Chemotherapy-induced alopecia (CIA) is widely regarded as the most traumatic side effect associated with cancer treatment and the associated stress can be detrimental to overall outcomes. Yet there has been little research into its pathobiology and no pharmaceutical intervention is available. CIA is caused by chemotherapy-mediated damage of the rapidly dividing cells of the hair follicle and although it is normally reversible, on regrowth the hair is often different in colour and/or texture and only grows gradually. The only effective treatment for CIA currently available is scalp cooling. It has been hypothesised that scalp cooling works by a combination of vasoconstriction, a reduction in the metabolic rate and/or reduced drug uptake by cells in the hair bulb. The ability of cooling to protect from CIA has been clinically demonstrated for years yet, to date, no cell biology is available to support its cytoprotective effects. The overall aim of this work was to for the first time provide a systematic investigation of the effects of cooling on chemotherapy-induced toxicity in human cells. The work established cellular models to determine the efficacy of cooling in rescuing from toxicity, investigate the temperature conditions providing maximal rescue and understand not only the mechanisms responsible for drug-mediated cytotoxicity, but also the way in which cooling regulates such mechanisms. Various human keratinocyte models were established, including normal (epidermal, NHEK, and follicular, HHFK) cells and adapted HaCaT (HaCaTa) cells. Viability, cell cycle and apoptosis assays were used, alongside Reactive Oxygen Species (ROS) detection, mitochondrial integrity assays and Western blotting, as well as functional pharmacological inhibition experiments. A panel of chemotherapy drugs commonly used in the clinic were employed, including doxorubicin, docetaxel and active metabolite of cyclophosphamide, 4-hydroxy-cyclophosphamide (4-OH-CP) and 5-FU, whilst a series of temperature conditions were tested, including 22°C as well as more severe cooling, particularly 18°C and 14°C (and even extreme cooling at 10°C). This study showed that cooling dramatically reduces or completely prevents the cytotoxic effects of docetaxel (T), doxorubicin (A), 5-FU (F) and particularly 4-OH-CP (C); however, optimal rescue was observed in conjunction with mono-therapy treatments (and substantial rescue with dual therapies, e.g. AC), whereas combinatorial treatment (TAC) showed relatively poor response to cooling, in agreement with clinical observations. Importantly, the work demonstrated that lowering the temperature below the widely accepted 22C threshold, even by a small number of degrees (e.g. 18C), resulted in significantly improved or even complete cytoprotection, a striking observation strongly suggesting that the scalp temperature achieved clinically is of critical importance in dictating the success of head cooling in CIA prevention. The panel of chemotherapy drugs tested caused differential effects on keratinocyte cell cycle progression and drug-mediated cell cycle arrest was significantly attenuated by cooling. Notably, cooling alone appeared to decelerate cell cycle progression, providing evidence for metabolic effects. More importantly, protective pre-conditioning (PPC) achieved either by growth factor removal or pharmacological inhibition of EGFR activation enhanced the cytoprotective effects of cooling and significantly reduced the effects of the chemotherapy drugs. As the ability of PPC to enhance protection from drug cytotoxicity could be attributed to its propensity to regulate the cell cycle progression, the work provided evidence that one mechanism via which cooling cytoprotects might be due to its ability to decelerate cell cycle progression. Disruption of mitochondrial membrane potential and elevation of ROS indicated the activation of an apoptotic pathway, which was confirmed by cell death-specific assays that confirmed a mitochondrial apoptotic pathway, as evident by plasma membrane disruption, caspase activation and DNA fragmentation. Importantly, cooling at a variety of temperatures (but mainly at or below 18C) attenuated drug-mediated apoptosis. To further investigate the precise mechanisms of growth arrest and/or cytotoxicity, activation/regulation of critical intracellular signalling mediators was investigated at the protein level. The majority of the drugs used induced activation of p53 and subsequent induction of p53-inducible mediators such as p21, as well as pro-apoptotic mediators associated with the mitochondrial pathway, such as Bak, PUMA and Noxa, whilst induction of pro-apoptotic FasL and Bid cleavage was detected, suggesting possible cross-talk with the extrinsic apoptotic pathway. Strikingly, cooling attenuated or blocked in a time- and, more importantly, temperature-dependent fashion induction of these pro-apoptotic mediators (an effect that became more marked as the temperature was reduced from 37C, to 22C, 18C and 14C); these results have provided for the first time a more detailed mechanistic explanation for the cytoprotective effects of cooling. As ROS appeared to be important in cytotoxicity, the hypothesis raised was that the cytoprotective effect of cooling might be enhanced via co-treatment with an antioxidant (e.g. NAC), aimed at enhancing the cytoprotective capacity of cooling at sub-optimal temperatures (such as 26°C). The findings presented here suggested that cooling plus topical treatment with antioxidants might represent a promising approach to improve the cytoprotective effects without compromising the anticancer effects of chemotherapy. Overall, despite their reductive nature, these in vitro models have provided experimental evidence for the ability of cooling to rescue from chemotherapy drug-mediated toxicity and shown that the choice of temperature may be critical in determining the efficacy of cooling in the clinic. This, whilst generating a novel combinatorial approach that has the potential to significantly enhance the ability of scalp cooling to protect against CIA in the clinic.

