OBJECTIVE: To determine the effects of vitamin C and insulin on osteoblasts harvested from neonatal mouse calvaria. To determine the effects of experimental media (vitamin C and insulin and a combination) on the ex-vivo live bone organ culture model and explore the capacity of addition of osteoblasts to allow for bone formation within a critical defect. To use the chick chorioallantoic membrane (CAM) model to explore bone formation within critical bone defect. METHODS: Osteoblasts were harvested from neonatal mice were tested using four types of experimental media: control DMEM, media prepared with 150 μg/ml vitamin C, 10 nM media, or a combination of both vitamin C, insulin, and a combination of vitamin C and insulin media. Cell were cultured for 18 days at 37°C. Neutral red was done to identify cellular activity and silver nitrate to detect calcium deposits. Two types of scaffolds were inserted into the defect: collagen membrane scaffold and NuOss (xenograft) with collagen scaffold. After 30 days the samples were collected for histological analysis. Neonatal mouse calvaria were harvested and a 2mm critical defect made on each calvaria. Each calvaria received a scaffold of collagen or NuOss with or without osteoblasts with one of three experimental media within the CAM model. After 7 days, the amnion membrane of the egg was dropped and a window was made. The calvaria with the scaffold samples were placed on the amnion membrane. The eggs were incubated for 10 days then the experiment was terminated. Calvaria were collected and processed for histological evaluation. RESULTS: Neutral red and silver nitrate of 2D in-vitro cells revealed calcium deposits in culture well using vitamin C media, cell cultured with insulin media showed calcium deposits and cell morphological change, and cell cultured with a combination of vitamin C and insulin media showed the most calcium deposits and morphological changed. Ex-Vivo samples with collagen scaffold had bone thickening but not enough nutrients for bone regeneration, despite the addition of cells. The collagen scaffold is a more suitable material than xenograft due to particle size. The CAM model showed new bone formation and new vessels were most abundant in areas closest to lining cells in collagen samples. Samples with additional osteoblasts added showed greater results. NuOss scaffold samples did not show the same bone formation or vessel growth. CONCLUSIONS: The results indicate and confirm the basic principles of tissue engineering. In order to have bone regeneration more cells allow for better results. The quality of the scaffold is important and should have stability as well as enough space for cellular migration and recruitment for new blood vessel to support regeneration of bone to its original state.

Cadmium (Cd) is a highly toxic metallic element to the human body such that prolonged occupational or environmental exposure produces renal, hepatic, pneumonic, and neurological disorders. Thus, as a consequence it is important to have a way of monitoring cadmium exposure as it has the potential to become an occupational health hazard. The primary uses of this element are in the mining and smelting industry in the manufacture of cadmium alloys and the manufacture of alkaline accumulators. Since the discovery of X-rays in 1895 by Wilhelm Conrad Roentgen, the science of X-ray analysis has become a cardinal tool in all domains of chemical identification and classification. X-ray fluorescence (XRF) has been shown to be an effectual technique for measuring trace quantities of heavy metals such as lead in various tissues within the body. This thesis stud:r elucidates a means of measuring Cadmium in bone. The study assesses the feasibility and practicality of the polarised XRF and source excited techniques. In the polarised cadmium concentration measurements, a gain in sensitivity due to improved background characteristics was perused by increasing the x-ray tube operating voltage of the system. It was found that an operating voltage of 175 kV, and a copper filter resulted in a significant gain in sensitivity for which a minimum detection limit (MDL) of 3.5 ± 1.4 ppm was determined with 3 mm of tissue equivalent overlay. Using the source-based technique, a MDL of 3.5 ± 0.2 ppm was estimated for the corresponding tissue equivalent overlay. / Thesis / Master of Science (MS)

No description available.

No description available.

