Integral membrane proteins often possess lipophilic a-helical regions of approximately 21 amino acid residues that are able to traverse the bilayer. These may either be amphiphilic or predominantly hydrophobic (such as those found in the photoreaction centres). Such helices are distinguished by low charge densities and large mean hydrophobicities. Several hydrophobicity measures (scales) and algorithms for identification of these segments have been developed. Here, a survey of currently available hydrophobicity scales and prediction techniques has been conducted and a brief description of each of the techniques is reported. The hydrophobic moment methodology, even after twenty years of existence, appears to be the most widely used technique for amphiphilic helical structure prediction. The reliability of this methodology at predicting structure and function relationship in membrane proteins was tested using proteins and peptides from different but known classes. In predicting structure, the hydrophobic moment appears to make predictions consistent with those from other researchers using other prediction techniques based on other alternative properties. However, the predictions for function were not consistent with the role of proteins in some cases. Scatterplots of mean hydrophobicity and mean hydrophobic moment for different window sizes revealed that there is a negative association between the two variates and, that mean hydrophobic moment decreases as window size increases. It was also deduced from 99% bootstrap confidence intervals for the correlation coefficients, that short window sizes (7-15) are more discriminative than tong ones (16-20). Angular frequencies between 95° and 107 0 were also investigated for all window sizes and it was observed that different window lengths have different optimum angular frequencies. Variation in both window size and angular frequency were seen to affect prediction with window sizes 7-15 residues giving better prediction. A data set of 403 transmembrane segments was assembled. The compositionat and distributional properties of constituent amino acids were investigated. It was observed that transmembrane segments tend to posses high occurrence of charged amino acid residues at the interface, large planar molecules just within the membrane interior and smalt hydrophobic residues in the central region. When the amino acid residue compositions of the boundaries of transmembrane a-helices were compared statisticatly, it was observed that at the 5% significant level there is no difference between the boundaries within, or between classes of transmembrane spans. It was also observed that the assumed length of twenty-one residues is, on average, reasonable for uncleaved sequences but that twenty-two may be appropriate for stop transfer sequences. When the presence of a hydrophobicity gradient in transmembrane a-hetices was examined, 25% of the sequences in the data set showed the distribution of hydrophobicity which is observed in typical transmembrane spans (tow-high-low). However, 13% showed the distribution of hydrophobicity which is similar to that seen in tilted peptides. It would appear that some transmembrane a-helices do have a hydrophobicity gradient, but this has not been related to any biological role. A new measure of amphiphilicity, which takes into account the third dimension of a helix, was also developed and compared to the conventional hydrophobic moment using bootstrap and regression modelling with a categoricat predictor. From the 95% bootstrap confidence intervals for estimates of model parameters, it was concluded though that there is no difference between the two measures, implying that the loss of spatial information in the conventional hydrophobic moment model, is not significant.

