Doctor of Philosophy / This work was digitised and made available on open access by the University of Sydney, Faculty of Dentistry and Sydney eScholarship . It may only be used for the purposes of research and study. Where possible, the Faculty will try to notify the author of this work. If you have any inquiries or issues regarding this work being made available please contact the Sydney eScholarship Repository Coordinator - ses@library.usyd.edu.au

Thesis (M.S.)--University of Michigan, Ann Arbor, 1978. / Typescript (photocopy). eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 46-48).

Indiana University-Purdue University Indianapolis (IUPUI) / A study was made to determine if heterogenous dentin, devoid of its antigenic potential, would stimulate reparative dentino-genesis in the dental pulp. The teeth of two monkeys were capped with bovine dentin mixed with methyl cellulose and histologic analysis was made at 21 and 42 days post-operatively. At the 21-day interval, seven of the 17 teeth capped with the experimental material were successfully repaired with atubular dentin. The remaining 10 teeth showed varying degrees of inflammation and repair. The teeth of the second animal (42 days) were stained for bacteria as an additional diagnostic tool. A direct correlation was found between delayed healing and inflammation and presence of bacteria in the pulp. No bacteria were found in pulps which were successfully repaired. It was concluded that anorganic bovine dentin seemed to induce calcific repair of the dental pulp in the absence of bacteria. Autogenous dentin chips appeared to have the same effect. The importance of including a bacteriologic stain in the histologic analysis of pulp capping studies was demonstrated.

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C-MYC is a transcription factor and a potent driver of many human cancers. In addition to regulating transcription, C-MYC promotes formation of the mRNA cap which is important for transcript maturation and translation. However, the mechanistic details of C-MYC-dependent mRNA capping are not fully understood. Since anti-cancer strategies to directly target the C-MYC protein have had limited success, enzymatic co-factors or effectors of C-MYC present attractive alternatives for therapeutic intervention of C-MYC-driven cancers. mRNA capping enzyme (CE) initiates mRNA cap formation by catalysing the linkage of inverted guanosine via a triphosphate bridge to the first transcribed nucleotide. The involvement of CE in C-MYC-dependent mRNA capping and C-MYC function has not yet been explored. Therefore, I sought to determine whether C-MYC regulates CE, and whether CE is required for C-MYC function. I found that C-MYC promotes CE recruitment to RNA polymerase II (RNA pol II) transcription complexes and to regions proximal to transcription start sites on chromatin. Consistently, C-MYC increases RNA pol II-associated CE activity. Interestingly, cells driven by C-MYC are highly dependent on CE for C-MYC-induced target gene expression and cell transformation, but only when C-MYC is overexpressed; C-MYC-independent cells or cells retaining normal control of C-MYC expression are insensitive to CE inhibition. C-MYC expression is also dependent on CE. Taken together, I present a bidirectional regulatory relationship between C-MYC and CE which is potentially therapeutically relevant. Studies here strongly suggest that inhibiting CE is an attractive strategy to selectively target cancer cells which have acquired deregulated C-MYC.

The production of methyl mercury, an acute neurotoxin which readily accumulates in the tissue of organisms, is a biologically mediated process facilitated by sulfate reducing bacteria in aquatic sediments. In-situ capping is a frequently considered risk management strategy for contaminated sediments. Since placement of an in-situ cap will induce anaerobic conditions that are known to be favorable for the growth of sulfate reducing bacteria, there is justifiable concern that capping could increase mercury methylation in underlying sediments. This research builds an understanding of the effects of in-situ capping on underlying biogeochemical processes and elucidates their importance in controlling methyl mercury production. Laboratory experiments and mathematical models were implemented to simulate mercury methylation in redox conditions likely to be induced by capping using sediment from different environments. Mathematical descriptions of processes known to be involved in methylation were incorporated into the model to quantify the effects of these processes. Observations in both well-mixed slurry conditions and intact sediment columns showed that methyl mercury concentrations are strongly dependent upon biogeochemical conditions. Results from experiments with sediment spanning a range of redox conditions and organic contents suggested that sulfate reduction rates, aqueous speciation, and solid phase partitioning are involved in limiting methylation depending on bulk geochemical characteristics. A model with a mechanistic basis that incorporates the effects of these processes provides a useful means of qualitatively and quantitatively considering their cumulative impact in limiting methyl mercury production. High methyl mercury concentrations observed in some lab experiments suggest that there is reason to be concerned about anoxic conditions induced by capping; however, not all anoxic conditions led to equivalent increases in methyl mercury. Experimental and modeling results suggest that in a high organic environment, in-situ capping may produce conditions which accelerate methylation in (formerly) surficial sediment while in a low organic environment, with an overall lower potential for methylation, capping can be expected to have a less dramatic effect. Over time, two processes will temper capinduced increases in methyl mercury. Increases will only last until sulfide builds up to inhibitory levels in underlying sediment or until organic carbon is depleted and overall bacterial activity slows. By providing a more fundamental understanding of the effects of capping on mercury methylation, the results of this research will aid in identifying situations and conditions in which cap-induced increases in methyl mercury have the potential to limit the effectiveness of the management strategy. / text