Apesar da introdução do controle da qualidade individual nas técnicas complexas de tratamentos, tem-se comprovado que, mesmo assim, é possível a ocorrência de erros na aplicação da dose no momento da aplicação. No entanto, ainda não estão bem estabelecidas as ferramentas de redundância a fim de controlar a dose no momento da terapêutica, além do que, as técnicas mais modernas de tratamento radioterápico desenvolvem as aplicações com feixes rotacionais e os dosímetros tradicionalmente utilizados em controle da qualidade oferecem limitações angulares. Assim, este trabalho vem contribuir para o desenvolvimento de uma metodologia de controle da qualidade de transmissão in vivo utilizando filmes radiocrômicos acoplados ao cabeçote do acelerador linear, durante aplicações radioterápicas nas técnicas de tratamento conformacional e IMRT. A metodologia de controle da qualidade desenvolvida neste trabalho baseia-se na obtenção da distribuição de dose in vivo de tratamentos radioterápicos com um filme radiocrômico EBT2 posicionado em um suporte acrílico, semelhante a uma bandeja, a uma distância fonte-superfície de 56,8 cm, acoplado ao acessório holder do acelerador linear durante a aplicação de todo o tratamento teleterápico. Posteriormente, foi realizada uma análise gama para comparação da distribuição de dose medida pelo filme com a esperada pelo sistema de planejamento, obtida no plano coronal e central de um objeto simulador, com dimensões semelhantes ao suporte acrílico, posicionado à distância de 100 cm, como resultado da transferência do plano em questão. Com os resultados encontrados na seção conformacional, avaliando tanto a simulação Monte Carlo quanto as irradiações, pode-se concluir que a diferença entre a distribuição de dose do sistema de planejamento, na distância foco detector de 100 cm, e do filme, na distância de 56,8 cm, é diminuta e, desta forma, é viável criar uma metodologia para verificação dosimétrica de transmissão utilizando o filme radiocrômico acoplado ao cabeçote do acelerador. O controle da qualidade proposto na técnica de IMRT concordou com o esperado em 24 das 25 situações testadas, apresentando apenas um resultado diferente, ou seja, uma concordância de 96% com o esperado. As avaliações in vivo concordaram com 98% dos controles avaliados. Desta forma, pode-se concluir que a metodologia proposta neste trabalho é factível para o controle da qualidade de transmissão in vivo, em tratamentos radioterápicos que usam a técnica de tratamento conformacional e IMRT e, como ela não oferece dificuldades para o deslocamento angular do gantry, ela poderá ser aplicada em técnicas teleterápicas mais modernas. / Even with the introduction of the individual quality control in the complex techniques of radiation therapy treatments, the occurrence of errors in the release of the dose at the time of application is possible. However, in order to monitor the dose at the time of therapy, redundancy tools are not yet well established, Besides that, the most modern techniques of radiation treatment use rotational beams to deliver the desired dose distributions and the dosimeters traditionally used in quality control of radiation therapy suffer angular limitations. In this way, this work aims to contribute to the development of a methodology of transmission quality control in vivo presenting a dose control technique using radiochromic film coupled to the headstock linear accelerator for radiotherapy applications to monitor conformational techniques and IMRT treatment. The quality control methodology developed in this work is based on obtaining the in vivo dose distribution of radiotherapy treatments with a radiochromic film EBT2, positioned on an acrylic stand, similar to a tray at a source-surface distance of 56.8 cm, coupled to the linear accelerator accessory holder during application of any treatment. It was subsequently performed a gama analysis for comparison of the dose distribution measured by the film with the expected dose distribution by the treatment planning system. The expected dose distribution was obtained in the coronal and central plane of a phantom, with similar dimensions to the acrylic stand and positioned on a source-surface distance of 100 cm as a result of the transfer of the plan in question. Based on the results presented in the conformational section, evaluating both, Monte Carlo simulation and irradiation results, it can be concluded that the difference between the distribution of the dose planning system, focus distance 100 cm detector, and the film, on distance of 56.8 cm, are small, and in this way it is feasible to create a methodology for dosimetry verification using radiochromic film coupled to the head of the accelerator. The proposed quality control in IMRT technique agreed with expected in 24 simulations of the 25 situations tested, showing only one different result, i.e., there was a 96% concordance with the expected. In this way, it can be concluded that the methodology proposed in this work is feasible for the in vivo quality control of radiation therapy treatments that use the conformational and IMRT treatment techniques, and also can be applied to the most modern radiotherapy techniques since, it does not offer difficulties with the angular displacement of the gantry.

