HPLC. However, the results showed that amygdalin exists in sweet kernels as well. Although the average amount of amygdalin in slightly bitter kernels (20.34 mg kg⁻¹ FW) was higher than sweet kernels (3.67 mg kg⁻¹ FW), some sweet kernels had higher amounts of amygdalin suggesting the impact of other components on slightly bitter kernel. The highest variability within the traits was observed in the percentage of double kernel, which showed the highest standard error. Strong environmental effects, particularly low temperature at pre-blossom time is speculated to produce much higher double kernels. Three genetic linkage maps, one for each parent and an integrated map were constructed by the addition of 54 new microsatellite markers to the previous dataset. All the data was scored and coded according to the coding system necessary by JoinMap3 which was used for map construction. 131 markers including microsatellite, ISSR, RAPD, SCAR and S-allele markers were placed on the integrated map covering 590.7 cM with the average density of 4.5 cM/marker. The minimum number of six microsatellite markers was placed on linkage group 8 and the linkage group 1 which is the longest linkage group has 14 microsatellite markers. Comparative mapping study with other Prunus maps, especially with the highly saturated reference map showed complete synteny and minor changes in the order of four markers on linkage groups compared with Prunus reference map. The conservation of molecular marker order observed in this study supports the idea of looking at Prunus genome as a single genetic system and practical application of this similarity would be in cross-transportability of microsatellite markers from well developed linkage maps to the less studied species in Prunus. Ten microsatellite loci placed on our map have not been reported before and could be used to improve the density of other Prunus maps, especially the reference map. This study contributed to the better understanding of the mode of inheritance and environmental effect on morphological traits and the effect of amygdalin on kernel taste. The most saturated microsatellite based almond linkage map developed in this study can serve as a framework for future almond breeding program in Australia and benefit Prunus improvement programs internationally. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1348850 / Thesis (Ph.D.) - University of Adelaide, School of Agriculture, Food and Wine, 2008

Diss. (sammanfattning) Uppsala : Univ., 2002. / Härtill 5 uppsatser.

Diss. (sammanfattning) Linköping : Univ., 2002. / Härtill 6 uppsatser.

Thesis (Ph.D.)--Biomedical Engineering, Georgia Institute of Technology, 2008. / Committee Chair: William D Hunt; Committee Member: Bruno Frazier; Committee Member: Dale Edmondson; Committee Member: Marie Csete; Committee Member: Peter Edmonson; Committee Member: Ruth O'Regan

Thesis (Ph.D.)--Memorial University of Newfoundland, 1999. / Bibliography: leaves 172-192.

The aim of this Master´s thesis is the contrastive analysis of discourse markers on a selected type of texts in Spanish and Czech language. The thesis is divided in two parts theoretical and practical part. Firstly, in the theoretical part of the thesis, is introduced the textual linguistic the field which studies discourse markers. For wholeness, are briefly mentioned all of the seven standards of textuality with profound focus on textual coherence. Other part of the thesis aims on terminology of discourse markers (especially on conectores), which is problematic, and various classifications according to Czech and Spanish authors. The last part of the theoretical part is dedicated to attitude markers the main theme of this thesis, their characteristics, functions and examples. The practical part of the thesis is introduced by contrastive analysis of attitude markers carried out on selected sample of texts. Firstly the attitude markers are identified and secondly described and classified. Also is mentioned the absolute and relative frequency and comparison of the contrasts and coincidences in both languages. The thesis is finally summarized in Spanish résumé.

Alzheimer's disease (AD) is the most common type of dementia characterized by intracellular appearance of neurofibrillary tangles, synaptic and neuronal loss; and extracellular accumulation of amyloid-β (Aβ) peptide in senile plaques. The initial causes leading to AD are unknown, and the available treatments are only effective at slowing the degeneration process. The accumulation of arginine in the brain of Alzheimer patients indicates a possible disruption of enzymes responsible for its metabolism. One such enzyme is neuronal nitric oxide synthase (nNOS) and controlling its activity by interacting with nanoparticles may lead to a delay in the onset of the disease. Neuronal nitric oxide synthase was purified using DEAE-Sephacel ion exchange resulting in 10 % yield, 0.43 fold recovery and specific activity 0.09 U/mg. The enzyme was found to be a dimer with a molecular mass of 150 kDa. Characterisation of the nNOS showed an optimum temperature and pH of 50°C and 7.5 respectively, and it was relatively stable at the optimum conditions (t½ = 100 min). The purity was analysed by SDS-PAGE followed by Western blot. Purified nNOS was challenged with 3-7 nm silver and 4-15 nm gold nanoparticles of between synthesized chemical using AgNO3 and either sodium borohydride or sodium citrate. Results showed that gold nanoparticles are more effective at low concentration (5 μM) than silver nanoparticles due to their size difference. Incubation of different concentration of nanoparticles (5, 15, 25, 50 μM) with the purified nNOS showed an initial decrease of 5% in enzyme activity which over time was restored to 80%. This suggests that different nanoparticles are produced in different sizes and interaction over a given time may result in enzyme association–dissociation mechanism. Inhibition studies showed a strong binding of both nanoparticles with Ki values of 1.4 μM and 0.2 μM for silver and gold, respectively. Both nanoparticles inhibited the activity of nNOS extensively as they bound strongly to the inhibition site on the enzyme and were more in contact with fluorophores nanoparticles. This was confirmed by fluorimetry with binding constants of 0.0084 μM and 0.01092 μM for silver and gold, respectively. Results of this study suggest that silver and gold nanoparticles competitively inhibit nNOS.

