Made available in DSpace on 2015-12-10T14:24:19Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-07-29. Added 1 bitstream(s) on 2015-12-10T14:30:31Z : No. of bitstreams: 1 000851698_20160729.pdf: 547839 bytes, checksum: 2adff6a515f107e4e977600d01ec7ba7 (MD5) Bitstreams deleted on 2016-07-29T12:53:57Z: 000851698_20160729.pdf,. Added 1 bitstream(s) on 2016-07-29T12:54:50Z : No. of bitstreams: 1 000851698.pdf: 3022510 bytes, checksum: a93d5ebfdfee8b2508562e1a0a8de9b2 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A zidovudina (AZT) é o fármaco mais utilizado isoladamente ou em associação para o tratamento da Síndrome da Imunodeficiência Adquirida (AIDS), causada pelo vírus HIV. A baixa biodisponibilidade do AZT é o grande desafio a ser vencido para otimizar seu desempenho na terapêutica por via oral, já que sua elevada taxa de metabolização e baixa permeabilidade resultam na necessidade de administração de elevadas doses do fármaco. Além disto, este fármaco ainda é substrato do mecanismo de efluxo mediado pela glicoproteína-P no intestino, o que diminui ainda mais sua biodisponibiidade. Desta forma, dispersões sólidas (DS) e nanopartículas poliméricas (NP) foram desenvolvidas por complexação polieletrolítica com diferentes proporções entre o fármaco e os polímeros quitosana (QS) e ftalato de hidroxipropilmetilcelulose (HP). O teor de incorporação de fármaco nas DSs (em torno de 98%) foi maior que nas NPs (cerca de 65%). Os ensaios de liberação in vitro realizados em HCl 0,1 mol/l (pH 1,2) demonstraram que a complexação entre os polímeros e compartimentalização do fármaco reduziu as taxas de liberação do AZT tanto para as DSs (26 a 50%) quanto nas NPs (aproximadamente 60%) e, em tampão fosfato 50 m mol/l (pH 7,4), ambos sistemas de liberação prolongaram a liberação do fármaco por até 240 minutos. A determinação da granulometria confirmou a obtenção de sistemas micro (até 100 μm) e nanoparticulados (até 400 nm) e o potencial zeta evidenciou a carga superficial negativa das DSs e positiva das NPs. Os dados de DSC e DRX mostraram que o AZT estava molecularmente disperso em ambos os sistemas e que as DSs apresentaram estrutura amorfa, enquanto as NPs apresentaram estrutura predominantemente cristalina.A espectroscopia na região do infravermelho mostrou a formação de ligações entre a QS e o HP tanto nas DSs quanto nas NPs, sem alterar a... / Zidovudine (AZT) is the most widely used drug alone or in combination with other antiretroviral agents for the treatment of AIDS, caused by the HIV virus. The low AZT bioavailability is the great challenge to be overcome to optimize its performance in oral therapy, since its high rate of hepatic metabolism and low permeability results in the need for high doses of the drug. In addition, this drug is substrate of the efflux mechanism mediated by P-glycoprotein in the gut, which further decreases its bioavailability. Thus, solid dispersions (SD) and polymeric nanoparticles (NP) were developed by complexation polyelectrolyte with different ratios between the drug and the polymers chitosan (CS) and hydroxypropyl methylcellulose phthalate (HP). The drug content in the SDs (around 98%) was higher than in NPs (about 65%). The in vitro release assays performed in HCl 0.1 mol/l (pH 1.2) showed that polymeric complexation and drug entrapment reduced the AZT rates release in both SDs (26 to 50%) and NPs (around 60%), and in phosphate buffer 50 m mol/l (pH 7.4), both modified the drug release until 240 minutes. The particle size determination confirmed obtaining micro systems (around 100 micron) and nanoparticles (around 400 nm) and zeta potential showed the negative surface of SDs and the positive charge of NPs. DSC and XRD data showed that AZT remained molecularly dispersed in both systems and the DSs had an amorphous structure, while the NPs showed structure crystalline predominantly. The IR spectroscopy showed in both SDs and NPs the formation of bonds between CS and the HP without changing the structure of AZT. The NPs showed higher liquid absorption capacity (until 260%) relative to SDs (until 160%) in different pH values, while SDs presented higher mucin adsorption capacity. The SDs and NPs mucin adsorption occurs according to the mechanism of Freundlich and Langmuir, respectively. Intestinal permeability assay showed the influence of SD to ... / FAPESP: 13/01670- 0 e 14/08044-0