1. Investigations have been carried out in two parts (a) in vitro studies into the roles of Mg2 in ACh and CCK3-evoked Ca 2 mobilisation assàciated with the secretory process and (b) in vitro and in vivo studies into the effects and mechanisms of action of phorbol esters (and related compounds) on secretagogueevoked 3H-protein release (ACh & CCK8) and pancreatic juice secretion (CCK8 & Secretin) in the rat pancreas. 2. Modifications in extracellular Mg 2 affected ACh-evoked 45Ca2 influx, efflux and intracellular free C a2+ mobilisation. Elevated extracellular Mg2+ (10 mM) depressed whereas, zero extracellular Mg2+ (0 mM) augmented these parameters in comparison to normal extracellular Mg2+ (1.13 mM). ACh also elicited simultaneous transmembrane movement of Ca2+ (influx) and Mg2+ (efflux). Parallel secretory effects under the same conditions were observed when monitoring ct-aniylase and total protein outputs. Similar results were obtained for CCK8. Mg2+ facilitates control over the secretory process which occurs primarily through modifications in Ca 2 mobilisation. 3. The phorbol ester TPA (12-0-tetradecanoyl phorbol-l3-acetate) alone induced 3 H-protein release from permeabilised pancreatic acini but when combined with ACh a synergistic secretory response resulted at an intracellular free [Ca 2+]i = 10 4M. Lowering intracellular free [Ca 2+]i to 10 7M removed the potentiatory action of TPA but did not abolish AC!, induced secretion. Whereas, at [Ca2t] of 104M combining TPA with CCK8 resulted in complete depression of the CCK8-induced 3H-protein release. The action of TPA on CCK8-evoked 311-protein release was mediated through protein kinase C activation as Polymyxin B, an inhibitor of protein kinase C, reversed the effects of TPA. However, the potentiatory action of TPA on ACh-elicited 3 H-protein secretion was not mediated through protein kinase C activation or increases in cellular cyclic GMP. Hence, another cellular mechanism or mediator independent of protein kinase C activation possibly exists to account for both ACh-evoked secretion and the potentiatory action of TPA. 4. ACh and CCK8-evoked dose-dependent increases in intracellular free [Ca 2 ]. The dose-dependency of the Ca2+ transients elicited by these secretagogues showed a degree of correlation with their known secretory activity. Incubation of the pancreatic acini with TPA inhibited CCK8-induced but NOT ACh-induced intracellular Ca2+ transients. Therefore, protein kinase C may facilitate to some extent its secretory modulatory activity through changes in intracellular C a2+ mobilisation. 5. In vivo administration of TPA to the anaesthetised rat resulted in increases in pancreatic juice secretion. Secretin administration caused marked increases in pancreatic juice flow but not of protein or u-amylase. Whereas, CCK8 administration induced marked increases in juice flow, protein and ct-amylase secretion. TPA combined with secretin or CCK8 differentially suppressed the secretory activity of these secretagogues. Inhibition of protein kinase C reversed to some extent the inhibitory activity of TPA. Hence, it is believed that protein kinase C can physiologically initiate and modulate the quantity and quality of secretagogue-evoked pancreatic juice secretion irrespective of the intracellular pathway utilised in the pancreas of the rat. 6. In conclusion, the present study has demonstrated that Mg2+ plays an important role in stimulus-secretion coupling and therefore requires greater consideration when evaluating secretory events. Protein kinase C has been shown to possess an essential modulatory role in the whole secretory process. Furthermore, the possibility exists that an as yet uncharacterised intracellular pathway may be responsible for the protein kinase independent actions of TPA.

This study investigates the effects of the neuropeptides, vasoactive intestinal polypeptide (VIP) and galanin, the islet hormones insulin, glucagon and somatostatin, the neurotransmitter acetyicholine (ACh), the gut hormone cholecystokinin octapeptide (CCK-8), sodium nitroprusside (SNIP), 8-Bromo guanosine 3'5' cyclic monophosphate (8-Br cOMP) and nerve stimulation on amylase secretion and intracellular free calcium concentration ([Ca 2 ]) in the isolated pancreas of normal and diabetic rats. The pattern of distribution of the islet hormones and neuropeptides in normal and diabetic rats. The results show that stimulation of isolated pancreatic segments and acinar cells with different concentrations of either glucagon, somatostatin, VIP, galanin, ACh or CCK-8 resulted in different effects on amylase secretion and [Ca 2']i Combining the islet hormones with either ACh or CCK-8 resulted in marked potentiation in amylase output and enhanced the [C2] in acinar cells. Genistein, had markedly inhibited the potentiation of the islet hormones with either ACh or CCK-8. Similarly, when rats were rendered diabetic either insulin, glucagon or somatostatin failed to potentiate the secretory response of ACh or CCK-8. The results indicate that both tyrosine kinase and cellular C2 seem to be the intracellular mediators involved with the enhanced secretory responses obtained with a combination of the islet hormones with either ACh or CCK-8. Immunohistochemiocal techniques confirm the presence of the islet hormones and neuropeptides in the endocrine and the neural elements of the pancreas. Moreover, the presence of viable pancreatic islets of Langerhans seems to be associated with the potentiation of the islet hormones with either ACh or CCK-8. The results also suggest that NO may modulate the release of the endogenous neurotransmitter(s) in response to EFS in the exocrine rat pancreas.

