Abstract Peptide surfactants developed in the Centre for Biomolecular Engineering at The University of Queensland are engineered to combine the advantages of traditional surfactants with biodegradability, biocompatibility, formation of a mechanically strong interfacial cohesive network, and reversible stimuli-responsiveness. In this project, the potential of peptide-stabilised emulsions as delivery systems for small interfering RNA (siRNA) was explored. In recent years, the potential of siRNA as a new class of therapeutics has attracted great attention. The ubiquitous nature of RNA interference (RNAi) implies that siRNA can be used to silence any disease-causing gene to treat any disease. The hurdle that needs to be overcome to turn siRNA therapy into clinical reality is its delivery into the cytosol, where gene silencing by siRNA occurs. Although numerous systems have been developed for the delivery of siRNA, safety and efficiency are major concerns associated with current formulations. Therefore this project aimed to prepare a stable peptide emulsion formulation and to conduct initial tests of its ability to deliver siRNA in vitro. The human tumour suppressor gene p53 and the human breast cancer MCF-7 cell line were used as the model gene and model cell line, respectively. The commercially available lipid-based transfection reagent Lipofectamine™ 2000 was used as the benchmark control. Sonication and membrane extrusion were used to formulate emulsions with droplet size (d=120 nm) suitable for intravenous applications using peptide surfactant in the presence of Zn(II). Although these peptide emulsions are stable by themselves and in bovine serum, emulsion stability was found to be strongly affected by the presence of salt, EDTA, and proteins. The instability of AM1 emulsion in cell culture media has been a concern when it was subjected to in vitro cell culture tests. AM1-stabilised emulsion droplets were shown to be taken up by MCF-7 cells. However, siRNA when coupled with AM1 emulsion was not delivered into cells. Cytotoxicity studies showed that peptide surfactants did not exhibit high-level toxicity to CHO cells at the tested concentrations (0.25-2 mg mL-1). AM1 peptide-stabilised emulsions were mildly toxic to CHO cells but no toxicity was observed with MCF-7 cells. Future work could include evaluation of peptide emulsion-siRNA complex formation, and exploring the effects of different cell culture media compositions on emulsion stability and their relation to cytotoxicity.

Mitochondria are the main sites for adenosine triphosphate (ATP) generation within most cells. Structural and functional alterations of mitochondria due to genetic abnormalities of mitochondria can cause respiratory chain dysfunction. In this study, the important role of mitochondria in energy metabolism was determined by comparing the effect of mitochondrial DNA (mtDNA) mutations on growth patterns and oxidative phosphorylation (OXPHOS) enzyme activities of six isolated clones (B5, B12, D4, D9, E1 and E8); as well as the effect of ATP supplement to culture using the slowest growing clone. The isolated clones had shown distinct growth pattern and morphology. The difference in proliferation rates among the clones was ascertained by the doubling times (B5=26.4h. B12=43.2h. D4=25.7h. D9=33.6h. E1=26.9h and E8=28.8h). The clone's slow growth rate was likely the result of mitochondrial mutations in the 16S rRNA gene, ND1, ND4, ND6 and COX III. Five heteroplasmic mutations were found in clone B12 (G2480T, C2513G, A2520T, C9527T and C14263G), one heteroplasmic mutation in clone D9 (A4137G) and one homoplasmic mutation in clone D4 (C11496). The mutations in clone B12 appeared to be deleterious to the cell by disrupting mitochondrial OXPHOS activities and reducing energy output. Additionally, extracellular ATP supplement to OXPHOS deficient clone B12 facilitated cell growth and enhances the gene expression. Increased expression of mtDNA-encoded respiratory chain complexes observed in clone B12 compared to clone D4 may reflect mitochondrial genomic adaptation to perturbations in cellular energy requirements. The stimulation of mitochondrial biogenesis may be a cellular response in compensation for defects in OXPHOS associated with mtDNA mutations. My data support the hypothesis that the variability in functional manifestations of mtDNA is attributed to the nature of the mutation, number of mutation and the gene specifically affected. These results will help to further our understanding of the relationship between mitochondrial mutation and cellular function.