Made available in DSpace on 2014-08-13T14:50:53Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-03-28Bitstream added on 2014-08-13T18:01:23Z : No. of bitstreams: 1 000765980_20150414.pdf: 404380 bytes, checksum: 96179c0575a35660857492c085ade65e (MD5) Bitstreams deleted on 2015-04-23T11:39:24Z: 000765980_20150414.pdf,Bitstream added on 2015-04-23T11:39:57Z : No. of bitstreams: 1 000765980.pdf: 4347682 bytes, checksum: 92fbfe6a7c4583f5c57af76211fc42f7 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Neste estudo foi proposta a utilização do processo antissolvente supercrítico (SAS), no qual o gás carbônico foi selecionado como agente antissolvente, para a obtenção de sistema de liberação controlada de fármaco. No delineamento experimental fatorial 32, a proporção entre zidovudina e poli(L-ácido lático) (AZT:PLLA) representou o fator X1, e as condições de temperatura e pressão representou o fator X2, sendo ambos variáveis independentes; o rendimento do processo e a morfologia macroscópica das partículas representaram as variáveis dependentes. No presente estudo, as variáveis dependentes determinaram os produtos dos lotes que foram selecionados para a caracterização do estado sólido, teor de AZT, tipo e cinética de liberação de AZT e permeação intestinal de AZT. A caracterização do estado sólido utilizou as análises de dispersão de raios X (DRX), microscopia eletrônica de varredura (MEV), espectroscopia de infravermelho com transformada de Fourier (FITR) e calorimetria exploratória diferencial (DSC). O teor de AZT foi determinado por metodologia analítica validada. O teste de dissolução forneceu dados para as avaliações da liberação e da cinética de liberação de AZT. O modelo de saco intestinal invertido de rato foi adotado para o estudo ex vivo de permeação intestinal do AZT. Os lotes L3 (91,54 % de rendimento e amostra com aspecto uniforme), L5 e L9 (59,06 % e 51,50 % de rendimento, respectivamente; ambos resultaram em amostra com aspecto não uniforme e sólido filamentoso) resultantes do planejamento fatorial 32 foram selecionados para os estudos analíticos. O lote L3 de proporção AZT:PLLA (1:2, m/m) resultou em rendimento de 91,54 % e teor de AZT 58,76 % elevados em comparação aos lotes produzidos e foi selecionado para o estudo de permeação intestinal. A permeabilidade do AZT a partir do lote L3 (9,87 ± 0,47 %) foi maior em relação ao AZT puro (3,84 ± 0,45 %). O AZT permaneceu ... / In this study we proposed the use of supercritical antisolvent process (SAS), in which carbon dioxide was selected as antisolvent agent, to obtain drug controlled release system. Factorial 32 experimental design use the of zidovudine and poly (L-lactic acid) ratio (AZT:PLLA ) as X1 factor, also the temperature and pressure conditions as X2 factor, both independent variables, the process yield and particles macroscopic morphology represented the dependent variables. In this study, the dependent variables determined the product that have been selected to solid state characterization, AZT content, type, release kinetics, and intestinal permeation. Solid state characterization used X-ray diffraction (XRD) analysis, scanning electron microscopy (SEM), infrared spectroscopy with Fourier transform (FTIR), and differential scanning calorimetry (DSC). The AZT content was determined by validated analytical methodology. The dissolution test provided data for AZT release and release kinetics evaluation. The rat everted gut sac model was take on for the ex vivo study of intestinal permeation of AZT. The L3 (91.54 % yield and sample with uniform appearance), L5 and L9 lots (59.06 % and 51.50 % yield , respectively , both of which resulted in samples with non-uniform and solid filamentous appearance) resulting from factorial 32 planning were selected for analytical studies. The L3batch ratio of AZT:PLLA (1:2, w/w) resulted in a high yield of 91.54 % and 58.76 % AZT content compared to batches produced, and was selected for the study of intestinal permeation . The AZT permeability from L3 lot (9.87 ± 0.47%) was higher than pure AZT (3.84 ± 0.45 %). AZT remained in crystalline form and PLLA remained in semi - crystalline form, in lots L3, L5, and L9 produced by the SAS process, when evaluated by SEM, XRD, DSC, and FTIR. This study demonstrated that it is possible to use the SAS process to obtaining modified release systems for poorly permeable drugs ...

