Master of Science

RAPD-PCR is a useful new molecular technique to characterize and study genomes. In this study, in order to ensure the repeatability and reproducibility of RAPD-PCR results within and between laboratories, various factors influencing the reproducibility of amplification were investigated systematically. Results indicate that template DNA and magnesium factors were important parameters which affected reproducibility. Variation in these factors could cause discrepancies in amplification of certain of certain RAPD bands. It was important to use relatively pure DNA samples for RAPD-PCR amplifications. Although a standard protocol for genomic DNA extraction was employed, purity of DNA extracts varied from sample to sample. Impure DNA samples had to be re-purified before they were used for amplifications.

Results indicated that higher template concentrations were favourable for stable amplification of higher molecular weight products. On the other hand, higher primer concentrations were favourable for stable amplification of lower molecular weight products. There were different optimal concentrations of magnesium in RAPD-PCR amplifications using long or short primers. The usage of high magnesium concentrations was necessary for longer primers.

By performing studies using mixture of templates, results suggests that competition for amplification by background or contaminating DNA could lead to discrepancy or error in scoring some bands. However, the results also implied that the effect of background or contaminating DNA was insignificant when the template of interest was present in a much higher proportion.

To test the potential use of RAPD-PCR in analysing genetic diversity in subpopulations, zebrafish families were used as artificial subdivided populations. Two sets of RAPD data were used. The first set was made up of 71 monomorphic and polymorphic bands which were detected by 4 primers. The second set comprised of 41 polymorphic bands detected by 12 primers. A similarity index was used to quantify genetic identity while a F_st index was used to measure the partitioning of genetic diversity in these "subpopulations". The F_st values calculated from both sets of data were comparably similar. Results indicated that about 46.5% of the genetic diversity was attributable to the restricted breeding groups or "subdivided populations". In comparing to a group of random fish, the between-groups diversity dropped from 37.3% to 6.9% when the three groups of families were taken as one group (no longer subdivided). The sharp drop indicated that RAPD-PCR assay was a sensitive method to detect partitioning of genetic diversity caused by population subdivision or inbreeding.

The RAPD technique was also applied in the identification of 3 species in the genus Danio. D. frankei was postulated to be not a true species by some fish biologists. Analysis of similarity index was performed for the banding profiles obtained from these 3 species with 7 primers. The genetic distance was then calculated. In this study, eight species-specific bands were also identified after screening with 32 primers. The RAPD results consistently implied that D. rerio and D. frankei were of the same species and D. albolineatus was a different species. This molecular identification was in agreement with the observation of extensive interbreeding between D. rerio and D. frankei. The origin of D. frankei is still unknown but nonetheless, D. frankei should now be considered as D. rerio.

Alpha thalassaemia is a major genetic disorder that affects the production of a-globin chain of the haemaglobin commonly found in Southeast Asia. As a result of defective α-globin chain synthesis, individuals with this disorder show varying degree of anaemia due to ineffective erythropoiesis and haemolysis.

The percentage of α-thalassaemia occurrence in this region is thought to be increasing if no proper preventive programmes are taken place. To date, rapid detection and prenatal diagnosis followed by genetic counseling are the effective means to prevent the elevated valency of a-thalassaemia spectrum in Southeast Asia.

The presence of Hb H inclusion bodies in a-thalassaemia subjects has enabled the detection of Hb H using immunologic methods. The objective of this study is to develop a simple, rapid and accurate immunological diagnostic kit for the detection of α-thalassaemia by means of an Enzyme-linked Immunosorbent Assay (ELISA).

A murine monoclonal antibody specific to Hb H (В₄) was developed by immunising mice with reconstituted В₄. The synthetic В₄ was generated by separating the α and В chains from normal Hb A and reconstituted the В chains to form В₄. A few hybridoma clones showed specificity to Hb H but three clones with the highest specificity were evaluated (2C65B/9E, 5E5 and 8E5). Meanwhile, polyclonal antibodies specific to Hb H were developed by immunising sheep with FPLC-purified Hb H.

These antibodies were employed in a sandwich enzyme-linked immunosorbent assay (ELISA) to detect and quantify Hb H in haemolysates from subjects with a-thalassaemia. Various designs of ELISA were tried but a three-layer sandwich-ELISA was found to be most suitable. The sandwich design consisted of sheep polyclonal antibodies that were specific to Hb H immobilized on microtiter plate as the first layer, followed by haemolysates as second layer; the third layer comprised of the murine monoclonal antibody specific to В₄ and conjugated to horseradish peroxidase; and lastly tetramethyl benzidine was used as substrate.

Using the third sandwich ELISA method , a total; of 768 blood samples, consisting of 511 anaemic samples with low haemoglobin count and low mean corpuscular volume, 223 samples from normal subjects, 34 samples with abnormal haemoglobins, in which 18 samples were confirmed to have Hb H disease and 16 samples with α-thalassaemia trait were screened. An OD value of 0.2 was chosen as the cut-off value for normal subjects. Samples with OD values between 0.2 and 0.4 would be diagnosed as positive for α-thalassaemia trait, and those with OD values above 0.4 would be diagnosed as Hb H disease. By using these criteria, the sensitivities for detecting the Hb H disease and α-thalassaemia trait were 100% and 68.8% respectively. Using the same criteria, the specificity of the assay was tested and found to be 98.9%.

A second group of samples were screened using the second sandwich ELISA design. This group of samples consisted of 28 Hb H disease, 36 α-thalassaemia traits and 31 normal subjects. An OD value of 0.14 was chosen as the cut-off for normal subjects. Samples with OD values between 0.14 and 0.2 would be diagnosed as positive α-thalassaemia trait, and those with OD values above 0.2 would be diagnosed as Hb H disease. By using these criteria, the sensitivities for detecting Hb H disease and α-thalassaemia trait were 89.3% and 61.1% respectively.

During the analysis, 8 samples which were diagnosed as normal by the conventional methods in the hospital were shown to have abnormally high OD values by the second sandwich ELISA design. These samples were further analyzed and confirmed as Hb Constant Spring carriers.

There was one sample which was diagnosed as Hb Constant Spring (αα/αα^cs) showed unexpected low OD value in the range of normal samples. This sample was further investigated by DNA analysis and confirmed to have concomitant inheritance of В-thalassaemia. Thus, the reason for the low sensitivity in detecting α-thalassaemia trait may be due to concomitant inheritance of В-thalassaemia

This method is very suitable for the large scale screening of Hb H disease and α-thalassaemia trait due to its simplicity and feasibility. In addition, this method may be useful for detecting carriers of (--^SEA/ ) deletion and deletions involving the complete ζ-α-globin gene cluster, such as (--^Fil/ ), (--^Thai/ ) and (--^HW/ ), which are the common deletional α-thalassaemias in Southeast Asians.

This thesis describes the development and study of electroanalytical procedures for the determination of trace metallic species in solution. Two major studies are included. In the first study, a square wave stripping voltammetric procedure, utilizing a thin film mercury electrode, was developed and optimized. The procedure was suitable for the determination of Cd, Pb and Cu at the μg/L concentration level in natural water. The second study included the construction and characterization of carbon paste electrodes which were modified with zeolite molecular sieves. In conjunction with a medium exchange procedure, electrode sensitivity to traces of Cu, Cd and Zn was achieved. Furthermore, the interference effects from Co(II), Pb(II), Ni(II), Al(III), Cd(II), Cu(II), Ag(1) and Hg(I1) on the Zn signals were studied. Most notably it was observed that the interference was stronger for species, which had the higher coordination number in solution. This suggests that electrode sensitivity and selectivity were partially governed by the coordination numbers of the metallic species.