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: The use of toxicity tests to evaluate the quality of streams affected by mixtures such as acid mine drainage (AMD), adds value to assessments whereby site-specific toxicological data may identify toxicants that pose a threat to humans. To successfully evaluate the risk of combined mixtures, an improved understanding of the individual components, their uptake, metabolism, excretion and mode of action is required. This study aimed to identify the extent of AMD toxicity in a dose dependant manner on the MCF-7 cell line. The first study site associated with gold mining was chosen as the Tweelopies Stream situated in the Gauteng province of South Africa. The AMD effluent (un-neutralised) contaminating the Tweelopies Stream had undergone pH-neutralisation using a reactor-bed limestone technology incorporating the use of both calcium carbonate (CaCO3) powder and limestone beds. The second study site, the Kromdraai River, is situated in the eMalahleni region of South Africa where a predominance of coal mining exists. The pH -neutralisation of the AMD (un-neutralised) contaminated Kromdraai River was performed using a caustic soda (NaOH) precipitation technique. This study demonstrated the rapid and effective application of the comet assay as a screening tool for AMD-associated DNA breakages in the human cell line, MCF-7. Moreover, the study analysed parameters of cellular survival, DNA fragmentation and variations in morphologies indicative of cellular death. Collectively, the cyto-genetic aberrations observed in the MCF-7 cells as a result of exposure to gold and coal mining associated AMD, confirms the urgency of incorporating high-throughput screening in ecological toxicity assessment to evaluate cellular damage at genetic levels in low dose exposures where detection might be missed. / AFRIKAANSE OPSOMMING: Die gebruik van toksisiteitstoetse om die gehalte van strome te evalueer wat geraak word deur mengsels soos suur mynwater (SM), gee waarde aan spesifieke toksikologiese data van gifstowwe wat 'n bedreiging vir die mens kan identifiseer. Om die risiko van gekombineerde mengsels en hul individuele komponente beter te begrip en suksesvol evalueer, is hul opname, metabolisme, uitskeiding en modus van aksie nodig. Hierdie studie het gepoog om die omvang van SM-toksisiteit in 'n dosis afhanklike wyse op die MCF-7-sellyn te identifiseer. Die eerste studie-area wat gekies is, hou verband met goudmyn-ontginning, en is die Tweelopiesspruit, geleë in die Gauteng-provinsie van Suid-Afrika. Die SM-uitvloeisel (on-geneutraliseerde) wat die Tweelopiesspruit besoedel, het pH-neutralisasie ondergaan met behulp van die integrasie van 'n reaktor-bed kalksorpsietegnologie wat gebruik maak van beide kalsiumkarbonaat (CaCO3) poeier en kalksteenbeddens. Die tweede studie-area, is die Kromdraairivier geleë in die eMalahleni-streek van Suid-Afrika, waar steenkoolontginning die oorheersende aktiwiteit is. Die pH-neutralisasie van die SM (on-geneutraliseerde) in die geval van die Kromdraairivier word met behulp van 'n bytsoda (NaOH) neerslag tegniek, uitgevoer. Hierdie studie het die komeet-toets getoon as 'n vinnige en doeltreffende toepassing vir SM-geassosieerde DNA-breekskade in die menslike sel lyn, MCF-7. Verder het die studie parameters van sellulêre oorlewing, DNA-fragmentasie en variasies in sel morfologieë wat ‘n aanduiding van sellulêre dood is, ontleed. Gesamentlik dui die resultate daarop dat die sitogenetiese afwykings wat in die MCF-7-selle waargeneem is, as 'n gevolg van blootstelling aan goud- en steenkool-geassosieerde SM is. Die studie het verder die dringendheid van die integrasie van hoë-deurset tegnologieë in ekologiese toksisiteitstoetse in selle wat genetiese skade mag ondergaan, na 'n lae dosis blootstelling waar opsporing dalk gemis word, ondersteun.