The widespread occurrence of thermophilous microfungi on wood is reported. Imported timber, in-service timber joinery and wood employed in colonization experiments were sampled and a list of cellulolytic fungi including thermophilous and soft rot fungi is submitted. The influence of insolation on the temperature-and moisture ranges in wood was investigated. Temperatures up to 160C above ambient were recorded as a result of insolation and data is submitted on the diurnal temperature cycle in a block of wood undergoing insolation. It is recorded that colour had no significant effect upon the moisture content of insolated wood in soil contact. Using a simple moisture gradient apparatus the surface growth and penetration of thermophilous fungi were investigated at above ambient temperatures, showing that thermophilous fungi could grow at the surface and within beech veneers at moisture levels below the 20% minimum recorded in the literature, it is considered that the biological activity of water is increased at above ambient temperatures. The effect of constant, alternating and fluctuating temperatures on the growth of microfungi was investigated. A temperature cycle simulator was devised producing a diurnal cycle similar to that occurring in insolated wood. Addition, stimulation and retardation of growth were recorded as a result of temperature alternation, whilst evidence is submitted that some thermophilous wood inhabiting-microfungi were better adapted to temperature fluctuations than mesophilic forms. Interaction studies between pairs of wood inhabiting microfungi were undertaken at above ambient temperature and under conditions of fluctuating temperature. Evidence that metabolic products influence interactions is offered, and that under âanditions of fluctuating temperature thermophilous fungi can play a significant role in ecological sequences. - Preliminary experiments into the tolerance of cellulolytic thermophilous microfungi to wood preservatives indicate that they, were more tolerant to preservatives at above ambient temperature than at 250C.

Distinguishing between burnt human and non-human bone fragments using macroscopic methods has proved challenging and it was seen in the literature review that the previous research did not all come to the same conclusions. The aim of the research was to determine if, using histological methods, it was possible to distinguish between burned human and nonhuman bone fragments. A literature review was compiled to provide an overview of the anatomy of bones, morphological characteristics of bone, general bone histology, a comparison between human and non-human bones and the effect of temperature on bones. Bones of five different species (human, baboon, wildebeest, pig and cow) were burned in a muffle furnace for twenty minutes at either 600ᵒC or 800ᵒC. Following the burning procedure, thin ground bone sections of the burned and unburned bone specimens were prepared for microscopic analysis and the minimum canal diameter, maximum canal diameter, minimum Haversian system diameter, maximum Haversian system diameter, area of canal, and area of the Haversian system were measured. A comparative analysis was then done across species and temperature. A total of 523 osteons in unburned bone and 147 in the burned bone samples were analysed. ANOVA testing found overall significance (p < 0.0001) for all parameters measured, which suggests that temperature does affect the size of microstructures. Most parameter sizes increased with an increase in temperature. A greater increase was seen at 600 degrees than 800 degrees. Qualitatively, carbonation within the burned bone, made the measurement of parameters difficult in some samples. Human bone can easily be differentiated from pig, cow or wildebeest bone due to no or very few osteons present. Pig bone consisted almost entirely of plexiform bone, while the cow and wildebeest presented with only a few osteons in some parts of the bone. Human and baboon bone appeared similar on a microscopic level. The study revealed that temperature did not, in general, hamper the ability to differentiate between burned human and non-human bone, but it did impact on the number of measurable data points for each parameter.

In South Africa, little is known about the presence of drugs in infant cases admitted for post-mortem medico-legal examinations,as toxicological investigations are not routinely performed. It was hypothesised that drugs would be detected in sudden and unexpected death ofinfant (SUDI) cases admitted to Salt River Mortuary (SRM), as infantsform a vulnerable population. Biological samples (blood, vitreous humour, urine and hair) were collected from 30 infants who were admitted as SUDIcasesto Salt River Mortuary,Cape Town,between 28 May 2019 and 17 October 2019. Samples were screened for at least 750 common drugs using a SCIEX X500R QTOF. Demographic variables, social circumstances and clinical historywere recorded from the medico-legal case folder. Of the 30 SUDI cases, drugs were detected in 25 (83 %) cases, with acetaminophen (61 %) and caffeine (54 %) being most prevalent. Methaqualone (32 %) and methamphetamine (11 %),two commonly abused drugs in the Western Cape,werealsoidentified, with the former only present in hair. There were significantly more drugs detected in hair samples compared to the other samples (p < 0.0001). Therefore, while challenging in its interpretation, hair analyses provideda wealth of information concerningpossiblelonger-termdrug exposure in infants.This was particularly valuable inrevealingmethaqualoneexposure, which may have otherwise gone undetected, and which may indicate an environment of neglect. While the cause of death in most cases was natural (infectious causes) (63 %), next-of-kin seldom declared that their infant exhibited symptoms of illness orthat medication was administeredprior to death. Therefore, theresults of this study illustrate the valueof toxicological testing in SUDIcases at SRM, as well as the need to analyse multiple samples. This study provides empirical data to motivate for the SUDI investigation protocolatSRM toinclude routine toxicological analysis. This is anticipated toadd value to the medico-legal investigation as well as add social value to the lives of siblings who may also be at risk for neglect.