This study was undertaken to introduce morphological variations in Fagraea fragrans Roxb. (a way-side tree), Heliconia psittacorum cv. 'Golden Torch' (a landscape herb) and Peperomia magnoliaefolia (Jacq.) Diert. (a potted herb) through in vitro mutagenesis. These species are commonly grown in Singapore either for landscape beautification or for indoor displays at homes and offices. Introduction of any phenotypic variation in these plants would enhance their ornamental values.

A stock culture of each plant species under study was developed for in vitro mutagenesis experiments. Since there was no known protocol for tissue culture of P.magnoliaefolia, a regeneration protocol was developed through the culture of leaf blade and petiole explants on MS medium supplemented with both BAP and NAA. The optimum medium for shoot regeneration from petiole is the one supplemented with 5.2 M BAP and 2.6 M NAA and those from leaf blades 11 M BAP and 5.2 M NAA respectively. A combined application of cytokinin and auxin was essential to induce a high rate of normal shoot regeneration and growth on both petioles (50%) and leaf blades (47.6%). Shoot elongation and maturation was improved by providing regenerated shoots, excised from petiole surface with a brief flush of a high concentration of GA3 solution (280 M for 90 seconds).

A protocol for in vitro mutagenesis using two chemical mutagens namely N-methyl-N-nitrosourea (NMU) and ethyl methanesulfonate (EMS), was established for each plant species under study. The effects of different mutagens on in vitro survival and growth or regeneration of plant species were studied and compared with the control. A workable dose range and LD₅₀ values were determined for each mutagen. Both mutagens had inhibitory effects on each plant species under study. These were very prominent from a threshold dose. The workable dose range of NMU established for F. fragrans, H. psittacorum and P. magnoliaefolia at 25^o to 26^oC were 1 to 10 mM (3 hours treatment), 0.5 to 5 mM (3 hours treatment) and 1 to 4 mM (1 hour treatment) respectively. Similarly, the dose range of EMS worked out for F. fragrans, H. psittacorum and P. magnoliaefolia at 25^o to 26^oC were 0.008 to 0.12M (3 hours treatment), 25 to 110mM (3 hours treatment) and 0.04 to 0.16M (1 hour treatment) respectively. It was observed that most of the aberrations were derived from dosages either close to or above LD₅₀ values. The mutagen treated explants of F. fragrans and H. psittacorum were subcultured for at least 3 cycles on appropriate medium in order to allow regeneration and proliferation and to eliminate chimeras. In P. magnoliaefolia, adventitious shoots were regenerated on mutagen treated petioles and then allowed to multiply and develop fully so that the variants can be screened.

Mutants were identified through visual screening during in vitro culture and after transfer to soil, based on morphological changes that were absent among the control plants. Some of the interesting morphological variations observed were :

● A high frequency of variegated shoots (52.1% of total MV4 shoots screened) and albinos (17.2%) was derived from cultures of F. fragrans treated with 5 to 10mM NMU. Eight lines of mutants were obtained that consisted of variegated, albinos and light green shoots. These mutant lines differed from each other by leaf shape, size, variegation patterns, stem coloration and growth rate. The rooting ability of different mutants lines was tested. Lines 1, 2 and 3 hold great promise as commercial variants.
● A number of 'root variants' (23% of total plantlets) were derived from cultures of H. psittacorum initially treated with 1 to 3 mM NMU. The variants displayed about 86.2% increase in the number of roots per rooted plantlet over the control.
● One dwarf and one vigorously growing plant of P. magnoliaefolia were obtained from 1 and 4 mM NMU, respectively. They constituted about 1.9% of the total NMU treated shoots that survived after transfer to soil.

NMU induced a broad spectrum of aberrations in the three species (chlorophyll variations, changes in leaf shape, growth habit and rooting efficiency). Whereas, only chlorophyll variations were induced by different EMS treatments. Therefore, NMU appears to be more effective on the three species than EMS. A definite conclusion in this regard can only be drawn after long term field trial of the mutagen treated plants as variations can also be appeared at later stages of growth.

The effects of EMS and NMU on the rooting efficiency of the plant species were studied in terms of percentage of rooted plantlets and mean number of roots per plantlet as compared to the control. In F. fragrans and H. psittacorum EMS reduced the percentage of rooted plantlets to 35% and 65% of control respectively. Similarly, NMU treatments reduced rooting efficiency in F. fragrans to 80% of control. However, a stimulatory effect on root formation (150% of control) was observed in H. psittacorum in plantlets derived from higher concentrations of NMU (1 and 3 mM). In the case of P. magnoliaefolia the mutagen treatments did not have any effect on rooting efficiency of the shoots derived from the treated cultures as compared to the control.

Over the past five years, there has been much research interest in the quantity and quality of physical activity experienced by children and adolescents. Different guidelines have been given regarding the appropriate physical activity for children and adolescents to maintain and/or improve cardiorespiratory health.

The purposes of this study were to assess the physical activity patterns of Singaporean adolescents, to determine the differences between the physical activity patterns of girls and boys, to determine the differences between self-reported physical activity and heart rate monitoring, and to relate their cardiorespiratory fitness and body fat measurements to their habitual physical activity patterns. Heart rate monitors (Sport Tester PE 4000; Polar Electro OY, Finland) were worn by 58 girls (mean + SD; 15.6 + 0.6 yrs) and 46 boys (15.6 + 0.8 yrs) on three school days and a Sunday for a minimum of 14 hours each day. On each day of heart rate monitoring, subjects recorded their physical activity on a self-report form provided. A 2.4 km walk/run test was administered on another day for all subjects. On a separate day, 11 girls and 10 boys were randomly selected from the 104 subjects to participate in the laboratory fitness test.

The number and length of time of sustained 5-minute and 10-minute periods of heart rates greater than or equal to 120 b.min^-1 (ACSM, 1991) and of at least 140 b.min^-1 (Armstrong et al., 1991) were the two criterion heart rate measures used to assess the intensity of effort during physical activity. In the assessment of self-reported data, a modified 4-point scale of activity index (Puhl et al., 1990; Dipietro et al., 1993) was used to categorize the different activities of the subjects. Values of 3 and 4 on the activity index scale which correlated to heart rates greater than or equal to 120 b.min^-1 were used in the determination of relationship between self-reported activity and heart rates.

Daily self-reported physical activity used together with heart rate monitoring was a suitable means of assessing the quantity and quality of physical activity experienced by Singaporean adolescents. This study found no differences in the physical activity patterns of boys and girls during schooldays and Sunday. All subjects were significantly more active on schooldays than on Sunday. Physical activity patterns of Singaporean adolescents on schooldays were similar to those found in other countries. Boys had significantly higher capacity than girls in the time taken to complete the 2.4 km walk/run test and the laboratory graded exercise test (VO_2peak ). There were no significant differences between percent body fat and the amount of time spent on physical activity.

This study provides further evidence that activity patterns for Singaporean adolescents do not attain sufficient intensity or duration to maintain or improve cardiorespiratory fitness. However, when the criteria of 120 b.min^-1 is applied, 71.1% of the boys and 79.3% of the girls achieved a minimum of three 5-minute blocks. This criteria may need further assessment to be considered as a minimum measure of habitual physical activity for adolescents for the maintenance of healthy lifestyles.

Distribution of light in biological media has interested many researchers especially with the advancement in medical applications of laser. It is well known that light distribution in tissues is governed by the absorption coefficient, μa, scattering coefficient, μa, and the anisotropy factor g. Many researchers have already developed models to map the light distribution in tissues in order to understand its behaviour better.

The models that are studied here are Beer's Law, Kubelka-MunkTheory, Wave Theory, Radiative Transport Theory and the Diffusion Approximation. The shortcomings and conditions of each theory is presented. Due to the nature of surface tissues, i.e. being very thin and multilayered, coupled with the fact that a perfectly diffused state is not achievable in such thin layer, direct application of these theories on the surface tissues is not possible.