Submitted by Maria Medeiros (maria.dilva1@ufcg.edu.br) on 2018-07-12T10:26:45Z No. of bitstreams: 1 MILENA COSTA DA SILVA - DISSERTAÇÃO (PPGCEMat) 2015.pdf: 4566309 bytes, checksum: ac360eb84cc411c27af09b604719adc6 (MD5) / Made available in DSpace on 2018-07-12T10:26:45Z (GMT). No. of bitstreams: 1 MILENA COSTA DA SILVA - DISSERTAÇÃO (PPGCEMat) 2015.pdf: 4566309 bytes, checksum: ac360eb84cc411c27af09b604719adc6 (MD5) Previous issue date: 2015-08-19 / Capes / A curcumina é um fármaco natural que apresenta propriedades medicinais dentre elas destaca-se a propriedade antineoplásica, nesta pesquisa a curcumina tem sido associada com a quitosana com a função de portador eficaz para a preparação de formulações na liberação de fármaco. Portanto o objetivo deste trabalho foi desenvolver arcabouços de quitosana/curcumina, pelo método de agregação de esferas, através da solução de gelatina (5%) com e sem reticulação. O fármaco foi incorporado na quitosana empregando a técnica de reação de soluções e adsorção. As esferas foram caracterizadas por Espectroscopia na Região Infravermelho com Transformada de Fourier (FTIR), Difração de Raios X (DRX), Microscopia Ótica (MO), Microscopia Eletrônica de Varredura (MEV), Termogravimetria (TG), Calorimetria Exploratória Diferencial (DSC) e Grau de Intumescimento (GI). E os arcabouços por MO, MEV, Porosidade por três diferentes métodos, propriedade mecânica de resistência a compressão e GI, também foram realizadas a validação do método analítico e a liberação da curcumina por UV/VIS, modelos matemáticos de cinética de liberação também foram aplicados, seguido dos ensaios de citotoxicidade com linha celular de fibroblastos (L929) e câncer de mama (MCF-7). As análises morfológicas de MO e MEV mostraram a presença do fármaco no interior das esferas, e nos arcabouços a adesão das esferas a partir da gelatina. Por FTIR sugeriu-se a interação da quitosana com a curcumina. Os resultados de DRX e TG mostraram que a presença do fármaco na matriz de quitosana, não alterou de forma significativa a cristalinidade e nem a estabilidade térmica do material, respectivamente. Por DSC verificou-se na amostra quitosana/curcumina, o desaparecimento do pico de fusão da curcumina. Constatou-se ainda que os arcabouços obtidos com gelatina reticulada apresentaram menor absorção de água no ensaio de GI, menor tamanho de poros e menor porosidade, como também um perfil prolongado de liberação de fármaco. A partir do ensaio de compressão observou-se que todos os sistemas apresentaram valores semelhantes de resistência a compressão do tecido adiposo humano. Na análise dos modelos matemáticos observou-se que os modelos de Korsmeyer-Peppas e de Higuchi foram os que mais adequaram aos sistemas e pelos resultados de citotoxicidade com a linha celular L929 verificou-se que os arcabouços não apresentaram toxicidade, já a amostra quitosana/curcumina(adsorção)-reticulada, apresentou perfil tóxico para a linha celular MCF-7. Dessa forma, constatou-se que o arcabouço de quitosana/curcumina (adsorção)-reticulada é promissor para o estudo em tratamento de câncer de mama, por apresentar, estrutura, tamanho de poro e perfil de liberação adequada para o tratamento, como também maior toxicidade nas células cancerígenas MCF-7. / Curcumin is a natural drug that has medicinal properties, among which are the anticancer property, thus, in this research, curcumin has been studied associated with chitosan that is an effective carrier for the preparation of formulations of drug delivery. Therefore the aim of this study was to develop scaffolds of chitosan/curcumin, by the ball aggregation method through gelatin solution (5%) with and without crosslinking. The drug was incorporated in chitosan employing the solutions reaction technique and adsorption. The beads were characterized by Fourier Transform Infrared Spectroscopy (FTIR), X-ray diffraction (XRD), Optical Microscopy (OM), Scanning Electron Microscopy (SEM), Thermogravimetry (TG), Differential Scanning Calorimetry (DSC) and Degree of Swelling (DS). And the scaffolds by OM, SEM, porosity by three different methods, mechanical properties by Compressive Strength and DS. The validation of the analytical method and the release of curcumin by UV / VIS were carried out, mathematical models of release kinetics were also applied, followed by cytotoxicity tests with fibroblast cell line (L929) and breast cancer (MCF-7). Morphological analysis of OM and SEM showed the presence of the drug inside the spheres, and in the scaffolds, the adhesion of the spheres from the gelatin. From the FTIR technique it was noticed the probable interaction of chitosan with curcumin. The results of XRD and TG showed that the drug present in the chitosan matrix did not significantly change neither the crystallinity nor the thermal stability of the material, respectively. By DSC,it was found in the chitosan / curcumin sample, the disappearance of the melting peak of curcumin. It was also found that the scaffolds obtained from cross-linked gelatin had lower water absorption in the GI assay, smaller pore size and lower porosity as well as an extended drug release profile. From the compression test it was observed that all systems had similar amounts of adipose tissue compression strength. Analyzing the mathematical models, it was observed that the Korsmeyer-Peppas and Higuchi models were the ones best adapted to most systems, and from the cytotoxicity results with the L929 cell line it was found that the scaffolds did not show toxicity, while the chitosan / curcumin (adsorption)-crosslinked sample showed toxicity profile for the cell line MCF7. Thus, it was found that the chitosan / curcumin (adsorption) - crosslinked scaffolds is promising for the study in treating breast cancer, because it presented structure, pore size and adequate release profile for the treatment, as well as greater toxicity in cancer cells MCF-7.

Depuis bon nombre d’années déjà, la question ne se pose plus ; l’utilisation des pesticides en agriculture est nécessaire puisque sans ces derniers les chances que les terres soient ravagées par les insectes, champignons ou petits rongeurs représenteraient un trop grand risque pour les agriculteurs. Cependant, cette utilisation massive de pesticides en agriculture apporte son lot de questionnements et d’inquiétudes face aux effets néfastes qu’ils pourraient avoir tant sur les humains que sur la faune et la flore. Bien que tous les pesticides utilisés au Canada soient homologués par Santé Canada, et sont donc jugés comme ne représentant pas de risques élevés ni pour l’environnement ni les humains, nul ne connait les effets d’un mélange de pesticides sur nos cellules, notre organisme. Ce projet de recherche vise donc à évaluer les effets d’un mélange de pesticides composé de cinq ingrédients actifs communément utilisés en agriculture sur les niveaux d’expression du gène CYP1A1, un gène cible du récepteur aux hydrocarbures d'aryle (AhR). Ce récepteur, bien qu’impliqué dans bon nombre d’autres réponses cellulaires, joue un rôle important dans la détoxification de l’organisme en activant la transcription de certains gènes dans la famille du cytochrome P450, dont le gène CYP1A1. Tel que mentionné précédemment, le but du présent projet de recherche est d’évaluer les effets d’un mélange composé d’au moins deux ingrédients actifs de pesticides sur l’activation de la voie de AhR. Les résultats du projet de recherche ont démontré que certaines combinaisons donnent lieu à une activation synergique de la voie AhR alors que d’autres donnent plutôt lieu à une activation additive. Dans le cas où la concentration des ingrédients actifs est élevée, on obtient plutôt un effet inhibiteur. N’est-ce pas paradoxal qu’à faibles doses, il y a un effet soit additif ou synergique alors qu’à de hautes concentrations, l’effet est plutôt inhibiteur ? Il ne faut alors pas croire que de fortes doses de pesticides sont bénéfiques puisque les effets sur les niveaux d’expression des gènes cibles de AhR ne signifient pas qu’il en sera de même pour les tous les autres gènes. Les résultats ont également démontré qu’en présence d’œstrogène, les ingrédients actifs seuls ou en combinaison ont le même effet que le 2,3,7,8-Tétrachlorodibenzo-para-dioxine (TCDD) sur l’interaction croisée entre AhR et le récepteur aux œstrogènes ; l’expression du gène CYP1A1 est réprimée alors que l’expression de CYP1B1 demeure inchangée. Maintenant qu’on comprend bien les effets que peuvent avoir une combinaison d’ingrédients actifs de pesticides sur l’activation AhR, il ne reste plus qu’à comprendre pourquoi certains mélanges donnent lieu à une activation synergique et d’autres additive. Une question bien simple, mais à laquelle il est si difficile de répondre.