Student Number : 9306227A - PhD thesis - School of Molecular and Cell Biology - Faculty of Science / It is well documented that N-capping motifs are stabilising local motifs for -helices. N-capping motifs have been identified within hGST A1-1 at the N-terminal ends of -helix 9 and helix 6. The conservational role of these two motifs in protein stability, folding and function was investigated. -Helix 9 is a unique structural feature to class Alpha GSTs that is important for its catalytic functioning. This amphipathic helix is highly dynamic, where upon ligand binding at the active-site, the delocalised C-terminal region becomes immobilised to form a structured helix forming a “lid” over the active-site. The specific role of the Asp N-cap motif toward the stability and dynamics of helix 9 was determined by substituting the Asp-209 for a Gly. ANS binding and urea-induced activity studies showed that by removing the N-cap motif of helix 9 in hGST A1-1, the helix 9 is destabilised rendering a less hydrophobic binding site compared to that in the wild-type. The helical content of the peptide, corresponding to helix 9 in the C-terminal region of hGST A1-1 (208 -222), decreased significantly upon the removal of the N-cap motif. The explanation for the conservation of the Asp N-cap residue can be found in its stabilising role of the C-terminal region of class Alpha GSTs. This stabilising role was however less apparent in context of the protein compared to that in the peptide. Majority of the atomic contacts owing to the stability of helix 9 appear to be governed by non-local tertiary interactions rather than local interactions, such as the N-cap motif. These tertiary interactions are likely to include short and long range contacts between residues on the surface of the protein that are already known to contribute towards the stability of the C-terminal region. In this study, the ligand displacement-studies and the molecular docking results strongly suggest that 8-aniline-1-napthalene sulfonate binds at the H-site in hGST A1-1. The N-capping motif of helix 6 identified in class Alpha GSTs is located within the core of domain 2. This motif is a common feature found amongst almost all GST-like proteins and is thought to be the folding nucleation site (Stenberg et al. J. Biol. Chem. 275 (2000), 10421-10428). The N-cap (Ser- 154) and N3 (Asp-157) residues were each substituted with an Ala in hGST A1-1 to investigate the role of this motif in the folding of hGST A1-1. Both substitutions resulted in thermal sensitive mutants compared to that of the wild-type. The N3 substitution (D157A) was however too disruptive, where the yields of this mutant were insufficient for any further studies to be carried out. For the N-cap mutant (S154A), the unfolding kinetic studies revealed a significantly destabilised core in domain 2 compared to that of the wild-type. The kinetic folding studies monitored by fluorescence spectroscopy, revealed that the N-cap motif contributes to the efficient folding and dimerisation of the subunits, and to a far lesser extent towards the final tight packing and reorganisation of tertiary interactions in hGST A1-1. Since no changes in the burst-phase of S154A was evident compared to that of the wild-type, it seems unlikely that this motif is a folding nucleation site in hGST A1-1. These results do not exclude the possibility that this motif contributes to the rapid formation secondary structure during the burst-phase of folding. Due to the highly conserved region surrounding helix 6 , the role of this motif contributing to the stability of hGST A1-1 could be a general feature for GSTs and GSTlike proteins. In this study, further insight into the mechanism of folding for hGST A1-1 was gained. The hydrophobic core packing surrounding helix 6 occurred as a late folding event, that is during the final packing and reorganisation of tertiary interactions of the protein. The N-cap motif is an important structural feature for the fast folding of domain 2. This N-cap motif is a unique structural feature important for the efficient folding of the monomers, which is exclusive to its role in stabilising helix 6 in hGST A1-1.