A method of measuring the lateral distribution profile on the epidermis is developed. Detailed theoretical considerations and calibrations of the system for both collimated and diffused light sources are conducted. Simultaneous measurements of lateral distribution profile for both the upper and lower surfaces are achieved. Samples used are from chickens, pigs, dogs and humans. The effects of beam diameter variations of incident light on lateral distribution is studied. It is found that when a collimated light source with a small beam diameter (-1.0 mm) is used, the distribution profiIe is closest to a normal distribution. With a larger beam diameter (-2.5 mm), the profile broadened at the centre but fell off more rapidly at larger lateral distances.

Comparison between a "high absorption" skin (Black Chicken) and "high scattering* skin (Normal Chicken) is conducted. The lateral distribution profile is also studied using a diffused light source. Across various beam diameters of incident light, the distribution profiles showed a lack of distinct shape when diffused light was used. In this case, the result show that the beam diameter has little effect in the profile whereas the intensity of the diffused light does. Experiments were also conducted with normal and carcinoma human rectal tissues. The difference in profile between normal and carcinoma tissues was observed. The results indicate a lower amount of absorption and a higher amount of scattering in carcinoma tissues.

A fundamental optical parameter, the refractive index is often used in light distribution studies. Its value, however, is usually assumed to be that of water. A simple yet accurate method for the measurement of refractive index on surface tissues is developed. The mismatch in refractive index between glass and tissue is exploited to achieve total internal reflection. Detailed theoretical studies of the measurement of the refractive index on skin is carried out. In-vitro experiments are conducted on porcine, chicken, and human abdominal epidermis. The refractive index of chicken and porcine epidermis at 633 nm are found to be 1.418 and 1.444 respectively.

Lasers of different wavelengths are used and correlations of refractive indices of abdominal epidermis between male and female, and across different age groups are studied. The results show that across all wavelengths studied, the refractive index of human abdominal epidermis is independent of age and sex.

In-vivo experiments are conducted on human palm epidermis at various wavelengths. Comparison of refractive index between Chinese and Indian palm epidermis is also performed. There is no significant difference between their refractive indices. Results also showed that the refractive indices of the palm are higher than the abdominal epidermis across all three wavelengths examined.

Light distribution in tissues is a problem that has baffled researchers of laser therapy. With the increased popularity of photodynamic therapy to treat certain tumours, the understanding of the light distribution in tissues becomes increasingly important. It is known that the light distribution in a tissue is governed by the absorption coefficient, p,, scattering coefficient, p,, and the anisotropy coefficient, g. Work has been done by many researchers to develop models to map light distribution in tissues in order to better understand its behaviour. The methods studied in this thesis were the Beer's Law, Transport Theory, Diffusion Approximation and the Monte Carlo simulations. The conditions and shortcomings of each method were presented. Recent developments have also indicated that the Monte Carlo method is effective in simulating light transport in tissues. As light impinges on a biological media, it undergoes a region of anisotropic distribution near the surface characterised by an increased fluence due to the backscattering of the incident light. This incident light, after passing through a region of tissue, causes the light distribution to go into diffusion characterised by a gradual drop in light intensity as the tissue depth is increased.

The 2-dimensional light distribution in a tissue was compared with a Monte Carlo simulation based on tissue optical parameters of p=, p, and g. This light distribution study was extended to 3-dimensions in bovine, chicken and porcine tissues. It was found that light distribution in the tissues depends considerably on the scattering and absorption in the tissue giving rise to distinct distribution patterns with the spread of the light intensity increasing with beam diameter. Chicken tissues having the greatest scattering, showed the least drop in intensity with increasing lateral displacement from the incident beam at a fixed tissue thickness, while bovine tissues, having low scattering, showed the greatest drop. The degree of beam spread in the tissue is also found to depend on the abeldo of the light distribution. The dependence of light transport on tissue refractive index, n, is always ignored in light distribution studies. An investigation on refractive index was carried out together with a simple method of determining its value in tissues. Values of the refractive index in simple biological materials, such as coagulated egg white, as well as in selected human tissues were found and compared. In selected human tissues, the refractive index of tissues from different patients as well as different wavelengths were compared.

This thesis describes the study of some novel piperazine derived amphiphiles and polymeric materials.

In the first chapter, the synthesis of a series of some long chain piperazine derivatives, Nalkyl-N'- methyl piperazine and their amphiphilic salts, N-alkyl-N'-ethyl-N'-methyl piperazinium bromide, the related N-alkyl-N,N-dimethyl piperazinium bromide, and the polymerizable counterparts, N-acryloyl-N'-alkyl-N'-methyl piperazinium bromide were described. The products were characterized by ¹H and ¹³C NMR FTIR spectroscopy; and elemental analysis. The NMR data showed unequivocally that the quaternization of Nalkjl-N'-methyl piperazine by the reaction with ethyl bromide, and the quaternization of Nacryloyl-N'-alkyl-N'-methyl piperazine by the reaction with n-alkyl bromide occurred exclusively at the nitrogen atom bearing the methyl group due to the steric hindrance of the substituents.

Critical micelle concentration (c.m.c.) and surfactant properties of the amphiphiles were investigated. Plots of surface tension y against log (concentration) were presented. It was found that y_c.m.c. increased progressively with the increase in the alkyl chain length for the three homologous series of amphiphiles, indicating that the piperazine moieties were more available for interaction with water as the chain lengh increased. N-acryloyl-N'-alkyl-N'-methyl piperazinium bromide were polymerized under both micellar and isotropic conditions. The polyelectrolyte behaviour was studied by viscometric method. The polymers prepared under micellar conditions had a higher intrinsic viscosity than the ones prepared under isotropic conditions. This was attributed to the aggregation of the monomers under the micellar environment.

The second chapter describes the synthesis of some polymeric materials of piperazine. N-acryloyl-N'-methyl piperazine (AcrNMP), homopolymer of AcrNMP, crosslinked polymers of AcrNMP with ethyleneglycol dimethacrylate, copolymers of AcrNMP with MMA, N-rnethacryloyl-N'-methyl piperazine, and N,W-diacryloyl piperazine were synthesized. The monomer ratios in the copolymer were analyzed by FTLR spectroscopy. The reactivity ratios of the copolymers of AcrNMPMMA were evaluated both by the Finernan-Ross (F-R) and Kelen-Tudos (K-T) methods. The monomer reactivity ratios were found to be r₁(AcrNMP) = 0.552 and r>(MMA) = 1.074. The swelling ratio of the crosslinked polymers decreased with an increase in the percentage of the crosslinker used. This was attributed to the hydrophobic nature of the crosslinker.

In the third chapter, the application of a piperazine based copolymer (AcrNMPmllMA) for the detection of Hg(I1) ions, using anodic stripping voltammetry was presented. A chemically modified electrode (CME), was fabricated by attaching the copolymer film to a glassy carbon electrode. The polymer swelled in aqueous solution, allowing easy access of Hg(I1) ion for binding. The effects of pH of the measurement and deposition solutions, deposition time and the Hg(I1) concentrations were studied. The limit of detection was estimated to be 0.23 μglml.

The management of turfgrass in Singapore has received much attention in recent years. It was given greater emphasis with the construction of more new golf courses. With higher expectations for quality of turfgrass management and the need to reduce maintenance cost, there arises a requirement to have better knowledge in maintaining good quality turfgrasses under local conditions and at a relatively low cost. As fertiliser programme, in particular nitrogen(N) and iron(Fe), constitutes the key factor in determining the quality of the turfgrass, this study was conducted to determine their optimal rates of applications under local conditions to achieve good quality turfgrass.