Magister Scientiae (Medical Bioscience) - MSc(MBS) / There is increasing concern worldwide by researchers with regards to the assessing of safety and therapeutic consumption of the plants used in traditional medicine. Date palm pollen (DPP) and flax seed have been used traditionally to improve fertility in Libya. DPP extracts have shown several reproductive beneficial effects. In vivo, studies have revealed the ability of DPP to increase sperm concentrations, ameliorate the testicular toxicity induced by cadmium and lead, raise testosterone, as well as LH and FSH hormone levels. Flax seed phytochemical analysis showed lots of valuable components such as lignans and α linolenic acid to which were attributed its positive health effects like antitumor, antioxidant and protective effects against coronary heart diseases. Moreover, flax lignans have both estrogenic and antiestrogenic potential. This study was aimed at testing the effects of Libyan DPP and flax seed on the Sertoli (TM4) cell line and human breast adenocarcinoma (MCF - 7) cell line. Different concentrations (0.01, 0.1, 1, 10, 100 and 1000 μg/ml) of ethanolic extracts of DPP and flax seed, respectively, were used to assess the morphology of TM4 and MCF - 7 cells after 24 and 72 hours exposure. Mitochondrial dehydrogenase activity as a marker of cell viability was measured by MTT assay after 24 and 72 hours exposure. Apoptotic effects were assessed by flow cytometeric APO percentage assay. TM4 cell production of Inhibin - B hormone and GGT enzyme activity under the effects of DPP or flax seed was determined by use of ELISA kits. Transepithelial electrical resistance (TEER) assay were used to detect the effect of DPP or flax seed on TM4 cell monolayer integrity. Finally the plants potential phytoestrogenic activity was determined by use of E - SCREEN assay in MCF – 7 breast cancer cells. Higher concentrations of DPP significantly increased the activity of mitochondrial dehydrogenase enzyme of TM4 cells after 24 hours associated with increasing cell number as detected in a microphotograph. Flax seed concentrations less than 100 μg/ml reduced TM4 cell viability but there were no morphological changes visible after 24 hours. MCF - 7 cells viability was reduced after 24 and 72 hours treatment with DPP and flax seed. DPP concentrations beyond 1 μg/ml significantly raised the TEER of TM4 monolayer over 72 hours while flax seed treatments caused a significant increase only after 72 hours of exposure. TM4 cells GGT activity increased significantly after exposure to higher concentrations of DPP and all flax seed concentrations. Significant stimulatory effects of all the concentrations of DPP or flax seed on TM4 inhibin - B hormone productions have been detected. Apoptotic studies showed no significant changes. E - SCREEN assay resulted in significant reduction in MCF - 7 proliferation rate under the effect of low concentrations of DPP or flax seed. Higher concentrations of the plant extracts, however, stated to increase MCF – 7 cell proliferation, this exerts weak estrogenic activities. In conclusion, the main finding of this study is that DPP and flax seed showed stimulatory effects on TM4 cells proliferation. The resistivity of TM4 cells monolayer which reflect the integrity of blood – testis barrier (BTB) was also significantly increased as well as inhibin - B production and GGT enzyme activity. In addition DPP and flax seed respectively showed inhibitory effects on MCF - 7 cells viability. This study indicated that DPP or flax seed may enhance spermatogenesis through their stimulatory action on Sertoli cells. Moreover, both plants could reduce breast cancer cells viability. However, further investigations are required to elucidate the exact mechanisms behind these obtained findings.

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This diploma thesis is focused on preparation of liposome by relatively new method called nanofluidisation. This method allows the controlled preparation of small unilamellar liposomes in one step. In my thesis I was dealing with optimalization of liposomes preparation which carry hydrophobic cytostatics using this method. Cytotoxic effect of liposomes carrying hydrophobic cytostatics in vitro on cell lines A549 and MCF-7 was determined. In cytotoxicyty test I compared the effect of hydrophobic cytostatics (paclitaxel and derivates of vitamin E specifically alfa-Tos, alfa-TEA) that were incorporated into liposomes prepared via nanofluidisation method and lipid film hydration method. Moreover, a technology of lyophilisation in the presence of cryoprotectants for preparation of liposomes using the method of nanofluidisation was developed.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.