A ocorrência de Listeria monocytogenes em alimentos vem sendo estudada desde o início dos anos 80, após seu envolvimento em vários surtos de doença de origem alimentar. Os frutos do mar são o grupo de alimentos que despertou menor atenção por parte dos pesquisadores, apesar de terem sido envolvidos em casos esporádicos de listeriose e mesmo em surtos da doença. A amostragem ambiental e de produto, ao longo de uma linha de processamento é uma forma de localizar áreas relacionadas à contaminação do alimento permitindo que sejam feitas correções para evitar a produção de bens que exponham o consumidor a doenças. Com a finalidade de avaliar a contribuição da matéria prima e fatores ambientais na ocorrência e distribuição de L. monocytogenes em uma indústria processadora de pescados, e mais especificamente, numa linha processadora de camarão rosa (penoeus brasiliensis), é que desenvolveu-se a presente pesquisa. Também buscou-se determinar as diversidades antigênica e genética das cepas de L. monocytogenes isoladas, e correlacionar esta diversidade à sua distribuição na indústria. Assim, um total de 363 amostras coletadas em diferentes pontos de uma linha de processamento de camarão rosa foram examinadas para a presença de L. monocytogenes, empregando-se a metodologia recomendada pelo Health Protection Branch, do Canadá. A seguir, 115 cepas de L. monocytogenes representativas das amostras positivas para o microrganismo foram sorotipadas e o polimorfismo do seu DNA cromossomico avaliado com o auxílio dos métodos RAPD (\"randon amplified polimorphic DNA\") e PFGE (\"pulsed-field MTDestro - Doutorado - Listeria monocytogeneses em camarao... 140 gel electrophoresis\"). Um grupo de 25 cepas foi também submetido a sorotipagem completa, fagotipagem e ribotipagem pelo sistema RiboPrint da DuPont. Do total de amostras examinadas, 64 (17,6%) apresentaram-se contaminadas por L. monocyfogenes. As amostras ambientais apresentaram 25,0% de positividade (14 positivas/56 examinadas). As amostras de utensílios 24,2% (8/33) e as de água 23,8% (5/21). As amostras de camarão apresentaram 18,0% de contaminação (32/178) e as de manipuladores 7,6% (5/66). Nenhuma das amostras de gelo foi positiva para L. monocyfogenes. Durante o processamento observou-se um aumento na percentagem de amostras positivas para L. monocytogenes, chegando esta percentagem a 35,0% em algumas etapas, mas reduzindo-se a 16,0% no produto final. O perfil composto gerado pela combinação dos resultados obtidos com a tipagem molecular das 115 cepas de L. monocyfogenes selecionadas, permitiu a sua divisão em 24 grupos, de acordo com seus perfis de DNA. A sorotipagem das 25 cepas selecionadas mostrou que 11 pertenciam ao sorovar 4b, 7 ao sorovar 1/2b, 2 ao sorovar 1/2c e 5 ao sorovar 1/2a. A fagotipagem permitiu que elas fossem divididas em 7 fagovares, sendo que a maior parte das cepas do sorovar 4b (81,8%) não foi fagotipável. A ribotipagem destas 25 cepas originou 6 RiboGroupsTM. Os resultados indicaram que cepas de L. monocytogenes de origem ambiental pertenciam a perfis compostos exclusivos para o ambiente, enquanto que cepas provenientes da água e de utensílios possuiam um perfil composto em comum. Amostras de camarão coletadas nas diversas etapas doprocessamento apresentaram L. monocytogenes com pelo menos um perfil composto em comum, perfil este também presente nas cepas isoladas das mãos dos manipuladores. / The importance of seafood in the spread of foodborne pathogens is well known, however, until the last few years, little attention has been paid to the role of seafood in disseminating L. monocytogenes. Two foodborne listeriosis outbreaks have been linked to the consumption of seafood. L. monocytogenes and other Listeria species have been isolated from different types of raw or processed seafood, but the main source of contamination is unknown. For this reason, it is important to monitor the potential sources of this pathogen in food processing plants, in order to minimize product contamination. The aim of this study was to evaluate the contribution of raw material and environment in the occurrence and distribution of L. monocytogenesin shrimp (Penaeus brasiliensis) processing plant. The antigenic and genetic diversities of L. monocytogenes strains were also determined. A total of 363 samples, collected in different areas of a shrimp processing plant in Santos, SP, were examined using the methodology recomended by the Health Protection Branch, Canada. One hundred and fifteen strains of L. monocytogenes representing the L. monocytogenes positive samples were first serotyped and then sub-typed by molecular typing (RAPD and PFGE). A group of 25 strains were also ribotyped and phage-typed. L. monocytogeneswas isolated from 64 (17.6%) of the total samples analysed. Environmental samples showed the highest positivity rate (25%) followed by utensils (24,2%) and water (23.8%) samples. Shrimp samples presented 18% of positivity for L. monocytogenes while food handlers samples presented 7.6%. None of the ice samples was positive for the microorganism. When the composite profile from \"both (RAPD-PFGE) methods was generated, the 115 strains could be separated in 24 groups, according to their DNA pattern. The results indicated that environmental strains of L. monocytogenes ali feel into composite profile groupings unique to the environment, while strains from both water and utensils shared another composite profile group. L. monocytogenes fresh shrimp iso/ates belonging to one profile group, were found in the different areas of the processing line. This same latter group was also present in food handlers from the processing and packaging areas of the plant.