Aberrant expression of short non-coding micro RNAs (miRNA) in many human diseases, along with remarkable stability in physiological media, has made them attractive clinical biomarkers. In particular, miRNA-122 is substantially elevated in plasma of patients with established drug-induced liver injury and can also be used to identify early liver injury when current markers, such as alanine aminotransferase (ALT), still show normal levels. The development of a rapid test for miRNA-122 e.g. in drug poisoning would allow earlier and more sensitive clinical diagnosis of liver injury. Nucleic acids are traditionally analysed by polymerase chain reaction (PCR), which has a high degree of sensitivity but suffers from high cost and is prone to sample contamination. The aim of this project is to develop a PCR free method to directly detect miRNA- 122 in biological samples using SMART technology. The SMART technology takes advantage of dynamic chemistry for sequence specific recognition of nucleic acids using aldehyde-modified nucleobases (SMART nucleobases), and target-complementary peptide nucleic acid (PNA) probe containing an “abasic” position (so called modified PNA probe). In this study, this unique detection method was used in a fluorescent detection with the use of light up probes, which are probes with an environmental dye as nucleobase; a FRET system was also designed to allow the discrimination between perfect match target and mismatched one. The SMART technology was also transferred onto magnetic beads to develop an ELISA like assay allowing sensitive and rapid detection of single stranded DNA mimic of the miRNA-122. With its potential PCR free approach, this easily adapted platform promises to transform and expand routine clinical diagnostic testing and screening for circulating miRNAs.

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Mycoplasmas conform most closely with the theoretical concept of 'minimum cells', existing as the smallest, free-living organisms capable of self-replication. They survive as parasites of plants, insects, animals or humans, with the most common human colonising species being Mycoplasma hominis. M. hominis has been characterised as a human pathogen responsible for a variety of infections, which pose a significant threat particularly to immunocompromised patients and neonates. However little has been elucidated about the cell physiology and molecular structure of this organism. Of interest to this study were the investigation of the heat shock response of M. hominis and the diagnostic assays used for its detection. The heat shock response is a ubiquitous physiological feature of all organisms and displays unprecedented conservation. This phenomenon is particularly evident in the 70 kDa family of heat shock proteins (hsp70) which exhibits a high degree of homology between different species. The hsp70 gene from M. hominis was cloned and preliminary partial sequencing indicated the similarity with other hsp70 homologs. The regulation of hsp70 expression at the transcriptional and translational levels was investigated. The level of hsp70 mRNA was found to increase correspondingly in response to heat shock, more visibly than the level of hsp70 protein. However imrnunochemical studies of the M. hominis hsp70 translation product demonstrated further the homology with other species. To facilitate rapid diagnosis of M. hominis infections, a PCR ELISA diagnostic assay was developed and optimised. The amplification of a conserved region of the M. hominis 16S rRNA gene was linked to subsequent hybridisation to an appropriate capture probe in a microtiter plate format. The sensitivity of the assay was comparable to other molecular assays although the PCR ELISA produces more rapid results and is less labour intensive. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1998.

Biological/biochemistry analyses traditionally require bulky instruments and a great amount of volume of biological/chemical agents, and many procedures have to be performed in certain locations such as medical centers or research institutions. These limitations usually include time delay in testing. The delays may be critical for some aspects such as disease prevention or patient treatment. One solution to this issue is the realization of point-of-care (POC) testings for patients, a domain in public health, meaning that health cares are provided near the sites of patients using well-designed and portable medical devices. Transportation of samples between local and central institutions can therefore be reduced, facilitating early and fast diagnosis. A closely related topic in engineering, lab-on-a-chip (LOC), has been discussed and practiced in recent years. LOC emphasizes integrating several functions of laboratory processes in a small portable device and performing analysis using only a very small amount of sample volume, to achieve low-cost and rapid analysis. From an engineer's point of view, LOC is the strategy to practice the idea of POC testing. This dissertation aimed at exploring the POC potentials of porous membrane-base LOC devices, which can be used to simplify traditional and standard laboratory procedures. In this study, three LOC prototypes are shown and discussed. First the protein sensor incorporating with silica nanofiber membrane, which has shown 32 times more improvement of sensitivity than a conventional technique and a much shorter detection time; secondly the bacteria filter chip that uses a sandwiched aluminum oxide membrane to stabilize the bacteria and monitor the efficacy of antibiotics, which has reduced the test time from 1 day of the traditional methods to 1 hour; the third is the sensor combining microfluidics and silica nanofiber membrane to realize Surface Enhanced Raman Spectroscopy on bio-molecules, which has enhancement factor 10^9 and detection limit down to nanomolar, but simple manufacturing procedures and reduced fabrication cost. These results show the porous-base membrane LOC devices may have potentials in improving and replacing traditional detection methods and eventually be used in POC applications.

Potato leafroll virus (PLRV) causes yield and quality losses in potato. PLRV is identified by plant symptoms and serological tests such as an enzyme-linked immunosorbent assay (ELISA). A similar serological test, direct tissue blotting assay (DTBA), was used to detect and monitor PLRV movement in field-inoculated Russet Burbank plants and plant tissues from Russet Burbank and Russet Norkotah seed tubers submitted by growers for winter certification tests. DTBA was as accurate as ELISA and easier to use for detecting tuber-perpetuated PLRV in stems and petioles of plants grown from grower-submitted seed tubers. ELISA detected twice as many PLRV positives as DTBA in leaflet tests. DTBA detected PLRV in tuber tissue but results matched ELISA in only 74% or less of the samples. Results of DTBA tuber tests were sometimes difficult to interpret while stem and petiole results were distinct and unambiguous. As inoculations were delayed later in the season and as plants matured, PLRV infection levels decreased sharply, most often within a two week period in early July. In same-age plants inoculated 43 days after planting but 18 days apart, early inoculation produced higher PLRV levels. Conversely, when same-age plants were inoculated 62 days after planting but 19 days apart, late inoculation produced higher PLRV levels. This discrepancy is not fully understood, but larger tuber size at the later inoculation probably produced a stronger sink for source-to-sink translocation of nutrients and phloem-limited viruses. Results of DTBA winter grow-out tests of summer-infected tubers approximated those of ELISA and visual inspections. Indirect DTBA testing of tubers utilizing stem and petiole tissues from winter growout plants detected more PLRV than directly testing tuber tissue 21 days post inoculation in summer. DTBA detected current season (primary) PLRV less reliably than secondary (tuber-borne) PLRV, similar to reported ELISA results. PLRV infection increased tuber numbers but decreased size. Size reduction was most evident in plants infected early in the season. Average tuber size in healthy plots was always larger than the average tuber size in infected plots. Within an infected plant, small tubers tended to be infected less often than large tubers. / Graduation date: 1993

Universiẗat, Diss., 2007--Giessen.

Thesis (Ph.D.). / Written for the Dept. of Biomedical Engineering. Title from title page of PDF (viewed 2008/01/12). Includes bibliographical references.

Thesis (M.S.)--Eastern Illinois University, 2009. / Includes bibliographical references (leaves 23-26).

Thesis (M.Sc. (Clinical Pathology))--Mahidol University, 1986.

Thesis (M.Sc. (Environmental Biology))--Mahidol University, 1988.