An experiment using specially designed and built lysimeters, was conducted to investigate the effects of different rates of N and Fe fertilisation on the growth of Tifdwarf bermudagrass (hybrid, Cvnodon dactvlon L. X C.transvaalensis L. BurttDavey) and Zoysia iavonica Steud. under local field conditions. Three N levels investigated for their effects on the growth of these two turfgrasses were NO = 4 g/m², N1 = 8 g/m² and N2 = 12 gm² while the Fe lcvcls investigated were Fe0 = 0.1 g/m², Fe1 = 0.3 g/m² and Fe2 = 0.5 g/m².

The effects of two sand particle sizes used as the rootzone media, on the rate of leaching of these two nutrients, were also studied. The sand particle size ranges were. P1 = 0.2 mm to 1.0 mm in diameter with 50% of thc sand at 0.5mm and P2 = 1.0 mm to 2.5mm in diameter with 50% of the sand at 1.5mm. The growth parameters measured were dry weight of the leaf clippings(DW), % N and Fe in the leaf tissue, relative chlorophyll content and visual ratings.

At the end of the experiment, the root length and density, the total biomass of the turf over the 1 m² plot and the organic layer accumulated were also measured. For N effects in Tifdwarf bermudagrass, the amount of DW of leaf clippings, % N in the tissue, biomass, relative chlorophyll content, organic layer and root density were all significantly increased at higher rates of application (N = 8 g/m² to 12 g/m²). For comparisons, DW were recorded as 68.24±8.58g under high N level, compared to 52.07±3.70g under low N level. % N in the tissue was 4.75±0.17% and 4.05±0.06% for high and Low N level respectively. Chlorophyll content was increased from 4.26±0.10 mg/g DW under low N level to 4.43±0.15 mgjg DW under high N level. There was an inverse relationship between N levels and root length, and there were no significant N effect on visual ratings.

Similarly for Zoysiagrass, the DW of leaf clippings, % N in the tissue, organic layer and root density were also increased with higher N fertilisation (N = 8 g/m² to 12 g/m²). There were significantly more DW under high N level at 69.48±7.42g compared to 58.01±3.11g under low N level. The % N in the tissue was higher at 3.02±0.17% under high N level compared to 2.73±0.05% under low N level. The relative chlorophyll content, biomass and root length however were different in terms of their response as compared to Tifdwarf bermudagrass, in that, no respouse towards different levels of N fertilisation was noted.

Generally the Fe effects were insignificant on the growth parameters measured for Tifdwarf bermudagrass. DW of leaf clippings, % N in the tissue, biomass, root length, organic layer, root density and visual ratings were not affected by the increased levels of Fe fertilisation.

Only the amount of Fe in the tissue and the relative chlorophyll content of Tifdwarf bermudagrass were significantly increased with higher levels of Fe fertilisation after 16 weeks. Relative chlorophyll content for turf under high Fe fertilisation (0.5 g Fe/m²) reached 4.42±0.11 mg/g DW at the end of the experiment as compared to 4.28±0.10 mg/g DW under low Fe fertiilisation (0.1 g Fe/m²).

Zoysiagrass was not affected by the different Fe levels, and showed similar response to Tifdwarf bermudagrass in the DW of leaf clippings, % N in the tissue, biomass, root length, organic layer and relative chlorophyll content. Amount of Fe in the tissue was increased with higher levels of Fe applications. Root density and visual ratings for Zoysiagrass were also significantly improved with the increased of Fe level from 0.1 g/m² to 0.5 g/m² . The Fe effect on visual ratings was significant for plots grown under medium N (8 g/m²) fertilisation but it was insignificant at the low N(4 g/m²) and high N (12 g/m²) fertilisation.As for leaching, the larger the sand particle size, the more leachate were observed to percolate through the sand profile. At commencement of the experiment, up to 5 1.97±0.68% of the total N applied, under the larger particle size range(P2) were lost through leaching compared to 49.29±0.74% under the smaller particle size range(P1). This effect were reduced with time from application of fertiliser as available nitrate and Fe within the soil were depleted or absorbed by the grass. The N effects on leaching for both Tifdwarf bemudagrass and Zoysiagrass werc noted to be insignificant after 16 weeks. Similar results were obtained for the percentage of Fe lost through leaching under the same conditions, for both Tifdwarf bermudagrass and Zoysiagrass.

Two systems, i) the HBV precore genes, and ii) the human β-globin gene were used as models to develop simple, reliable and fast methods for the detection of point mutations. Three PCR-based methods were employed to detect the HBV precore stop codon mutant. They are RG-PCR, SSCP analysis and non-radioactive direct DNA cycle sequencing. To test the feasibility of RG-PCR for detection of this HBV mutation, wild-type and mutant control templates which were generated through site directed mutagenesis were used. Amplification of these templates with a modified primer will create a new Dde I restriction site for the wild-type DNA but not the precore mutant DNA. Results showed that the mutant sample could be distinguished from the wild-type after restriction digestion with Dde I. RG-PCR was then used to detect the presence of the mutant viral DNA in patients' serum samples. Of forty eight serum samples which are HBsAg positive but HBeAg negative, 27 samples displayed the mutant pattern. Wild-type pattern was observed in only 8 samples, while another 7 samples showed a mixture of wild-type and mutant patterns. No result was obtained for the remaining 6 samples due to unsuccessful amplification. Non-radioactive direct DNA cycle sequencing was subsequently employed for the detection and confirmation of the presence of the mutation in viral DNA in the serum samples. Sixteen samples tested with RG-PCR were sequenced. Nine samples had mutant sequence at nt 1896 and 3 samples had wild-type sequence. One sample showed both wild-type and mutant sequences. No bands were obtained for the remaining 3 samples. Other mutations were also found in the HBV precore/core gene. One was a frameshift mutation caused by the insertion of an A between nt 1836 and nt 1837. All the rest were base substitution mutations and were found in 13 different positions (nt 1846, 1848, 1899, 1916, 1933, 1938, 1975, 1977, 1984, 1992, 1993, 2002 and 2047). A mini gel system was used for SSCP analysis. The optimal condition was first determined with four different sets of PCR products generated through site directed mutagenesis. Each set consists of a wild-type and a mutant sequence. Electrophoresis was carried out on gels of various acrylamide concentrations (6%, 10% and 18%) and different percentages of cross-linking (5%C, 3.3%C, 2.0%C, and 1.0%C). The best result for SSCP analysis was observed in gels with 18% acrylamide (2.0%C) and 5% glycerol. SSCP analysis was carried out on samples which were previously characterized by RG-PCR. Two types of band patterns were observed for the wild-type samples, 8 for the precore stop codon mutant samples and 1 for the sample which has a mixture of wild-type and mutant. The many different patterns obtained showed that besides the stop codon mutation at 1896, there must be other base changes in the region. As results from DNA sequencing showed that this region of the HBV precore/core gene was highly polymorphic, SSCP analysis is therefore not suitable for the detection of this particular mutation. SSCP analysis was also used to detect the presence of point mutations in the β-globin gene of 46 samples. Four pairs of primers and four different gel conditions (1X and 1.5X MDE gel), 18% acrylamide [2.0%C] with and without 5% glycerol} were tested. The best separation was seen on 18% acrylamide (2.0%C) gel with 5% glycerol. All but one of the mutations could be distinguished from the wild-type and from each other. Unfortunately, the mutation which showed no mobility difference with the wild-type was the codon 41/42 (-TTCT) mutation, the most common in the local population. To detect this mutation by SSCP analysis, further optimization could be carried out with a shorter PCR fragment. In most cases, the heterozygous can be distinguished from the homozygous mutant. By using only four different sets of primers, 12 other mutations including the next 3 most common mutations nt -28 [A → G], codon 17 [A → T], and IVSI nt 5 [G → C] could be detected. In conclusions, RG-PCR is useful for the detection of the HBV precore stop codon mutant although other mutations in the region will not be detected. SSCP is sensitive but it is not suitable for the detection of the particular HBV mutant as this region of the viral genome is highly polymorphic. On the other hand, SSCP is useful for screening mutations in β-globin gene when it is complemented by other methods. Non-radioactive direct DNA cycle sequencing can be used to detect and confirm mutations in the HBV genome.

