Thesis (Ph.D.) -- McMaster University, 2000. / Includes bibliographical references (leaves 215-223). Also available via World Wide Web.

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Forensic analysis of evidence consists of the comparison of physical, spectroscopic, or chemical characteristics of a questioned sample to a set of knowns. Currently, decisions as to whether or not the questioned sample can be associated or grouped with the knowns are left up to the discretion of the forensic analyst. The implications of these outcomes are presented as evidence to a jury in a court of law to determine if a defendant is guilty of committing a crime or not. Leading up to, and since, the publication of the National Academy of Sciences (NAS) report entitled “Strengthening Forensic Science in the United States: A Path Forward,” the inadequacies of allowing potentially biased forensic opinion to carry such weight in the courtroom have been unmasked. This report exposed numerous shortcomings in many areas of forensic science, but also made recommendations on how to fortify the discipline. The main suggestions directed towards disciplines that analyze trace evidence include developing error rates for commonly employed practices and evaluating method reliability and validity. This research focuses on developing a statistical method of analysis for comparing visible absorption profiles collected from highly similarly colored textile fibers via microspectrophotometry (MSP). Several chemometric techniques were applied to spectral data and utilized to help discriminate fibers beyond the point where traditional methods of microscopical examination may fail. Because a dye's chemical structure dictates the shape of the absorption profile, two fibers dyed with chemically similar dyes can be very difficult to distinguish from one another using traditional fiber examination techniques. The application of chemometrics to multivariate spectral data may help elicit latent characteristics that may aid in fiber discrimination. The three sample sets analyzed include dyed fabric swatches (three pairs of fabrics were dyed with chemically similar dye pairs), commercially available blue yarns (100% acrylic), and denims fabrics (100% cotton). Custom dyed swatches were each dyed uniformly with a single dye whereas the dye formulation for both the yarns and denims is unknown. As a point for study, spectral comparisons were performed according to the guidelines published by the Standard Working Group for Materials Analysis (SWGMAT) Fiber Subgroup based on visual analysis only. In the next set of tests, principal components analysis (PCA) was utilized to reduce the dimensionality of the large multivariate data sets and to visualize the natural groupings of samples. Comparisons were performed using the resulting PCA scores where group membership of the questioned object was evaluated against the known objects using the score value as the distance metric. Score value is calculated using the score and orthogonal distances, the respective cutoff values based on a quantile percentage, and an optimization parameter, ?. Lastly, likelihood ratios (LR) were generated from density functions modelled from similarity values assessing comparisons between sample population data. R code was written in-house to execute all method of fiber comparisons described here. The SWGMAT method performed with 62.7% accuracy, the optimal accuracy rate for the score value method was 75.9%, and the accuracy rates for swatch-yarn and denim comparisons, respectively, are 97.7% and 67.1% when the LR method was applied.

Indiana University-Purdue University Indianapolis (IUPUI) / Automotive paints have used an ultraviolet (UV) absorbing clear coat system for nearly thirty years. These clear coats have become of forensic interest when comparing paint transfers and paint samples from suspect vehicles. Clear coat samples and their ultraviolet absorbers are not typically examined or characterized using Raman spectroscopy or microspectrophotometry (MSP), however some past research has been done using MSP. Chemometric methods are also not typically used for this characterization. In this study, Raman and MSP spectra were collected from the clear coats of 245 American and Australian automobiles. Chemometric analysis was subsequently performed on the measurements. Sample preparation was simple and involved peeling the clear coat layer and placing the peel on a foil-covered microscope slide for Raman or a quartz slide with no cover slip for MSP. Agglomerative hierarchical clustering suggested three classes of spectra, and principal component analysis confirmed this. Factor loadings for the Raman data illustrated that much of the variance between spectra came from specific regions (400 – 465 cm-1, 600 – 660 cm-1, 820 – 885 cm-1, 950 – 1050 cm-1, 1740 – 1780 cm-1, and 1865 – 1900 cm-1). For MSP, the regions of highest variance were between 230 – 270 nm and 290 – 370 nm. Discriminant analysis showed that the three classes were well-differentiated with a cross-validation accuracy of 92.92% for Raman and 91.98% for MSP. Analysis of variance attributed differentiability of the classes to the regions between 400 – 430 cm-1, 615 – 640 cm-1, 825 – 880 cm-1, 1760 – 1780 cm-1, and 1860 – 1900 cm-1 for Raman spectroscopy. For MSP, these regions were between 240 – 285 nm and 300 – 370 nm. External validation results were poor due to excessively noisy spectra, with a prediction accuracy of 51.72% for Raman and 50.00% for MSP. No correlation was found between the make, model, and year of the vehicles using either method of analysis.