Orientador : Maria Palmira Daflon Gremião / Coorientador: Marco Vinícius Chaud / Banca: Victor Manuel Cardoso Figueiredo Balcão / Banca: Clovis Augusto Ribeiro / Banca: Ana Luiza Ribeiro de Souza / Banca: Beatriz Stringhetti Ferreira Cury / Resumo: Neste estudo foi proposta a utilização do processo antissolvente supercrítico (SAS), no qual o gás carbônico foi selecionado como agente antissolvente, para a obtenção de sistema de liberação controlada de fármaco. No delineamento experimental fatorial 32, a proporção entre zidovudina e poli(L-ácido lático) (AZT:PLLA) representou o fator X1, e as condições de temperatura e pressão representou o fator X2, sendo ambos variáveis independentes; o rendimento do processo e a morfologia macroscópica das partículas representaram as variáveis dependentes. No presente estudo, as variáveis dependentes determinaram os produtos dos lotes que foram selecionados para a caracterização do estado sólido, teor de AZT, tipo e cinética de liberação de AZT e permeação intestinal de AZT. A caracterização do estado sólido utilizou as análises de dispersão de raios X (DRX), microscopia eletrônica de varredura (MEV), espectroscopia de infravermelho com transformada de Fourier (FITR) e calorimetria exploratória diferencial (DSC). O teor de AZT foi determinado por metodologia analítica validada. O teste de dissolução forneceu dados para as avaliações da liberação e da cinética de liberação de AZT. O modelo de saco intestinal invertido de rato foi adotado para o estudo ex vivo de permeação intestinal do AZT. Os lotes L3 (91,54 % de rendimento e amostra com aspecto uniforme), L5 e L9 (59,06 % e 51,50 % de rendimento, respectivamente; ambos resultaram em amostra com aspecto não uniforme e sólido filamentoso) resultantes do planejamento fatorial 32 foram selecionados para os estudos analíticos. O lote L3 de proporção AZT:PLLA (1:2, m/m) resultou em rendimento de 91,54 % e teor de AZT 58,76 % elevados em comparação aos lotes produzidos e foi selecionado para o estudo de permeação intestinal. A permeabilidade do AZT a partir do lote L3 (9,87 ± 0,47 %) foi maior em relação ao AZT puro (3,84 ± 0,45 %). O AZT permaneceu ... / Abstract: In this study we proposed the use of supercritical antisolvent process (SAS), in which carbon dioxide was selected as antisolvent agent, to obtain drug controlled release system. Factorial 32 experimental design use the of zidovudine and poly (L-lactic acid) ratio (AZT:PLLA ) as X1 factor, also the temperature and pressure conditions as X2 factor, both independent variables, the process yield and particles macroscopic morphology represented the dependent variables. In this study, the dependent variables determined the product that have been selected to solid state characterization, AZT content, type, release kinetics, and intestinal permeation. Solid state characterization used X-ray diffraction (XRD) analysis, scanning electron microscopy (SEM), infrared spectroscopy with Fourier transform (FTIR), and differential scanning calorimetry (DSC). The AZT content was determined by validated analytical methodology. The dissolution test provided data for AZT release and release kinetics evaluation. The rat everted gut sac model was take on for the ex vivo study of intestinal permeation of AZT. The L3 (91.54 % yield and sample with uniform appearance), L5 and L9 lots (59.06 % and 51.50 % yield , respectively , both of which resulted in samples with non-uniform and solid filamentous appearance) resulting from factorial 32 planning were selected for analytical studies. The L3batch ratio of AZT:PLLA (1:2, w/w) resulted in a high yield of 91.54 % and 58.76 % AZT content compared to batches produced, and was selected for the study of intestinal permeation . The AZT permeability from L3 lot (9.87 ± 0.47%) was higher than pure AZT (3.84 ± 0.45 %). AZT remained in crystalline form and PLLA remained in semi - crystalline form, in lots L3, L5, and L9 produced by the SAS process, when evaluated by SEM, XRD, DSC, and FTIR. This study demonstrated that it is possible to use the SAS process to obtaining modified release systems for poorly permeable drugs ... / Doutor