O desenvolvimento de biomateriais com aplicações na área da saúde mostram-se cada vez mais importantes e a procura por novos polímeros com propriedades bioativas, biodegradabilidade, atoxicidade são o foco das principais pesquisas em diferentes aplicações médicas e odontológicas. Os materiais capeadores pulpares evoluíram rapidamente na ultima década, sendo que são disponibilizadas atualmente diversas alternativas para uso clínico odontológico. Este trabalho teve como objetivo o desenvolvimento de um novo produto bioestimulador e capeador dentino/pulpar que poderá ser base para o desenvolvimento e recobrimento de scaffolds para reparo das diferentes estruturas dentárias. O desenvolvimento das bandagens BBio e os resultados obtidos nos testes das propriedades físico-químicas (absorção de água, perda de massa e pH), bem como as análises biológicas da morfologia celular e viabilidade celular com MTT a BBio apresentaram dados favoráveis e desejáveis para sua aplicação clínica. A propriedade de liberação de cálcio foi bastante promissora, sendo esta uma condição que dará a diferenciação positiva da BBio como um produto bioestimulador pulpar. Com esses dados pode-se concluir que a mesma se encontra dentro dos parâmetros desejados para o produto final e com propriedades semelhantes aos produtos existentes no mercado, de qualidade e aprovados pelas agências reguladoras. / The development of biomaterials with applications in the health area are increasingly important and the search for new polymers with bioactive properties, biodegradability and toxicity are the focus of the main researches in different medical and dental applications. The pulp capping materials evolved rapidly in the last decade, and several alternatives are now available for clinical dental use. This project aimed to develop a new biostimulating and dentin / pulp capping product that could be the basis for the development and recoating of \"scaffolds\" for repair of different dental structures. The development of the BBio bandages and the results obtained in the physical-chemical properties tests (water absorption, loss of mass and pH), as well as the biological analyzes of the cellular morphology and cell viability with MTT to BBio presented favorable and desirable data for its clinical application. The calcium release property was quite promising, and this is a condition that will give BBio a positive differentiation as a pulp biostimulator product. With this data it can be concluded that it is within the parameters desired for the final product and with properties similar to the products on the market, of quality and approved by the regulatory agencies.

>Magister Scientiae - MSc / During the course of this study iron oxide nanoparticles, which have been researched for drug-targeted delivery, were synthesised via the co-precipitation method and characterised using various methods. This study focused on the role of relevant capping agents for the inhibition of agglomeration of the particles; chitosan, polyvinyl alcohol (PVA) and poly lactic glycolic acid (PLGA) were the capping agents of interest. The study is an assessment of the effects brought about the different capping agents used for this work. The prepared particles were then capped with the different capping agents followed by the loading of the drug curcumin. Various analytical methods were used to analyse the particles such as High resolution transmission electron microscopy (HR-TEM), Superconducting quantum interference device (SQUID), Fourier Transform Infrared spectroscopy (FT-IR), X-ray diffraction (XRD), Thermogravimetric analysis (TGA) and zeta potential. PVA, chitosan and PLGA capped SPIONS were successfully prepared and verified by FT-IR spectrometry, various sizes were prepared almost ranging the same for the successfully prepared particles verified by XRD. The resultant particles were found to be spherical with an average particles size between 13- 22 nm. From the study it was concluded that the addition of the different capping agents resulted in the reduction of the intensity of the peaks in XRD, it was also found out the presence of the capping agents did not alter the crystalline phase of the particles. From the study it was also observed that higher saturation magnetization was experienced where PVA was used as the capping agents.

Capping is typically used to control contaminant release from the underlying sediments. However, the presence of conventional caps often eliminates or slows natural degradation that might otherwise occur at the surface sediment. This is primarily due to the development of reducing conditions within the sediment that discourage hydrocarbon degradation. The objective of this study was to develop a novel active capping method, an electrode enhanced cap, to manipulate the redox potential to produce conditions more favorable for hydrocarbon degradation and evaluate the approach for the remediation of PAH contaminated sediment. A preliminary study of electrode enhanced biodegradation of PAH in sediment slurries showed that naphthalene and phenanthrene concentration decreased significantly within 4 days, and PAH degrading genes increased by almost 2 orders of magnitude. In a sediment microcosm more representative of expected field conditions, graphite cloth was used to form an anode at the sediment-cap interface and a similar cathode was placed a few centimeters above within a thin sand layer. With the application of 2V voltage, ORP increased and pH dropped around the anode reflecting water electrolysis. Various cap amendments (buffers) were employed to moderate pH changes. Bicarbonate was found to be the most effective in laboratory experiments but a slower dissolving buffer, e.g. siderite, may be more effective under field conditions. Phenanthrene concentration was found to decrease slowly with time in the vicinity of the anode. In the sediment at 0-1 cm below the anode, phenanthrene concentrations decreased to ~70% of initial concentration with no bicarbonate, and to ~50% with bicarbonate over ~70 days, whereas those in the control remained relatively constant. PAH degrading gene increased compared with control, providing microbial evidence of PAH biodegradation. A voltage-current relationship, which incorporated separation distance and the area of the electrodes, was established to predict current. A coupled reactive transport model was developed to simulate pH profiles and model results showed that pH is neutralized at the anode with upflowing groundwater seepage. This study demonstrated that electrode enhanced capping can be used to control redox potential in a sediment cap, provide microbial electron acceptors, and stimulate PAH degradation. / text