Developmental studies on elasmobranchs are challenging due to the difficulties in obtaining sufficient numbers of animals of different age cohorts. The brown banded bamboo shark, Chiloscyllium punctatum is a good model in this regard as it is abundant and readily available in the wild, is quick to mature, is frequently housed and bred in captivity and is a relatively small species of shark. Whilst there are important factors that must be considered when comparing the retinal development of animals raised in captivity to those caught from the wild, the use of C. punctatum represents an outstanding opportunity to study the development of the elasmobranch visual system from pre-hatching embryonic to adult life stages. In this study, the developing eye and retina of C. punctatum were studied using light and electron microscopy, electroretinography (ERG) and microspectrophotometry (MSP). To provide a source of early-stage animals, and to investigate the effects of environmental factors (such as temperature) on physical development, a captive breeding program was established at the University of Queensland. Sharks sourced from this facility were supplemented with animals bred at UnderWater World, Sea World and caught from the wild. Monitoring the fecundity, embryonic development, growth and viability of captive C. punctatum showed that females lay an average of 115.3 eggs, 38 of which were viable and 21.4% of which hatched. Embryos have an average gestation of 153 days post deposition (dpd; temp: 21 - 25º C) and embryonic growth is most rapid from 99 dpd until hatching. The eye of C. punctatum develops early in embryogenesis, with visible optic vesicles bulging at 27 dpd. Recent advances in fixation and processing techniques for transmission electron microscopy (TEM) have yielded improved levels of ultrastructural detail in a variety of tissue types. Consequently, in addition to conventional chemical fixation (CF) methods, the retina of C. punctatum was also processed using microwave chemical fixation (MCF) and high pressure freezing (HPF), and the resulting ultrastructure compared. Both MCF and HPF produced superior retinal ultrastructure compared to conventional CF, evidenced by higher resolution of ultrastructural detail and fewer artefacts. MCF provided the best, consistent ultrastrucutral results. By examining the time-course of retinal cell differentiation, it was found that ganglion and Müller cells are the first to differentiate, at approximately 81 dpd. The interneurons differentiate next, beginning with the amacrine cells (81 dpd), followed by the bipolar cells (101 dpd) and horizontal cells (124 dpd). The adult retina is duplex and rod and cone photoreceptors are differentiated and synaptic connections are formed by 124 dpd. Topographic analysis of retinal neuron sub-types reveals that C. punctatum undergoes rapid changes in ganglion cell distribution during embryogenesis. High levels of apoptosis, especially around the retinal periphery, result in relatively higher cell densities in the central retina, which progressively extend nasally and temporally to form a meridional band. C. punctatum develops a horizontal streak and shows only minor changes in topography during growth. Only basal levels of apoptosis are seen post-hatching. In the adult shark, the total ganglion cell number reaches 547,881. The mean and highest retinal ganglion cell densities reach a peak around hatching (3,228 cells mm-2 and 4,983 cells mm-2, respectively). Using measurements of lens focal length and ganglion cell density, the calculated maximum spatial resolving power (assuming a hexagonal mosaic) increases from 1.47 cycles degree-1 during embryogenesis to 4.29 cycles degree-1 in adults. The addition of a high ganglion cell density area within the visual streak and an increasing spatial resolving power over post-hatching development suggest an increased prey targeting and capture ability for this species. Using ERG, it is shown that C. punctatum becomes responsive to light at 127 dpd and light sensitivity peaks around the time of hatching, with a slight decrease post-hatching. C. punctatum maintains a flicker fusion frequency (FFF; an indicator for temporal resolution) at 7 - 22 Hz through juvenile stages), which is relatively low compared to other marine predators. ERG results suggest that this species is adapted to low light vision with low temporal resolution. The early differentiation, development and functionality of the visual system in C. punctatum allows for a period of synaptic maturation and potentially the ability of embryonic predator avoidance. The retina of C. punctatum contains a rod visual pigment with a wavelength of maximum absorbance (λmax) at 500 nm and cone visual pigment with a λmax at 532 nm; the max values of these pigments do not change during development. Rod and cone outer segments differentiate at 113 days post deposition (dpd), lengthen during embryogenesis and accumulate pigment throughout life. Although the photoreceptors develop and differentiate well in advance of hatching, there is considerable variation in outer segment length and pigment density during embryogenesis, which suggests that these cells are developing up until hatching. C. punctatum does not appear to have the potential for colour vision based on the lack of two cone photoreceptor types each containing a visual pigment maximally sensitive to different parts of the visual spectrum, but appears specialised for dim-light contrast vision.

