Philosophiae Doctor - PhD / Variation in sympatric and allopatric populations is believed to be a precursor to eventual speciation. The dispersion of genes from one gene pool into another is prevented by various processes, including the founder effect, sexual selection, ecological differences and random genetic divergence. Examining patterns of intraspecific variation in phenotypic and genotypic traits may thus provide valuable insights into the processes that govern species origination. Bladder grasshoppers (Orthoptera; Pneumoroidea) are an ideal model system to investigate patterns of geographic and ecological divergence due to their high host plant specificity, low dispersal and distinctive acoustic signals. This dissertation investigates intraspecific diversification in the bladder grasshopper Bullacris unicolor (Orthoptera: Pneumoroidea). Recent research on this species has shown significant intra- and inter-population variation in male advertisement calls and morphological characters. However, the exact cause of this variation has remained unclear. Furthermore, a previous study showed that the calls of B. unicolor from one particular population are highly differentiated from other populations, possibly due to the effects of anthropogenic noise. Here we aim to examine the drivers of diversity within Bullacris unicolor by embracing a multidisciplinary approach that encompasses the effects of environmental factors, genetics, anthropogenic noise and host plant associated divergence.

Bladder cancer (BlCa) is the second most common genitourinary cancer, affecting both men and women. Most (70%) cases present at the superficial stage; 20% of these recur with muscle-invasive disease. Major genetic alterations associated with BlCa include: loss/gain in expression or mutations in Retinoblastoma (RB) gene, human epidermal growth factor receptors (HERs), H-ras, p53 and FGFR3. Only p53 mutations are well correlated with invasive BlCa; other changes show variable correlations with disease status. To understand the progression of BlCa, a model of nine human BlCa cell sublines derived from a single parent but differing in in vivo characteristics, has been developed previously. These cells represent a heterogenous population from a single tumour and a model of different stages of BlCa progression, from non-tumourigenic to invasive. Two sublines were selected for further investigation: C3 (non-tumourigenic) and B8 (invasive). These were transfected with green (C3-GSP-2) and red fluorescent reporters (B8-RSP-gck) respectively to investigate the effects of their co-injection in vivo, specifically, promotion of C3 tumour growth by B8 cells. Surprisingly, B8 tumour growth was inhibited by C3 cells in vivo at different cell numbers and proportions of cells injected. Microarray analysis of C3 and B8 cells revealed differential expression of 1367 genes with dramatic differences in the transforming growth factor-?? and integrin-mediated pathways. Gene expression of BMP2, INHBB, FST, NOG, ID4 and TGF- ??1, in the TGF- ?? pathway was further analysed with qRT-PCR in all nine sublines. Expression of BMP2 was significantly related to tumourigenic potential (p=0.0238, Mann-Whitney) and INHBB to invasive ability (p=0.0476, Mann-Whitney). The BlCa model did not include a metastatic component. To broaden the model, cell lines were established from an invaded lymph-node (B8-RSP-LN) and a bone-metastasis (B8-RSP-BN) after subcutaneous and intra-cardiac injection of B8-RSP-gck cells. No significant differences were observed in the migratory capability and anchorage-independent colony formation of these metastatic cells compared with B8 cells. Evaluation of expression of the panel of TGF-beta genes (BMP2, INHBB, FST, NOG, ID4 and TGF- ??1) and metastasis-related genes (MMP9, MMP2 and KAI1) indicated that expression of BMP2, FST, ID4 and MMP9 was decreased or lost in the metastatic sublines.