Magister Pharmaceuticae - MPharm / Background: Zidovudine (AZT) is a very useful drug for the management of Human Immunodeficiency Virus (HIV) infection. Its optimal use is limited by its bitter taste, sparing solubility (20.1 mg/ml) and acid lability. Cyclodextrins (CD) are a class of compounds which can be used to form inclusion complexes with drugs such as AZT to improve it is taste, solubility and palatability. Purpose: This study complexed hydroxypropyl-beta-cyclodextrin (HPβCD) with AZT. The formulated inclusion complex was evaluated for suitability as a dosage form and as a tool for improving AZT’s palatability, solubility and acid liability. Method: AZT was complexed with HPβCD using the lyophilisation method. The binding constant for the formulation was determined by the phase solubility method, and complex formation between AZT and HPβCD evaluated using proton nuclear magnetic resonance (1H NMR), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and hot stage microscopy (HSM). Tablets of the inclusion complex were formulated by direct compression, using the least possible amount of excipients, and the dosage form evaluated for hardness, friability, durability, disintegration time and dissolution. Results: The binding constant of the formulation was 3.919, and the degree of incorporation was 4.0 mg AZT/g of CD per complex. 1H NMR showed significant chemical shifts between the inclusion complex and AZT. DSC and TGA analyses showed significant differences in the curves for the pure AZT and HPβCD. Values for tablet hardness, friability, durability and disintegration time were 236 ± 20 N, 0.7 %, 1.02 % and 10.25 minutes, respectively. The solubility of the formulation was 148.08 mg/ml, and its dissolution profile was different from that of the branded formulation. Conclusions: AZT-HPβCD inclusion complex, with a 7.4-fold increase in AZT solubility, was successfully prepared using the lyophilisation method. The binding constant and friability of the formulation were within acceptable limits. Although the hardness value is high, the tablet still disintegrated within acceptable specified times. This study has significant implications for anti-retroviral complex formulations.

AZT (3'-azido-2’, 3’-dideoxythymidine) has been shown to prolong the survival of patients infected with human immunodeficiency virus (HIV) and decrease the severity of opportunistic infections. Such studies have prompted the use of AZT to treat symptomless individuals infected with HIV in the hope of delaying or even preventing the progression to acquired immunodeficiency syndrome (AIDS). However, before chronic use of AZT in symptomless individuals is initiated, it is important to establish whether this anti-viral drug would directly alter the phenotype and functions of the cells involved in the immune system. In the current study, we observed that AZT when administered orally for 7 -14 days into DBA/2 mice at 500 - 1000 mg/kg body weight induced a dose-dependent decrease in cellularity of the thymus. AZT caused significant alterations in the thymus resulting from a significant decrease in the number of double-positive (CD4⁺CD8​​⁺) cells and an increase in the number of double-negative (CD4⁻CD8⁻) cells. Interestingly, after the i.p. administration of interleukin-2 (IL-2) simultaneously with AZT, the total cellularity of the thymus was completely reconstituted. We also observed that AZT effectively suppressed the in vivo T cell response to conaibumin and gp120 of HIV. Furthermore, the addition of AZT to in vitro cultures caused a dose-dependent decrease in T and B cell proliferative responses to mitogens at 50μM or greater concentrations. Also, AZT inhibited the generation of cytotoxic T lymphocytes when added to the culture and this inhibition was reconstituted by the addition of exogenous IL-2. Together, our studies demonstrate that AZT modulates the phenotype and function of cells of the immune system which, in turn, could have marked repercussions on immune responses of the host toward infections and cancers. Also, our data demonstrating that AZT can suppress T cell responsiveness against HIV antigens caution against chronic use of AZT in asymptomatic HIV-infected individuals. / Master of Science