Syngnathidae (seahorse, pipefish, pipehorses & seadragons) are highly visual feeders with different species feeding on specific types of prey, a behaviour that has been related to snout length. Worldwide, many species have become threatened by habitat destruction, collection for the aquarium trade and exploitation for traditional medicine, as well as recreational and commercial bycatch. Attempts to establish aquaculture programs have been of limited success. Little is known about their visual capabilities in detail. The visual systems of fishes are known to have evolved specific adaptations that can be related to the colour of water in which they live and specific visual tasks such as predator detection and acquisition of food. This study examined the ocular and retinal morphology, photoreceptor structure and spectral sensitivity of adult individuals of a local pipefish (S. argus), local seahorse (Hippocampus subelongatus) which both inhabit green water seagrass beds, and a tropical species of seahorse (Hippocampus barbouri) from blue water coral reefs. Some juveniles were also investigated. Accordingly, we developed an understanding of the features that are common to all syngnathids and those that have evolved for specific environments. Cryosections of the eyes were taken to determine morphological distinctions of this group. Lens characteristics measured using a spectrophotometer determined 50% cut-off wavelengths below 408nm for all 3 species, hence no transmission of UV light to the retina. Histological examination determined a cone dominated fovea in the ventro-temporal retina and very large rods concentrated in the peripheral retina and adjacent to the optic nerve. Microspectrophotometry measured the absorption characteristics of the visual pigments within the photoreceptors showing the presence and maximum sensitivity (λmax) of rods, SWS single cones, and a broad, complex array of LWS double/twin cones. The results are discussed in relation to the light environment inhabited by each species and their feeding requirements. The implications for the design of suitable light environments for aquarium and aquaculture programs for the Syngnathidae are also discussed. Rearing success of this family of fish, for both the aquarium trade and re-stocking programs, would be advised to take lighting regimes and specifics of the animals’ vision into account

Ray-finned fish from a diversity of distantly related lineages have remarkably large visual opsin repertoires. Starry flounder (Platichthys stellatus) development, morphology, life history, and behavior make this species especially suitable for experiments designed to determine why fish have so many opsins. Human and bird colour vision uses three and five opsins, respectively. Fish often have many more opsins. We sequenced an eye transcriptome to determine the starry flounder opsin repertoire, and used high performance liquid chromatography to determine the chromophore content of the retina. We found eight visual opsins that utilize only 11-cis-retinal (vitamin A1). This species’ entire visual opsin toolkit appears to be functional. The number of distinct cone and rod cell absorbance profiles determined using microspectrophotomery are consistent with the number of visual opsins in the transcriptome. RH2 transcripts were more abundant and SWS1 and SWS2 transcripts were less abundant in the dorsal retina, where cone density was highest, outer segments the longest, and where we observed double cones with outer segments that differed in their wavelength of maximum absorbance. Regions of fish retinas appear to be specialized and I predict that this fine-tuning is enhanced by photoreceptor plasticity and opsin gene duplication and divergence. Studies that compare opsin expression patterns among individuals, populations, or species typically assume that the differences observed influence vision. Direct connections between opsin expression and quantitative behaviours are rare. This thesis aimed to test whether varying opsin expression affects vision by modifying opsin expression and characterizing vision in starry flounder. We held starry flounder in aquaria exposed to either broad spectrum sunlight or green-filtered light. We tested vision by quantifying the visually-mediated camouflage response and we measured opsin expression using digital-PCR. Granularity analysis of photographs of the camouflage response revealed higher overall pattern energy at each of the seven spatial frequency bands in fish exposed to broad spectrum sunlight compared to the green-filtered fish. However, no statistical difference in typical measurements of pattern or contrast (e.g., maximum filter size, the standard deviation of pattern energy, and the proportional power) was observed between the two groups. Opsin expression was different between fish held in the green light environment compared to those exposed to broad spectrum light. SWS1 (UV sensitive) and SWS2B (blue sensitive) were significantly down regulated in response to the green light environment. Surprisingly, this difference was lost after only three hours under a white LED light, suggesting rapid changes in opsin expression in response to the light environment. We found tantalizing, albeit not statistically significant evidence that fish with higher expression of UV- and blue-wavelength sensitive opsins could see more contrast in colour on blue-green checkerboards. / Graduate

Patterns of DNA synthesis and mitosis in the coenobial alga Pediastrum boryanum (Turp.) Meneghini were analyzed by cytophotometric measurements of individual, Feulgen‐stained nuclei from swarming zoospores aggregating into colonies, and cells in colonies varying in age from 12 to 96 h after their initial transfer to fresh culture medium. A haploid genome size of 0.2 pg DNA (corresponding to roughly 11 × 1012 daltons, or 1.64 × 105 kb) was estimated by comparative measurements of nuclei from zoospores or young colonies and chicken erythrocyte (RBC) nuclei which were included with each set of Pediastrum slides as an internal reference standard of 2.5 pg DNA/cell. Although nuclear morphology and extent of chromatin condensation vary with different stages of colony development, nuclear division in P. boryanum appears to follow each cycle of DNA replication with no accumulation of DNA beyond the 2C level. Cytoplasmic cleavage resulting in the formation of individual zoospores is delayed until completion of mitosis, as is the demise of the pyrenoid. After 96 h of culture, 40% of all colonies have cells that are 8‐ or 16‐nucleate and some colonies have 32 nuclei/cell. Release of zoospores within vesicles occurs at this time to complete a cycle of asexual reproduction.