Although previous studies have addressed the issue of the health of overweight adolescents, few researchers have comprehensively examined the body composition and physiological profile of this population. The purpose of this study was to determine the body composition and selected physiological characteristics of overweight adolescent Singapore Chinese males and to examine the relationship of these parameters with that of an age, gender and race matched non-overweight adolescent group. This study addressed the following specific questions : what are the differences in the results of measurement of body composition using skinfolds and bioelectric impedance; and what are the differences (if any) between overweight and non-overweight male adolescent in body composition, resting blood pressure, heart rate, blood chemistry, pulmonary function variables and functional aerobic capacity?

Forty boys of ethnic Chinese background and whose age ranged from 15 to 16 years from 10 different schools throughout Singapore were assessed in this study. One-half (n=20) of the subject met the criteria of being overweight and the other (n=20) as non-overweight, based on the Singapore standard height for weight charts. Body composition was evaluated using height, weight, sum of skinfolds, circumference of girths, bone widths and bioelectrical impedance. Blood chemistry was determined using the dry chemistry desktop analyzer for total cholesterol, low and high density lipoprotein and triglycerides. A spirometer system was used to measure pulmonary function variables. A graded exercise test was administered to obtain maximal oxygen uptake. Resting and exercise heart rate was determined using a heart rate monitor.

The overweight subjects had significantly higher (p < 0.001) Body Mass Index (BMI), waist/hip ratios, and in all 8 skinfolds, 10 circumferential girths and 2 skeletal widths measurements compared to the non-overweight subjects. There was a significant difference (p < 0.001) in percent body fat between the two groups using four formulae for skinfolds (eight sites) and two formulae for bioelectric impedance. Results showed that body fat was higher in all overweight than non-overweight subjects for all techniques with the Slaughter (1988) 2-site skinfold formulae and Houtkooper (1992) bioelectric impedance equation providing the most realistic data (that is, overweight subjects had predicted body fat greater than 25%.) Other skinfold and bioelectric impedance formulae tended to under-estimate percent body fat in the overweight group (that is, body fat was predicted to be less than 21%). The overweight subjects also showed significantly higher (p < 0.001) systolic blood pressure, triglycerides and very low density lipoproteins but significantly lower (p < 0.001) relative VO₂ max compared to the non-overweight subjects. There were no significant differences in the pulmonary function variables of the two groups. These data provide vital information to the practitioner in planning programmes and identifying precisely where overweight subjects differ physiologically from their non-overweight counterparts.

Nasopharyngeal carcinoma (NPC) is a malignant tumor that occurs is prevalent among Chinese in southern China, and in places wherever these Chinese migrate to. Studies on factors affecting the prognosis of NPC have revealed that early diagnosis and treatment are very important in disease management. One of the factors associated with the etiology of NPC is the Epstein-Barr virus (EBV), and antibodies towards this viral antigen have commonly been used in the diagnosis of this disease.

Various antigen complexes of EBV which are useful as serological markers in the diagnosis of NPC are broadly categorized as viral capsid antigen (VCA), diffuse early antigen (EA-D), restricted early antigen (EA-R), membrane antigen (MA) and Epstein-Barr nuclear antigen (EBNA). Each of these antigens has several polypeptides. For the diagnosis of NPC using the whole antigen complex extracted from the cell lines, immunofluorescence assay (IFA) has been shown to be the most sensitive and specific assay compared to other techniques. Enzyme-linked immunosorbent assay (ELISA) is less sensitive but has the advantage of being more suitable for screening populations at high risk for NPC. This test is easily automated, quick to perform and does not involve much skill. The sensitivity and specificity of using ELISA for the detection of NPC can be improved by using gene cloning technology to obtain pure polypeptides from the EBV antigen complex. Polypeptides are used to coat microtiter plates for ELISA.

The cDNA inserts, which were confirmed to have high potential for NPC diagnosis by immunoblotting, were recloned in the plasmid expression vector, pMAL-C2, and over-expressed in E. coli. The fusion protein was easily purified by affinity chromatography on an amylose column. However, out of the eight cDNA inserts studied, only the cDNA inserts from clones R29-3, R29-2, B19-4 and P24-1 were successfully expressed in large quantities. Large-scale purification of the MBP-fusion proteins was carried out and ELISA tests were done using the polypeptides as target antigens.

DNA sequencing indicated that the EBV antigens expressed from clone R29-3 and R29-2 belonged to the large subunit of the EBV ribonucleotide reductase, which was encoded by the carboxyl-terminal end of the EBV BORF2 reading frame. The recombinant protein p28 expressed from clone R29-3 was 28.1 kDa in length, which was encoded by 759 bp of DNA. The recombinant protein p15 from clone R29-2 was 14.9 kDa in length, which was encoded by 405 bp of DNA. In addition, a 13.1 kDa EA-D antigen p13, which was expressed from clone B19-4, was encoded by the 352 bp carboxyl-terminal end of the BMRF1 reading frame of the EBV genome. Recombinant antigen, p18, expressed from clone P24-1, was a thymidine kinase (TK) coded by the carboxyl-terminal end of the EBV BXLF1 open reading frame. This recombinant antigen was 17.6 kDa in length and was encoded by 480 bp of DNA.

A total of 156 sera from patients with NPC and 100 sera from healthy donors were tested for IgA and IgG antibodies to native VCA and EA, as well as recombinant polypeptides p28, p15, p13 and p18 by ELISA. IgA-VCA antibodies were detected in 80% (125/156) of the NPC samples, whilst only 4/100 (4%) of the sera from healthy donors were positive for IgA to VCA. In the IgA-EA assay, however, sensitivity was calculated as 64% (100/156), but this test was more specific with a specificity of 99% (1/100 positive).

In the diagnosis of NPC, detection of anti-EBV RR IgG antibodies had a higher sensitivity of 80% by p28-ELISA and 69% by p15-ELISA. The detection of anti-EBV RR IgA antibodies had a sensitivity of 60% by p28-ELISA and 50% by p15-ELISA. However, it was more specific for the detection of IgA anti-EBV RR antibodies with a specificity of 100%. The detection of IgG anti-EBV RR antibodies had a specificity of 98%.

Results showed that IgG-ELISA test with recombinant p13 (EA-D) could detect 71% of NPC patients' sera, whereas only 1% of normal individuals gave positive results. IgA-ELISA was less sensitive with a sensitivity of 67% but more specific with a specificity of 100% compared with IgG-ELISA.

The TK-IgG ELISA showed a sensitivity of 57% and a specificity of 91%. The Tk-IgA Elisa, however, was not as good and gave a low sensitivity of 43%, but showed a higher specificity of 100%.

We have also tried to test the suitability of combining different recombinant antigens as target antigens in ELISA for the diagnosis of NPC. Recombinant antigen p28 belonging to EA-R and the recombinant antigen p13 belonging to EA-D were combined at a ratio of 2:1 and coated onto ELISA plates. It was found that 133 out of 156 NPC patients (85%) had IgG antibodies to the combined antigens, whilst only 2 out of 100 normal individuals (2%) had such antibodies. 111 samples from the 156 NPC patients (71%) had IgA antibodies against the combined antigens whereas only 1 out of 100 normal samples (1%) had such antibodies. The ELISA using combined antigens was more sensitive than the ELISA in which individual recombinant antigens as well as the native antigens were used separately.