Made available in DSpace on 2014-06-11T19:29:32Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-06-22Bitstream added on 2014-06-13T18:39:11Z : No. of bitstreams: 1 figueiredo_as_me_botfmvz.pdf: 290159 bytes, checksum: 38ae46f390705b8d387a7ef9c78d6040 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O Vírus da Imunodeficiência Felina (FIV) é um lentivírus que causa uma síndrome de imunodeficiência em gatos domésticos. O FIV tem sido particularmente utilizado em estudos de resistência viral aos análogos de nucleosídeos devido a Transcriptase Reversa (TR) apresentar propriedades físicas, catalíticas e sensibilidade às drogas semelhantes à TR do HIV. Os objetivos desse trabalho foram tratar com AZT gatos naturalmente infectados com o FIV, fazer o monitoramento da carga viral e DNA proviral por PCR em tempo real e monitoramento genético por seqüenciamento. Dos 12 animais infectados, 6 receberam o AZT na dose de 10mg/kg/dia e 6 receberam placebo. Durante 96 dias de tratamento, o plasma e sangue destes animais foram analisado com relação à carga viral e concentração relativa de DNA proviral utilizando-se a técnica de quantificação relativa por PCR em tempo real com SYBR Green, desenvolvida por nossa equipe. Além disso, foi realizado o sequenciamento genético da região que codifica a TR de 3 dos animais. Foi realizada com sucesso a padronização da PCR em tempo real para quantificação relativa do FIV. Não houve diferença estatisticamente significativa da carga viral ou do DNA proviral entre os grupos tratado e controle. O seqüenciamento genético revelou a presença de lisina na posição 41 do sítio ativo da TR. A presença deste aminoácido confere até 4 vezes menor sensibilidade ao AZT em mutantes do HIV. Por possuir alta estabilidade genética, supomos que os vírus dos demais animais não sequenciados possuem também a 41-lisina A presença da 41-lisina pode ser uma das possíveis explicações para a falha do tratamento com AZT. Outra hipótese é a de que a dose fornecida não foi adequada. / Feline Immunodeficiency Virus (FIV) is a lentivirus which causes a progressive disruption of the host's immune functions. FIV has been particularly used as a model for studies in retroviral resistance to nucleoside analogs because its similarities in physical properties, catalytic and sensitivity in comparison with HIV/RT. The aims of this work were to treat cats naturally infected with FIV, quantify viral load and proviral DNA by real time quantitative PCR with SYBR Green and analyze the viral nucleotide sequence. From 12 animals naturally infected, 6 received AZT at a dose of 10mg/kg/day and 6 received placebo. During 96 days of treatment, viral load and concentration of proviral DNA were measured by relative quantitative real time PCR developed by our staff. The nucleotide sequence of the RT encoding region was also achieved for 3 animals. The real time PCR relative quantification was successfully standardized for FIV. There was no significant statistical difference between treated and control groups. The nucleotide sequence revealed a lysine at position 41 on the enzyme active site. This lysine confers 4-fold decreased sensitivity to AZT in HIV RT-mutants. FIV subtype B has high genetic stability and we purposed that the other virus not sequenced have the same amino acid and hypothesized that this mutations can be one of the reasons determining the failure of the treatment. The other hypothesis is that the dose was not adequate.