Regulatory toxicology in the 21st century is faced with the challenge of having to replace its use of experimental animals in chemical risk assessment with alternative methods. This is due to the introduction of the REACH legislation and the seventh amendment to the cosmetics directive. Such alternative methods include the use of in vitro (cell culture/tissue etc.), in chemico (chemical experiments e.g. determination of reactivity) and in silico (computational) approaches. Importantly, it is envisaged that data from all these alternative sources will be required for the prediction of the animal-based endpoints used in regulatory toxicology. One of the key computational approaches used for data gap filling is category formation and read-across. When using this approach to assess the potential toxicity of a chemical, a chemical category is best defined based on a common molecular initiating event e.g. the formation of a covalent bond with biological nucleophile via the same chemical mechanism. The structural features that define a chemical's membership of such a category can be encoded computationally as structural alerts, which in turn, can be grouped together to form an in silico profiler. The work discussed in this thesis addresses the key shortcoming of traditional in silico profilers, this being that current in silico profilers provided no information about the rate of covalent bond formation for chemicals containing the same structural alert but with different substituents. The research within this thesis addresses this problem through the introduction of a fragment-based approach to in silico profiler development. This fragment-based approach introduces the use of calculated activation energies determined through the use of quantum mechanics calculations which enable chemical reactivity to be predicted. Chapter 3 outlines the development of the approach for α,β-unsaturated aldehydes, ketones and esters which form covalent bonds through Michael addition. Chapter 4 extends the work outlined in Chapter 3 demonstrating how the fragment-based profiler can be used to predict both chemical reactivity and skin sensitisation and toxicity to Tetrahymena pyriformis. Finally, Chapter 5 extends the approach to chemicals capable of reacting with proteins via an SN2 mechanism demonstrating the approach can be applied to any mechanistic domain for which data exist. Overall, this thesis outlines an approach for the development of novel fragment-based in silico profilers capable of quantitatively predicting chemical reactivity and by extension toxicity. It is envisaged that the work outlined in this thesis will be of use primarily in regulatory toxicology, within such tools as the OECD QSAR toolbox.

Cell-based therapeutic interventions are being developed for a variety of clinical indications, including irreversible retinal pathologies and stroke. Numerous cell therapy procedures use injection-based administration to deliver high density cell preparations, either systemically or directly. The mode of delivery of fragile cells can compromise treatment efficacy, which is dependent on cell viability and functionality post-injection. Reviewing current literature, there is a lack of comprehensive testing of the effects of injection-based cell delivery on the various parameters of cell function. This study investigated the effects of the administration process on a range of cell characteristics, and aimed to answer critical questions regarding possible reasons for failure to deliver a sufficient numbers of viable cells. Biomaterial-assisted cell delivery was also investigated to determine improvement of cell recovery and possible influence on cell fate. Primary human mesenchymal stem cells (hMSCs) and Swiss mouse embryonic fibroblast cell line (NIH 3T3) suspensions were drawn up into 100 μL Hamilton syringes with 30- and 34G needles. They were then ejected at rates ranging from 10-300 μL/min. A comprehensive toolset was employed to assess the effects of various injection parameters, including ejection rate and needle size. Cell dose recovery, viability, apoptosis, senescence and other parameters of cellular health were evaluated using various standard and multiplex assays. Trilineage differentiation potential of ejected hMSCs was also assessed. Moreover, various injectable cell carriers were explored in terms of improvement of cell recovery and potential influence on differentiation capacity. Ejections at slower flow rates resulted in a significantly lower percentage of dose delivered as viable cells, with ejections at 300 μL/min showing the maximum percentage of hMSCs dose delivered at 77.6 ± 11.7%. Lower cell numbers delivered at slower ejection rates were mainly attributed to cell retention within the delivery device. Normalised caspase-3/7 activity measurements ejected at 10 μL/min were also significantly higher than control. Quantification of differentiation of ejected hMSCs revealed that both ejection rate and cell carrier employed may exert an effect on differentiation capacity. The use of biomaterials as cell carriers significantly improved cell recovery. Ejection of hMSCs in gelatin solution resulted in 87.5 ± 14% of the cell dose being delivered as viable cells, in comparison with 32.2 ± 19% of the dose ejected in phosphate buffered saline (PBS) at 10 μL/min. This study shows that ejection rate, needle size and cell carrier have a significant impact on the percentage of cell dose delivered as viable cells, cellular health and differentiation potential post-ejection. Optimal delivery strategies for injectable cell-based therapeutics are required to enhance their efficacy and reproducibility. This study emphasises the importance of careful consideration of administration protocols, according to the nature of the administered cells and cellular responses post-ejection. The combination of the investigated factors, among others, may also influence the fate of stem cells injected, thereby affecting the success of cell-based therapies.