The IgG ELISA with combined antigens also detected 9 out of 49 NPC sera which were IFA-VCA and IFA-EA negative for IgA antibodies. These results implied that combined antigens might be a good complementary diagnostic test to IFA. Based on the results of 72 patients with other diseases, some of them were having early primary symptoms of NPC, 18 cases were tested positive indicating EBV infection. A follow up study of these samples to see whether the EBV positive cases would finally lead to any EBV related carcinomas would be very useful in ensuring early detection and early treatment.

Conventionally, IgA was the antibody used in both IFA and ELISA for the diagnosis of NPC and this is supported by many research publications. In our study, when pure recombinant protein of different antigen complexes (recombinant ribonucleotide reductase (p28 and p15), EA-D (p13) and thymidine kinase (p18)) were analysed systematically by ELISA, we found that in fact IgG against the different recombinant antigens is much better than IgA in the diagnosis of NPC. In comparison, use of IgG as serological markers was not recommended when the conventional method of native VCA and EA, extracted from the whole cell lines, were used.

The IgG antibody titers against the combined antigens determined by ELISA were found to correlate well with the IgA anti-EA titers determined by IFA. Hence this assay might also be suitable for use in monitoring the recovery of NPC patients after radiation or other therapies. In addition, there was also a positive and significant correlation between the values of the OD readings of IgG anti-p28/p13 combined antigens determined by ELISA and the IgA titers against EA (r = 0.37) and VCA (r = 0.33) determined by IFA. Therefore the ELISA IgG test using combined antigens could be considered as a complementary screening assay to the IgA-IFA for the detection of NPC. Using the p28/p13 combined polypeptides as target antigen, there was also a significant correlation (r = 0.34) between the ELISA IgG antibody titers and the dOD readings determined by ELISA.

In summary, for the diagnosis of NPC, it is strongly recommended to use a mixture of several recombinant antigens in detecting antibodies to EBV by ELISA. Sensitivity is greatly improved. The ELISA with combined antigens p28/p13 is recommended for rapid and inexpensive serodiagnosis of populations with high risk of NPC. The combined recombinant antigens anti-EBV ELISA described here represent an innovative assay system for the diagnosis of NPC.

This thesis is organized into seven chapters :

Chapter 1 outlines what was done in this thesis and how it was implemented.

Chapter 2 introduces the typical architecture, general features and characteristics of multilayer neural nets (MLNNs) studied in this research.

Chapter 3 focuses on the most popular learning algorithm, the standard Backpropagation (BP) method. The standard steepest descent algorithm is studied in detail and a fast adaptive steepest descent (FASD) algorithm, is proposed. The objective of the FASD is to maximize the stepsize every step, for the steepest descent algorithm. Also, the standard conjugate gradient algorithm and its extension to nonquadratic problems are studied while the Scaled Conjugate Gradient Algorithm (SCG) is studied in detail and a Modified Scale Conjugate Gradient Algorithm (MSCG) is proposed and implemented. The MSCG is an effective and feasible method to implement the principle of the standard conjugate gradient to nonquadratic problems and it can make the learning fast and has less calculation complexity and memory usage involved in training the neural networks.

Chapter 4 presents a heterogeneous model of the ANNs, an alternative method to train the multilayer neural nets faster which has different activation functions among different hidden neurons.

Chapter 5 discusses a global minimum algorithms, i.e. the simulated annealing algorithm in detail.

In Chapter 6, a novel application of artificial intelligence, forecasting force-field parameters and lattice energies using neural networks, is discussed and it provides a complementary alternative to traditional methods and demonstrates the innate strength of neural networks and their potential as forecasting techniques in solid state physics and condensed matter physics.

Chapter 7 summarizes the student's research work and recommends a Multiple Conjugate Gradient Algorithm.

In this study, the effects of ultra sound on some heterogeneous chemical systems have been investigated.

The results of ultrasonic irradiation of some metal and non-metal systems show that ultrasound can promote the related reactions at lower temperature and that the ultrasonic effect depends on the frequency and the power of the sonicators, the nature of the reactants, and the solvents used.

The findings from the ultrasonication of copper/lead with dilute hydrochloric acid indicate that the mechanical effects of sonication will depend mainly on the structural morphology and the size of the solid reactant(s).

The dehydration of copper(II) sulphate pentahydrate by ultrasound is probably attributed to thermal effects caused by microjet impacts and short waves. It is believed that the nature of the sonicating solvent used also plays a critical role in the process.

The increased reactivity of magnesium in cold water by ultrasound and the enhanced kinetics of aluminium-copper(II) chloride (aqueous) system under the sonication are probably associated with the depassivation of the oxide coating by ultrasound.

The experience from the ultrasonic extraction of chlorophyll reveals that ultrasound can be a viable alternative extraction technique which offers advantages over other conventional methods.

The optimal performance of deinking process under sonication depends on a number of factors, e.g. the type of solvent, operating parameters of the ultrasonic source, nature of the inks, and type of paper used, etc.

It has also been discussed that the outcome from some of the sonochemistry experiments conducted in this study can also serve as discrepant events which will capture the curiosity of learners and thus promote their learning interest. The justification as well as the procedure of bringing sonochemistry into high school chemistry curriculum have also been illustrated.

Thus, this study has resulted in some interesting and useful results. It has also led to the publication of three papers in the international refereed journals, and the presentation of two papers at the international conferences.

This thesis describes the synthesis of several new N-(2'-dimethylaminoethyl)arylidene-imines (L) of the formula 2-HOC₆H₃(X)C(R)=N(CH₂)nNR'2 and their reduced products (LH) with the formula of 2-HOC₆H₃(X)CH(R)NH(CH₂)nNR'₂ (where X = H, 3-OCH₃, 5-Br and 5-NO₂; R = H and CH₃; n = 2 and 3; R' = CH₃ and C₂H₅). The coordination behaviour towards diphenyltin dichloride and triphenyltin chloride of these newly synthesized Schiff bases, together with their saturated analogues, was also investigated. For the sale of convenience, the work embodied in this thesis is presented in the following chapters.

In the first chapter of the thesis, a critical review of the organic reactions as well as the complexation reactions of Schiff bases with organotins was presented.

The second chapter incorporates the details of suppliers, purity and other specifications of materials and instruments used in the present study.

The third chapter describes the preparation and characterization of a series of N-(2'-dimethylaminoethyl)arylideneimines (L) derived from the condensation reaction of substituted salicylaldehydes and N,N-dialkylethylenediamines. These newly synthesized Schiff bases were found to exist as zwitterions with a strong 6-membered intramolecular hydrogen bond between the phenolic proton and the azomethine N atom. The strength of this hydrogen bond is very much dependent on the nature of the substituents (X) attached to the phenyl ring.

The fourth chapter concerns the reactions of L with NaBH4. The results derived from the C, H, N elemental analyses, IR, ¹H NMR, ¹³C NMR and X-ray crystallographic studies of the products (LH-1-LH-7) show that this series of saturated Schiff base analogues exists in zwitterionic forms via the formation of a 6-membered intramolecular hydrogen bond. The study on these hydrogen bonds indicates that there is a gradual proton transfer process of the phenolic proton to the secondary amine N atom (O-H...N<->O...H-N) as the phenolic proton acidity increases due to the substituents at the phenyl ring. It is also found that for LH-5 (5-nitro substituted), the most acidic in the LH series of compounds, the intramolecular hydrogen bonding is broken and replaced by an intermolecular hydrogen bonding.

Two compounds, S1 and S2, were isolated as the major products for the two reactions involving LH-4 and LH-6 respectively. These two compounds were also characterized using C, H, N elemental analyses, IR, ¹H NMR and ¹³C NMR. An X-ray crystal structure determination of S1 was performed to confirm the proposed structure for S1 and S2 on the basis of their physico-chemical and spectral studies.