Continuous monitoring of physiological parameters such as pH and oxygen (O2) are of great importance in determining the health status of a patient. Arterial blood gas analysis is a current clinical method used to measure pH, PCO2, PO2, and the concentration of variety of ions, typically with blood withdrawn from an artery. The need for robust, and a rapidly responding technology to enable bed-side monitoring has driven considerable efforts to produce better sensor devices. Optical sensing systems have experienced rapid growth, with drivers including low-cost optical fibres, and the availability of miniature optical set-ups (light sources, detectors, etc.). Herein, polymer-based optical fibre sensors for pH and O2 sensing were developed. The pH and/or oxygen reporters were immobilised at the end of an optical fibre by photo-polymerisation, and their performance in measuring pH and O2 concentration investigated. pH sensing was based on fluorescence detection using single excitation/single emission (Chapter 2), and single excitation/dual emission (Chapter 3). O2 sensing was based on the luminescence quenching of metalloporphyrins by oxygen (Chapter 4). In the last chapter, the in vivo applicability of an O2 sensor was investigated by measuring O2 level changes inside an ex vivo lung.

Le centre médian/parafasciculaire (CM/Pf) du thalamus a récemment émergé comme un élément d'intérêt dans le contexte de la maladie de Parkinson. Ainsi le fonctionnement normal et pathologique des GB ne peut pas être pleinement élucidé sans qu'il ne soit pris en considération. Dans ce contexte, nous avons analysé le transfert des informations thalamiques dans les GB en enregistrant, in vivo, les réponses évoquées au niveau de la structure de sortie des GB, la substantce noire pars reticulata (SNr) soit par la stimulation électrique ou optogénétique du CM/Pf. Ensuite, nous avons étudié les composantes des GB impliquées dans ces réponses en analysant les réponses évoquées par l'activation optogenetique spécifique des voies thalamo-striée, thalamo-subthalamique ou thalamo-nigrale. À la fois l'activation électrique et optogenetique du CM/Pf évoquent des réponses complexes dans la SNr qui sont composées d'une inhibition qui peut être précédée et/ou suivie d'excitations. L'inhibition et l'excitation tardive dépendent de l'activation des voies trans-striatales, alors que les premières excitations mettent en jeu les voies thalamo-subthalamique et thalamo-nigrale. Nous avons également étudié l'impact des interneurones cholinergiques du striatum ainsi que les afférences dopaminergiques sur le transfert des informations thalamiques dans les GB. Pour ce faire, nous avons enregistré les réponses évoquées au niveau des neurones de projection du striatum suite à la stimulation électrique du CM/Pf avec ou sans l'inhibition optogénétique des CINs. Nous serons alors en mesure de déterminer comment les CINs sont impliqués dans le transfert des informations thalamiques au sein des GB. / The centre median/parafascicular (CM/Pf) of the thalamus has recently emerged as a component of interest in the context of Parkinson’s disease. Thus normal and pathological dynamics of BG cannot be fully understood unless it is taken into account. Here, we analyzed the transfer of CM/Pf information through BG by recording, in vivo, the evoked responses of BG output neurons in the substantia nigra pars reticulata (SNr) to either electrical or optogenetic CM/Pf stimulations. Then, we investigated the BG components involved in these responses by analyzing the responses evoked by specific optogenetic activation of the thalamo-striatal, thalamo-subthalamic or thalamo-nigral pathways. Both electrical and optogenetic activation of CM/Pf evoke complex responses in SNr that are composed of an inhibition that can be preceded and/or followed by excitations. The inhibition and the late excitation rely on the activation of the trans-striatal pathways, whereas the early excitations involve thalamo-subthalamic and thalamo-nigral projections. We are currently analyzing whether and how the striatal cholinergic interneurons (CINs) and the dopaminergic afferent system modulate the transfer of thalamic information within the BG. For the second part of my project, we analyzed the treatment of thalamic information from CM/Pf at the level of the striatum. To do this, we recorded the evoked responses of striatal projection neurons by the electrical stimulation of the CM/Pf with or without the inhibition of the CINs by optogenetics. We will then be able to determine how CINs are involved in the transfer of thalamic information at the level of the striatum.