The large number of unidentified, decomposed and skeletonised remains found in South Africa (SA) necessitates relevant and reliable methods to assist in victim identification. Ancestry estimation from unknown skeletal remains is essential when reconstructing a demographic profile of a missing person. In the SA population, estimating ancestry is problematic as standards developed internationally rarely apply to the local, biologically heterogenous population. Craniofacial morphology is known to be ancestrally distinct and studies are yet to explore shape and size variation in the zygomatic bone of the SA population. The aim of this study was to assess ancestral variation in zygomatic shape and size in a SA population using three-dimensional geometric morphometric analyses. A sample of 158 individuals were analysed from Bantu-speaking (BA), European (EA) and Mixed Ancestral (MA) South African groups. Males were larger in size than females, but no size differences were observed between ancestral groups. Significant shape differences were observed between ancestral groups, while none were observed between males and females. BA and MA individuals had narrower, shorter and more anteriorly projecting zygomas than EA individuals. The zygoma was shown to accurately distinguish EA (84%) from BA (81%), and MA (80%) from EA (68%) individuals, but unreliably distinguished BA (60%) from MA (66%) individuals. This is likely correlated to the historical peopling of SA and historical forced racial classification. Age-related changes and antemortem tooth loss did not confound the ancestral variation in size, despite minor changes in zygomatic shape being associated with these two factors. These confounders did not impact ancestry estimation accuracies, further suggesting a minor impact on overall zygomatic shape. Furthermore, the patterning of ancestral variation in the zygoma revealed the need for further research to distinguish between the biologically heterogenous ancestral groups in SA.

A retrospective study of deaths attributable to anaphylaxis at the Salt River Forensic Pathology Laboratory was undertaken, with a view to determine if eosinophilia was present in tissue samples of the spleen, in accordance with previously published research. Suitable cases of non-anaphylactic death were used as controls. Use was made of two commonly available fabric dyes as alternative stains to the traditional Haematoxylin -Eosin ["H&E"].

The aim of this project is to develop a single quantification method for certain benzodiazepines, opiates and antidepressants in whole blood, serum and urine by LC-MS/M5 and to consequently validate the analytical method for official use in the Division of Pharmacology at the University of Cape Town.

Animal sexual abuse (ASA) involves the sexual molestation of animals by humans. The identification of semen provides a legally-accepted indicator that sexual activity occurred, while forensic DNA analysis provides a lead to a potential suspect. After conducting a systematic literature review, no previous research investigating semen and/or DNA recovery from animals over time was found. Therefore, this pilot study aimed to assess the recovery of human semen and DNA from animal fur over a two-week period to establish baseline data pertaining to evidence retention in the ASA context. This pioneer study also attempted to contribute towards the development of a suitable animal fur model on which to perform experiments. Daily swabbing and testing of semen from three fur models (unpreserved baboon fur, preserved nyala hides and faux fur) showed that semen could still be detected at 14 days using standard presumptive and confirmatory tests. Although DNA degradation showed a statistically significant increase over time, forensically usable DNA profiles (≥ 12 fully typed short tandem repeat loci) were consistently obtained. There was significantly higher DNA degradation in samples from the baboon fur compared to the others, while DNA concentrations were significantly different between each fur model. These differences highlight that future research must consider the choice of fur model to best represent the animal of interest; e.g. dissected fur from a recently deceased animal would best mimic a fatal ASA case. The insight regarding the choice of animal model hopes to be of benefit for future research, which should focus on the influence of more realistic variables (e.g. movement and body heat) on semen and DNA retention on animal fur. Overall, this study successfully generated baseline data, and provides a foundation for additional research, which hopes to eventually assist in the interpretation of forensic evidence in the global burden of ASA.