O objetivo desta investigação foi analisar a percepção do doente de tuberculose sob tratamento supervisionado em uma Unidade Distrital de Saúde do município de Ribeirão Preto-SP. Optou-se pela pesquisa de natureza qualitativa. A população do estudo constituiu-se 6 doentes de tuberculose inscritos no Programa de Controle de Tuberculose sob o regime de tratamento supervisionado (TS). Para a coleta de dados utilizou-se o prontuário do doente e a Ficha Epidemiológica de Notificação da doença e a entrevista semi-estruturada como tendo como questões orientadoras "o significado da tuberculose" e "o tratamento supervisionado na vida do doente". Para a análise dos dados utilizou-se a técnica de análise de Conteúdo, modalidade Temática. A unidade temática central é o tratamento supervisionado na tuberculose: a percepção do doente foi conformada a partir dos seguintes núcleos de sentido: Enfoque da ação terapêutica no Tratamento Supervisionado (A ingestão medicamentosa e Fortalezas e Debilidades do Tratamento Supervisionado ); e Singularidades do doente sob tratamento supervisionado, (Percepção sobre a doença, Convivência com a doença, As repercussões do tratamento supervisionado na vida do doente). As fortalezas percebidas no processo da doença e no tratamento: medicação gratuita; a cesta básica, o vale transporte; a supervisão através da visita domiciliária permite o vínculo entre os trabalhadores de saúde e os doentes e suas famílias. As debilidades do TS percebidas foram: a fiscalização na tomada da medicação; a dependência do horário da visita para ingerir a medicação. A família e a equipe de saúde (na figura da visitadora) são reconhecidos como fator de adesão ao tratamento. / This study aimed to analyze the perception of tuberculosis patients under supervised treatment (ST) in a District Health Unit in the City of Ribeirão Preto - SP. A qualitative analysis was selected as the methodological approach. The study population consisted of 7 tuberculosis patients participating in the Tuberculosis Control Program under the a regimen of supervised treatment. The patients' medical records, the Epidemiological Form of Disease Notification and semi-structured interviews were used as instruments for data collection. The following were used as guiding questions: "the significance of tuberculosis" and "the supervised treatment in the patient's life". The Content Analysis technique - Thematic Modality - was used for data analysis. The main thematic unit "Supervised treatment in tuberculosis: the patient’s perception" was found from the following meaning units: the focus of ST as a therapeutic action (medication ingestion and the weaknesses and strengths of ST); and the Singularities of patients under supervised treatment (The perception of the disease, Living with the disease, The consequences of ST on the patient's life). The following were perceived by patients as strengths in the disease and treatment process: the provision of free medication; the availability of other forms of incentive such as the basic food basket and transportation vouchers; supervision through home visits allowed the link between health workers, the patients and their families. The weaknesses of ST were perceived as: the surveillance of medication ingestion and the dependence on the visiting hours for medication ingestion. The social actors involved in the treatment, such as the family and the health care team (in the visiting figure), were recognized as a factor of adherence to the treatment.

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"First published 1935." / Includes index. / 223 p. : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (M.D.)--University of Adelaide, 1935

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Thesis (M. Dip. Tech.)--Technikon Witwatersrand, 2002. / Supervisor: Dr. Harold Humphries. Also available via World Wide Web.