Als Prüfung auf das genotoxische Potenzial einer Substanz ist die Entstehung von Micronuclei in proliferierendem Gewebe als genetischer Endpunkt wissenschaftlich und behördlich anerkannt. Zugrunde liegendes Experimentalmodell für diesen Test sind zumeist Mäuse und Ratten, deren Knochenmark das Zielgewebe dieses Tests darstellt. Dieses Testmodell geht mit dem Tod der verwendeten Tiere einher. Die vorliegende Arbeit greift den von Wolf & Lüpke (1997) sowie Wolf (1999) vorgestellten HET-MN (Hen’s Egg Test for MicroNucleus induction) auf, der angebrütete Hühnereier als Experimentalmodell verwendet (Bebrütungsdauer 11 Tage, d11). Zielorgan ist das periphere Blut der extraembryonalen Membranen. Das entnommene Blut wurde nach modifizierten hämatologischen Standard-verfahren angefärbt und die Zellen im Hellfeld-Durchlicht-Mikroskop bei 1000-facher Vergrößerung ausgezählt. Anhand der nucleosidanalogen Reverse Transkriptase Hemmer Zidovudin, Stavudin, Zalcitabin, Lamivudin und Didanosin fand ein Abgleich mit den durch Tierversuche gewonnenen Daten statt. In den versuchsreihen zu Zidovudin, Stavudin Zalcitabin, Didanosin und in den Kombinationsversuchen mit Zidovudin und Didanosin konnten die Daten aus der Literatur reproduzierbar bestätigt werden; wobei der HET-MN sensitiver reagierte als das Testmodell am Tier. Für Lamivudin konnte reproduzierbar eine biologisch und statistisch relevante, dosisabhängige Erhöhung der Micronucleusfrequenz erzielt werden. Damit steht der HET-MN in Kontrast zu den in der Literatur verfügbaren Informationen. Die im HET-MN gewonnenen Daten ließen in allen Versuchsreihen auf vergleichbare oder höhere Sensitivität als im Micronucleustest an Nagern schließen. Damit stellt der HET-MN eine kostengünstige und methodisch leicht durchführbare Alternative zum Tierversuch dar und wird trotz seiner hohen Komplexität und den damit verbundenen in-vivo-ähnlichen Bedingungen allen Belangen des Tierschutzes gerecht.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal. / Les promédicaments amphiphiles du 3-azido-31-désoxythymidine (AZT) ont préalablement été synthétisés dans le but d'augmenter l'activité antivirale et d'améliorer la biodisponibilité de la molécule mère dans le sang. Bien que plusieurs de ces promédicaments ont déjà été incorporés dans des formulations de liposomes, leur stabilité dans le plasma ainsi que leur potentiel hapténique restent encore à déterminer. Dans ce mémoire on décrit: a) la synthèse, la caractérisation ainsi que la rétention d'une série d'esters aliphatiques d'AZT (Cu-CIO (laurate d'AZT, myristate d'AZT, palmitate d'AZT et stéarate d'AZT) dont la synthèse a déjà été publiée par Kawaguchi et al. (J. Pharm. Sci., 79, 531-33, 1990) (chapitre I), b) la synthèse du [3E11-palmitate d'AZT, son incorporation, sa rétention ainsi que sa libération induite par le plasma à partir des porteurs liposomaux (chapitre II) et c) la capacité du palmitate d'AZT d'agir comme un haptène suite à son encapsulation dans les liposomes (chapitre Ill). Les caractéristiques des liposomes formulés avec du DPPC/DMPG et contenant l'ester de l'AZT étaient différentes pour les quatre sortes de promédicaments suite à une incubation dans du PBS à 4, 22 ou 37°C. Les liposomes avaient un potentiel Ç négatif variant entre (-8,91 et -10,23). Une meilleure rétention pour le palmitate d'AZT ainsi que pour le laurate d'AZT a été observée, et ce, aux trois différentes températures. De même, une augmentation du taux de libération, en allant de 4 à 37°C, était observable dans ces deux formulations, due à l'augmentation de la fluidité membranaire qui induit une augmentation de la libération des promédicaments à travers les bicouches phospholipidiques suite à une élévation de la température. L'incorporation liposomale du [311]-palmitate d'AZT a été améliorée lors de l'utilisation d'une chaîne de palmitate lipophile. D'une part la rétention du palmitate d'AZT dans les liposomes en présence de PBS est excellente avec 5 à 10% de perte sur une période d'incubation de 24 heures. D'autre part, l'incubation du [31-1]-palmitate d'AZT liposomal dans le plasma résulte en une libération significative avec le temps. Des injections répétées de liposomes contenant le palmitate d'AZT résulte en une induction d'anticorps du type 1gM et IgG chez les souris. L'absence d'une réponse immunitaire contre l'AZT non estérifié incorporé dans les liposomes montre que la molécule est présentée sur la surface des liposomes plutôt qu'a l'intérieur de ceux-ci.

No description available.

Thesis (MSc.(Anatomy)--Faculty of Health Sciences)-University of Pretoria, 2008.] / Includes bibliographical references.