Streptococcus pneumoniae is a nasopharyngeal commensal in most healthy individuals. However, it can translocate from this niche to deeper tissues, causing diseases such as otitis media, meningitis, sepsis and pneumonia, which are responsible for significant morbidity and mortality worldwide. At the commencement of this work, inherent difficulties in harvesting sufficient bacterial numbers from experimental animals restricted the examination of pneumococcal gene expression during pathogenesis, and thus virulence gene transcription patterns were largely unknown outside of an in vitro environment. This thesis aimed to investigate such transcriptional patterns in vivo, and to hence gain a better understanding of pneumococcal behaviour during colonisation and disease. This work describes refinement of an intranasal S. pneumoniae infection model in CD-1 mice that enables pneumococci to be harvested from multiple niches with low contamination by nasopharyngeal microflora or host tissue, and minimal crosscontamination with circulating pneumococci in the vascular system. The challenge route simulates the acquisition of S. pneumoniae in the human population, and progression to IPD occurs naturally. RNA extraction, enrichment and linear amplification procedures were optimised so that RNA could be obtained from in vivo site in sufficient quantities and with sufficient integrity to be used in semi-quantitative assays. Linear amplification allowed the examination of gene expression in niches where low bacterial numbers had previously prevented such analyses. Real-time RT-PCR and microarray analyses were used to examine bacterial RNA samples recovered from the nasopharynx, lungs, blood and brains of CD-1 mice, providing the first comparative transcriptional data for pneumococci during carriage and disease, within the same animal model. Two pneumococcal serotypes were examined; a type 2 (D39) and a type 6A (WCH16) strain. CbpA, Ply, and SpxB were shown to be important for carriage in both strains, with pneumococci up-regulating the expression of the genes encoding these virulence proteins in the nasopharynx. This provides in vivo evidence supporting the ascribed roles of these proteins in reducing the level of competing microflora and promoting nasopharyngeal adherence. Similarly, D39 nanA and pspA transcription levels were up-regulated in the nasopharynx. The level of pspA mRNA was also higher in the blood than the lungs, suggesting an increased requirement in the bloodstream, where PspA is involved in reducing complement-mediated opsonisation. Despite the antiphagocytic role of the pneumococcal polysaccharide capsule in the bloodstream, D39 cpsA mRNA was present in similar quantities in the nasopharynx, lungs and blood, which may support previous studies indicating post-transcriptional regulation of capsule expression. However, cpsA expression was up-regulated in the blood for WCH16. These results may indicate the existence of strain-specific differences in virulence gene regulation. Microarray analysis of in vivo-harvested S. pneumoniae D39 found that mRNAs encoding components of phosphotransferase systems, CbpA, a putative neuraminidase, and v-type sodium ATP synthase subunits were significantly higher in bacteria involved in carriage than bacteraemia. Conversely, the expression of genes involved in competence, and dinF (present on a competence-induced operon), were up-regulated in the blood compared to the nasopharynx, providing evidence that competence is induced during bacteraemia. Pneumococci also showed increased expression of genes involved in fatty acid metabolism, pgdA, lytB and cbpG in the blood compared to the nasopharynx. This study used a single pneumococcal strain and infection model and, therefore, overcomes inherent issues of serotype/strain- and animal model- specific gene expression that may have complicated interpretation of data in previous studies. This thesis reports some of the first in vivo pneumococcal gene expression data gained using a single animal model and pneumococcal strain. The data reinforce the putative roles of several virulence factors, and provides novel transcription data for pneumococci during carriage. Results suggest the existence of core genes that are essential for infection in multiple pneumococcal serotypes, whereas other genes appear to have strain-specific roles. / http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1287056 / Thesis (Ph.D.)-- University of Adelaide, School of Molecular and Biomedical Science, 2007