The synthesis and characterization of the triphenyltin chloride and diphenyltin dichloride adducts of L were described in Chapter 5. L compounds reacted with triphenyltin chloride or diphenyltin dichloride in zwitterionic forms and the coordination was through the phenolic oxygen atom resulting in a shift of the phenolic proton to the azomethine N atom (O-H...N<->O...H-N) in the 6-membered intramolecular hydrogen bond found in the free ligand. The structural implications derived from spectroscopic studies were also confirmed by the X-ray results for [Ph₃SnCI:L-1]0.5C₆H₆The last chapter of the thesis deals with the synthesis and structural features of the triphenyltin chloride and diphenyltin dichloride adducts of LH compounds. It was found that all the LH ligands reacted with organotins via its phenolic oxygen atom as this atom was polarized because of the presence of the zwitterions in LH-1-LH-6. Thus the Ph₃SnC1 tin atom in these adducts is pentacoordinated with the three phenyl rings in the equatorial positions while the chloride atom and the oxygen of the ligand occupying the axial positions. An X-ray structural crystallography for [Ph₃SnC1:LH-3]CH₃OH reveals the presence of methanol beside confirming the proposed structure for the adducts..

KTiOPO₄ (KTP), KTiOAsO₄ (KTA), β-BaB₂O₄ (BBO), LiB₃O₅ (LBO) and LiNbO₃ are important nonlinear optical materials for second harmonic generation (SHG) and optical parametric oscillation (OPO). We present a comprehensive investigation on the two-photon absorption (TPA) and bound electronic Kerr nonlinearity in these crystals using a picosecond, 532-nm-wavelength laser beam. By using Z-scan technique the nonlinear refractive indexes and two-photon absorption coefficients in the z-cut crystals are measured, as well as in KTP, BBO and LBO along the phase-matching angles for the SHG of 1064-nm radiation. The obtained TPA-coefficient and nonlinear-refractive-index data for the SHG crystals have extended the database of third-order optical nonlinearities in nonlinear optical crystals. The microscopic origin of the observed refractive nonlinearity can be understood in terms of bound electronic effects, and the theoretical predictions are in agreement with our measurements. Finally, we introduce an experimentally simple method to study the laser-induced damage in these crystals by extending the Z-scan technique. The measured damage threshold is inversely proportional to the nonlinear refractive index and the thickness of a crystal. The effect of self-focusing on laser-induced damage in these crystals is also discussed.

We begin Chapter 1 with a brief introduction to nonlinear optical materials. The third order-optical nonlinear processes in nonlinear optical crystals are described, and the past research on the third-order nonlinearities of SHG crystals is reviewed in brief. The purpose of our research work and the outline of the thesis are also given.

In Chapter 2 we describe the technical details of the Z-scan method used for the evaluation of the optical nonlinearities in the SHG crystals, including the experimental set-up and theoretical background directly related to the experimental data analysis.

Chapter3 presents our experimental study on the third-order optical nonlinearities of KTP, KTA, BBO, LBO and LiNbO3 crystals. We first measured the nonlinear refractive indexes and two-photon absorption coefficients in the crystals using Z-scan technique with linearly polarized 25-ps, 532-nm laser pulses. The anisotropy of the measured nonlinearities in the crystals is also determined by analyzing our experimental data through computer simulation. The microscopic origin of the measured refractive nonlinearity is compared with a theoretical model based on bound electronic effects, indicating that bound electronic Kerr effect associated with the two-photon absorption should be responsible for the refractive nonlinearity in SHG crystals under picosecond laser radiation.

In Chapter 4, we introduce a new method for measuring laser-induced damage in nonlinear optical crystals by extending Z-scan technique, and determine the optical damage thresholds in KTP, BBO, LBO and LiNbO3 crystals. We also discuss the effect of self-focusing on laser-induced damage in the crystals.

Chapter 5 summarizes all the important experimental findings and the conclusions.

Migratory shorebirds flying southwards to escape the harsh winter may make use of one of the routes, the Asian-Australian Flyway. Successful migration requires that the birds make regular feeding stops, such as at the mudflats of coastal Malaysia and Singapore. I studied one such feeding site in Singapore, at the Sungei Buloh Nature Park, to investigate the effects of artificial manipulation of mudflats on the quantity of the birds' food supply.

Benthic samples from four transects in differently-managed disused prawn ponds of Sungei Buloh Nature Park were investigated. Family diversity, abundance and biomass of macroinvertebrates in each station were recorded at monthly intervals for 14 months. A higher family diversity in all the stations was observed in the non-migratory season. Temporal variation was only observed in the two transects where removal of vegetation took place, resulting in a decrease in family abundance and biomass. Ponds subjected to tidal influence, and with vegetation intact, were richest in terms of number of families, the highest abundance and biomass of benthic macroinvertebrates. The transect in which the pond was managed, and vegetation removed, had the poorest benthic macrofaunal diversity, abundance and biomass.

Shorebirds were observed to feed mostly in the tidal pond without any vegetation, and roost mainly in the managed pond without vegetation. To increase the attractiveness of the disused prawn ponds to shorebirds, a management strategy is proposed, including altering the present water management regime and removing mangrove seedlings and grasses from all the tidal ponds.

DNA carries biological information in a form that must be precisely copied and transmitted to all progeny cells. Occasionally, changes in the sequence of a gene may occur during the replication process. These "genetic mistakes" are known as mutations. Mutations can be identified by physical, chemical or enzymatic means. In this study, mutations in the human B-globin gene and HBV pre-core gene were used to evaluate the sensitively of Denaturing Gradient Gel Electrophoresis (DGGE). DNA sequencing was then performed to verify and confirm the unknown mutations in the human B-globin gene as well as the HBV pre-core gene.

Different regions of the B-globin gene were obtained via Polymerase Chain Reaction (PCR) amplification and the products were subjected to DGGE. Modifications to standard protocols were made to establish better electrophoretic and gel conditions to improve the resolution of bands as well as to increase the sensitivity of the system. All the different mutations were detected and the band pattern for each mutation was different. The presence of polymorphisms was also detected by DGGE as different band patterns. Results were found to be highly reliable. Changes in samples with unknown B-globin mutations were detected by DGGE and were subsequently verified by sequencing. DGGE was also used in antenatal diagnosis. Results from DGGE analysis of antenatal samples were found to be highly reliable.

For the detection of HBV pre-core mutant (mutation at nt 1896) using DGGE, results were not promising. The region of interest for DGGE analysis was obtained via PCR. Bands at different positions were observed. The pre-core region of these samples were sequenced and changes in other nucleotides were found. As a result, band positions that were obtained from control samples could not be used as references to identify the presence of nt 1896 mutation in serum samples.

In conclusion, DGGE has proven to be a highly sensitive method in the detection of mutations with an efficiency approaching 100%. It is suitable for the detection of single substitution mutation which occurs in any position along a DNA fragment which is highly conserved. Its application in prenatal diagnosis shows promise in future as a screening method. However, it might not be useful if the region is highly polymorphic.

The discovery that the use of PCR with an arbitrarily selected primer to amplify a specific set of arbitrarily distributed loci in any genome laid the foundation for high output of genetic markers that can be used for a variety of purposes. In this study, RAPD was utilized as a method in the identification, interrelationship analysis and the construction o f a basic linkage map of medaka (Oryzias latipes). The optimized RAPD-PCR protocol obtained, reveal a moderate degree of genetic diversity among the strains of medaka studied.