There is much debate on the carcinogenic potential of disinfection by-products (DBP) in chlorinated water supplies. Until recently, epidemiological studies have been limited in their ability to examine accurately the risk of cancer with exposure to environmental carcinogens. This has largely been due to the long latency periods associated with cancer development, and the difficulties in accurately estimating chronic exposure. Although there is evidence, from predominantly case-control studies, of increased bladder cancer with exposure to chlorinated water supplies, the evidence is inconclusive. ¶ In an attempt to determine the carcinogenic potential of trihalomethanes (THMs) in chlorinated water, this study utilises DNA damage to bladder cells, evident as micronuclei, as a pre-clinical outcome measure. Using a pre-clinical marker helps overcome some of the limitations associated with long latency periods. The study improves on previous studies by estimating exposure to DBP at an individual level, and takes into consideration ingestion, inhalation and dermal exposure. ¶ A cohort study was undertaken in three Australian communities. The Bungendore (NSW) water supply was not chlorinated thereby providing a community unexposed to DBPs from chlorinated water. Canberra (ACT) and Adelaide (SA) had intermediate and relatively higher (but still within NHMRC guideline levels) of DBPs in the reticulation system. Trihalomethane levels in reticulated water (external dose) and in urine (internal dose) were used as exposure indices. As well, intake dose was computed by adjusting external dose for individual variations in ingestion and bathing. The primary outcome measure was the prevalence of micronuclei in bladder epithelial cells. A DNA index derived from flow cytometry was also used to estimate DNA damage in bladder cells. Associations between exposure and outcome were estimated using Poisson regression models, having identified and adjusted for interaction effects and confounders. ¶ A total of 529 participants were eligible to participate, of which 348 (65.8%) completed all aspects of the study. Analysis was limited to the 228 participants (65.53% of those who completed the study) who had slides suitable for micronuclei scoring. One hundred and forty three (63%) of the 228 participants were from the exposed communities, while 85 (37%) were from the unexposed community. This sample exceeded the estimated 50 per group required to detect a relative risk of 1.4, with a significance level of 0.05 and 80% power. ¶ External dose for total THM for the two chlorinated (exposed) communities ranged from 37.75 to 157.25 mg/l. Intake dose estimated by fluid intake diary ranged from 2.9 to 469.5 mg/l, while a retrospective questionnaire estimated intake dose to range from 0 to 409.4 mg/l. Internal dose (urine levels) of total THM for the same two communities ranged from 0 to 6.82 mg/l. Adjusted risk estimate for DNA damage to bladder cells (using the micronuclei assay) when total THM was assessed by available dose was 1.0002 (0.997 to 1.003), by intake dose estimated by fluid intake diary was 1.0001 (0.998 to 1.002), by intake dose estimated by questionnaire was 1.001 (0.999 to 1.003), and by internal dose was 1.05 (0.89 to 1.24). Using DNA index from flow cytometry as the outcome measure also did not identify significant associations, except when exposure was assessed as available dose of total THM (RR=1.0042; 1.0003 to 1.0081). ¶ The results suggest that THM levels are not significantly associated with DNA damage to bladder cell. This supports suggestions of THMs being non-genotoxic. Further work is required to assess the relationship between THM and the more mutagenic compounds, and to assess the carcinogenicity of the more mutagenic compounds at concentrations occurring in drinking water.

Abstract: Introduction: Partial bladder outlet obstruction (pBOO) is a ubiquitous problem that results in renal damage. We hypothesize that change in the bladder progresses over time. Methods: Fischer rats underwent surgical pBOO for 2, 4, 8, or 13 weeks and were compared to shams. Urodynamic measurements were taken, bladders weight and thickness recorded, and tissue analyzed with microscopy. RT-PCR was performed for inflammatory mediators and spectrometry used to quantify collagen. Results: Urodynamics demonstrated an increased capacity and deterioration into high-pressure. H+E demonstrated an initial inflammatory response, and increased mRNA levels of TGF-, CTGF, HIF-1, and PDGF. Muscle hypertrophy was evident on H+E and increased bladder mass and thickness. Massons Trichrome and mass spectrometry showed an increase in collagen. Conclusion: We believe that this represents distinct phases of bladder decompensation: inflammation, hypertrophy, and fibrosis. This could lead to improved preventative strategies, with respect to biochemical pathways and the time course of their initiation. / Experimental Surgery