The objective of the current research project relies on implementation of solvent-free, green and environmentally friendly solid phase microextraction (SPME) sample preparation alternative in the area of complex sample characterization. The advantages that the technique offers in comparison to traditional methods of sample preparation including solvent-free implementation, short sample preparation times, small sample amount requirements, advanced automation capability and minimization of matrix effects are effectively employed during ex vivo and laboratory investigations of complex samples. More important, the underlying features of the technique including miniaturized format, nonexhaustive extraction recoveries and on-site compatibility were fully exploited in order to investigate the metabolome of biological systems directly on the site. Hence, in vivo SPME extraction format was employed in direct immersion SPME sampling of biological systems, hence eliminating the crucial prerequisites associated with multiple preparative steps and incorporation of metabolism quenching that are encountered during implementation of traditional sample preparation methods in global metabolite analysis. Furthermore, in vivo sampling format was hyphenated to comprehensive two-dimensional gas chromatography – time-of-flight mass spectrometry (GCxGC-ToFMS) for high-resolution sampling of volatile and semivolatile metabolites in ‘Honeycrisp’ apples. The initial stages of the project involved evaluation of performance characteristics of commercial SPME extraction coatings in terms of extraction selectivity, extraction sensitivity and desorption efficiency by employing headspace SPME analysis of both aqueous standards spiked with representative volatile and semivolatile metabolites as well as the apple homogenate. DVB/CAR/PDMS coating was selected on the basis of optimum metabolite coverage and extraction sensitivity and was consequently employed during ex vivo and in vivo sampling assays corresponding to determination of volatile and semivolatile metabolites. The former extraction methodology incorporated appropriate sample preparation steps for quenching metabolic activity so that the relevant metabolome profile is not biased against unstable metabolites and those that are susceptible to inter-metabolite conversions which adversely impact preservation of metabolite identity. The two sample preparation assays were compared in terms of metabolite coverage and analytical precision in order to identify SPME route toward characterization of more representative metabolome and determination of instantaneous and more ‘true’ metabolism snapshoot. This is the first report illustrating the implementation of in vivo direct immersion SPME assay for non invasive determination of endogenous fruit metabolites whose profiles and contents are highly correlated to a multitude of influential fruit quality traits.

The objective of the current research project relies on implementation of solvent-free, green and environmentally friendly solid phase microextraction (SPME) sample preparation alternative in the area of complex sample characterization. The advantages that the technique offers in comparison to traditional methods of sample preparation including solvent-free implementation, short sample preparation times, small sample amount requirements, advanced automation capability and minimization of matrix effects are effectively employed during ex vivo and laboratory investigations of complex samples. More important, the underlying features of the technique including miniaturized format, nonexhaustive extraction recoveries and on-site compatibility were fully exploited in order to investigate the metabolome of biological systems directly on the site. Hence, in vivo SPME extraction format was employed in direct immersion SPME sampling of biological systems, hence eliminating the crucial prerequisites associated with multiple preparative steps and incorporation of metabolism quenching that are encountered during implementation of traditional sample preparation methods in global metabolite analysis. Furthermore, in vivo sampling format was hyphenated to comprehensive two-dimensional gas chromatography – time-of-flight mass spectrometry (GCxGC-ToFMS) for high-resolution sampling of volatile and semivolatile metabolites in ‘Honeycrisp’ apples. The initial stages of the project involved evaluation of performance characteristics of commercial SPME extraction coatings in terms of extraction selectivity, extraction sensitivity and desorption efficiency by employing headspace SPME analysis of both aqueous standards spiked with representative volatile and semivolatile metabolites as well as the apple homogenate. DVB/CAR/PDMS coating was selected on the basis of optimum metabolite coverage and extraction sensitivity and was consequently employed during ex vivo and in vivo sampling assays corresponding to determination of volatile and semivolatile metabolites. The former extraction methodology incorporated appropriate sample preparation steps for quenching metabolic activity so that the relevant metabolome profile is not biased against unstable metabolites and those that are susceptible to inter-metabolite conversions which adversely impact preservation of metabolite identity. The two sample preparation assays were compared in terms of metabolite coverage and analytical precision in order to identify SPME route toward characterization of more representative metabolome and determination of instantaneous and more ‘true’ metabolism snapshoot. This is the first report illustrating the implementation of in vivo direct immersion SPME assay for non invasive determination of endogenous fruit metabolites whose profiles and contents are highly correlated to a multitude of influential fruit quality traits.