Coefficient of similarity based on RAPDs between individuals from each strain ranged from 93.2% to 99.7% with the two inbred strains, HO5 and HNI, having similarity coefficients of 99.78% and 99.42% respectively. Matching coefficients between strains based on RAPDs ranged from 0.7 to 0.95 suggest a narrow genetic base for the medaka studied. Cluster analysis of RAPDs employing UPGMA grouped the medaka strains examined into 3 main groups. The HNI strain, the local fish obtained from Singapore, and the clustering of HO5 with all the mutant strains examined in one group. Medaka species specific DNA markers were obtained but strain-specific markers were only observed in HNI strain while markers specific for all the mutant strains derived from HO5 strain were also observed. The optimized RAPD-PCR protocol for medaka was also suitable for the analysis of two other organisms.

Using MAPMAKER, 13 linkage groups were generated with 53 segregating fragments falling into one of the linkage groups that covered 938.1 cM. However, 26 of the total 79 markers still remained unlinked. The two loci investigated, Wy and Red, were successfully included into linkage groups that covered 472.7 cM.

The cytoplasmic injection into fish egg is performed at the first cell stage, in order to maximize the chance of early integration into all cell lineages. Microinjection technique was employed for transferring the chimeric gene pOBZ-109 into the early stage of zebrafish embryos. Both the linearized and supercoiled forms of the injected DNA were shown to be replicated and expressed in the zebrafish, albeit in a mosaic fashion. Expression of lacz was observed in microinjected fish, with the expression concentrated in the regions surrounding the yolk of the fish suggesting transient expression. The assaying for transgenesis by PCR has made it feasible to screen for large number of sample in this study. Circular and linearized pOBZ109 were detected in 3 day old and 12 week old fish by PCR using the primer pair X-galC and OBA-15 which amplifies a 603 bp DNA fragment. However, variation in the intensity of the band were observed among the fish indicating that the initial concentration of the transgene sequence in the template used in PCR varies among the different fish samples. This result shown that foreign genes which were introduced at the beginning of fish embryogenesis, were not just present but were also replicated through the gastrula stage.

This thesis described the complexation reaction of selected ligands with organotin chlorides.

In the first chapter, the basic concepts of organotin compounds, ligands and multi-dentate ligands were described. Organotin complexes, with examples, were classified into several categories according to their structures. The second part of this chapter is a review on the factors that affect the formation of adducts.

In the second chapter, the reactions of 8-aminoquinoline (Aq) with diorganotin dichlorides (R₂SnCl₂, R = Ph, Me, Bu) were described in the first part. With Ph₂SnCl₂, a stable adduct, Aq-Ph₂SnCl₂, was obtained. However, the reaction of BU₂SnCl₂ with Aq, irrespective of the solvent used, gave hydrolytic products, [(BU₂SnCl)₂O]₂ and HCI, which subsequently protonated the ligand to form Aq-HCI. Depending on the solvent used, Me₂SnCl₂ reacted with Aq and yielded either an adduct, Aq-Me₂SnCl₂, or hydrolytic and protonated products, [(Me₂SnCl)₂O]₂ and Aq-HCI. Additionally, the adduct, Aq-Me₂SnCl₂ was found to decompose into Aq-HCI and [(Me₂SnCl)₂O]₂ upon recrystallisation in a commercial solvent. The adducts, Aq-Ph₂SnC1₂ and Aq-Me₂SnCl₂, were characterised with various physical methods including X-ray crystallography. These results confirmed that the decreasing sequence of acceptor strength for diorganotin dichlorides with respect to Aq is Ph₂SnC1₂ > Me₂SnC1₂ > Bu₂SnC1₂.
,br>The second part of Chapter 2 was concerned about the reactions of 8-methyl-aminoquinoline (MeAq) with R₂SnC1₂ (R = Ph, Me, Bu) in commercial chloroform. The reaction of Me₂SnC1₂ with MeAq gave (HMeAq)₂Me₂SnC1₄, together with [(Me₂SnC1)₂O]₂ while MeAq-HCI and [(Bu₂SnC1)₂O]₂ were isolated from the reaction with Bu₂SnC1₂. Diphenyltin dichloride reacted with MeAq and afforded an unstable adduct, MeAq-Ph₂SnC1₂, which was easily decomposed into (HMeAq)₂Ph₂SnC1₄ and [(Ph₂SnC1)₂O]₂ upon recrystallisation in hot CHC1₃. The adduct, MeAq-Ph₂SnC1₂, and the complex salts, (HMeAq)₂R₂SnC1₄ (R = Ph, Me), were characterised. All the products isolated revealed that the hydrolytic rate of R₂SnC1₂ followed the order of Ph₂SnC1₂ < Me₂SnC1₂ < Bu₂SnC1₂ . In addition, results derived from product studies showed that the electron-donating ability of MeAq was reduced compared to that of Aq. It indicated that the methylation on donor atom (N) of a ligand reduced the coordination ability of this ligand.

The reactions of 8-methoxyquinoline (Mq) with R₂SnC1₂ (R = Ph, Me, Bu) and Ph₃SnC1 were discussed in the third chapter. All these RSnC1₂ reactions yielded not adduct but complex salts and protonated compounds, together with the corresponding distannoxanes. The reactions of Bu₂SnC1₂, irrespective of the solvent used, gave Mq-HC1 and [(Bu₂SnC1)₂O]₂ while (HMq)₂Me₂SnC1₄ and [(Me₂SnC1)₂O]₂ were isolated from the Mq reaction with Me₂SnC1₂. When Mq reacted with Ph₂SnC1₂ in chloroform and it yielded (HMq)₂Ph₂SnC1₄ and [(Ph₂SnC1)_2O]2 . However, when the same reaction was carried out in cyclohexane, an additional complex salt (Mq-HMq)Ph₂SnC1₃, besides (HMq)₂Ph2SnC1₄ and [(Ph₂SnC1)₂O]₂, was isolated. Furthermore, (Mq-HMq)Ph₂SnC1₃ was found to decompose into (HMq)₂Ph₂SnC1₄, [(Ph_2MSnC1)₂O]₂ and Mq-HCI. All the protonated products wre characterised, including X-ray structural analyses of (Mq-HMq)Ph₂SnC1₃ and (HMq)₂Ph₂SnC1₄. Isolation of these products reaffirmed that the hydrolytic rate of R₂SnC1₂ followed the sequence of Ph₂SnC1₂ < Me₂SnC1₂ < Bu₂SnC1₂. Additionally, the composition of (Mq-HMq)Ph₂SnC1₃ displayed that the [Ph₂SnC1₃] anion disproportionated into [Ph₂SnC1₄]^2-, a dianion, and Ph₂SnC1₂.

An outer-sphere coordination adduct, Mq-H₂O-Ph₃SnC1, with a different structure as usual, was prepared and characterised with various physical methods through the reaction of Mq and Ph₃SnC1.

The reactions of 3-methyladenine (3-MeAd) and N,N,9-trimethyladenine (Me3Ad) with R₂SnC1₂ (R = Ph, Me, Bu) were depicted in the fourth chapter. Dimethyltin dichloride reacted with 3-MeAd and produced a 2:1 adduct, (3-MeAd)₂Me₂SnC1₂, irrespective of the ratio of the donor and acceptor used. The reaction of Bu₂SnC1₂ with 3-MeAd gave [(Bu₂SnC1)₂O]₂ and 3-MeAd-HCI. However, the reaction of Ph₂SnCI₂ with 3-MeAd only afforded, besides Ph₃SnC1 and 3-MeAd-HCI, the disproportionated product, (3-MeAd)₂PhSnC1₂, which cannot be characterised probably due to its hydrolysis. From the structural characterisation of (3-MeAd)₂Me₂SnC1₂, the coordination site of 3-MeAd was confirmed to be at the N7 atom.

Me3Ad reacted with R₂SnC1₂ (R = Ph, Me, Bu) and yielded Me₃Ad-HCI, together with the corresponding distannoxanes, [(R₂SnC1)₂O]₂. These results reaffirmed that the methylation on donor atom (N) of a ligand reduced the coordination ability of this ligand.