The fishes of the family Sciaenidae have been known to vocalize during the reproductive season, and it is known that in most species only the male calls. Sounds are produced by vibration of the sonic muscles, which set the swim bladder into resonance. In addition, they also emit sounds by the same mechanism while being disturbed. The large yellow croaker, Larimichthys crocea (Perciformes, Sciaenidae) is one of the important commercial fish species distributed in South China Sea, East China Sea and southern Yellow Sea. In the past years, they have been overfished because of their high fishery value. Many aquatic farms started to culture economically important sciaenids because of the established artificial propagation technique. Now large yellow croakers have been successfully cultured in Fuchien Province, China. Unlike most sciaenids, the sonic muscles are only possessed by male, both male and female large yellow croakers have sonic muscles. This species provides the best opportunity to investigate the characteristics of sounds produced by different genders. The aims of this study were 1) to describe the hand-held disturbance sounds in large yellow croakers, 2) to describe the sounds produced during courtship and spawning in large yellow croakers being injected hormone (LHRH-A3), 3) to investigate the phylogenetic position of large yellow croaker in relation to other sciaenid fishes, and 4) to understand the evolutionary path of swim bladder morphology in the family Sciaenidae. The results show that 1) the pulse numbers of hand-held disturbance sounds in large yellow croaker can reach 23; 2) The reproductive sounds consisted of 1 to 7 pulses which started at about 1400 hr, and both vocal activity (no. sounds/min) and pulse numbers per call would increase with time. However, spawning occupied at the time slot when pulse numbers per call reached 7 in the unisex pond. The sounds in the ponds with only males or females can only recorded the sounds with 1 to 2 pulses; 3) The genus Collichthys was the sister taxon of large yellow croaker. Morphology of the swim bladder in Collichthys is similar to large yellow croaker; 4) Morphology of swim bladder evolved from simple to complex forms. Finally, Larimichthys, Collichthys, and other sciaenid genera distributed in the Indo-western Pacific Ocean including Miichthys, Boesemania, Bahaba, Panna, Atrobucca, Otolithes, Pterotolithus, Chrysochir, Paranebris, Protonibea, Pennahia, Nibea, Dendrophysa and Johnius form a monophyletic group with a bootstrap value of 100. Most of the members have the complex swim bladders with many appendages except Boesemania, Bahaba and Paranebris. Presence of simple form of the swim bladder in these three genera is inferred as a result of morphological reversal.

The swim bladder of Glaucosoma hebracium is specialized by having two side membranes extending dorsally from the side to the vertebrae. This characteristic supports the morphology of the Glaucosomatidae. There is a pair of reddish muscles connecting the skull and the swim bladder of the Glaucosoma hebracium. This type of muscle is likely to involve with sound production. However, vocalization has not been reported in the Glaucosomatids. As direct recordings for sound production in live specimens of this species could not easily be carried out, function of these swim bladder muscles was assessed by means of biochemical assay. Activities of Citrate synthase (CS) and L-lactate dehydrogenase (LDH), known to involve with energy supply during muscle contraction were assessed. Two-dimensional gels for the white muscle, tail region red muscle and swim bladder muscles were compared to reveal difference of protein expression. Results show that CS activity of the swim bladder muscle was higher than the white muscle. But LDH activities in these two types of muscles were not significant different. As high activities in CS reflecting aerobic nature of the tissue, the present dataset suggests that the swim bladder muscles should involve with sound production. Proteomics analyzes identified 14 proteins from the swim bladder muscle, one of them are related to energy production (ATP synthase beta chain). Two-dimensional gels show some different spots from these three type muscles. These results help us to find some different proteins in these three types of muscles of this fish. In conclusions base on these evidence in morphology, histology, enzyme activity assay and two-dimensional gels, the swim bladder muscles should involve with sound production in G. hebracium.

The thesis is composed of two topics : a low-noise instru-mentation amplifier (IA) for neural signal sensing and a low-power implantable bladder pressure monitor SOC (system-on-chip). A low-noise instrumentation amplifier for bio-medical appli-cations is proposed in the first topic. It is designed for sampling vague neural signals thanks to its high gain, high CMRR in a pre-defined bandwidth. A low-power implantable bladder pressure monitor system is presented in the next topic. The system contains several parts : a commercial pressure sensor, an IA, an analog to digital converter (ADC), a parallel to serial converter (PtoS), an RF transmitter and a sleep controller. The IA with 1-atm canceling is designed for high resolution and linearity in the pre-defined bladder pressure range. For low power and low speed applications, a successive approximation ADC (SA ADC) is employed in the system. A clear flag is added to the PtoS to enhance reliability. Our chip saves a great portion of power to extend the processing time owing to the novel sleep controller.

Thesis (Ph.D.)--Virginia Commonwealth University, 2009. / Prepared for: Dept. of Mechanical Engineering. Title from title-page of electronic thesis. Bibliography: leaves 98-106.

Thesis--M. Med. Sc., University of Hong Kong, 1982.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences