Yes / The gold standard substrate for the ex vivo expansion of human limbal stem cells (LSCs) remains the human amniotic membrane (HAM) but this is not a defined substrate and is subject to biological variabil-ity and the potential to transmit disease. To better define HAM and mitigate the risk of disease transmis-sion, we sought to determine if decellularisation and/or c-irradiation have an adverse effect on culture growth and LSC phenotype. Ex vivo limbal explant cultures were set up on fresh HAM, HAM decellularised with 0.5 M NaOH, and 0.5% (w/v) sodium dodecyl sulfate (SDS) with or without c-irradiation. Explant growth rate was measured and LSC phenotype was characterised by histology, immunostaining and qRT-PCR (ABCG2, DNp63, Ki67, CK12, and CK13). Ƴ-irradiation marginally stiffened HAM, as measured by Brillouin spectromicroscopy. HAM stiffness and c-irradiation did not significantly affect the LSC phe-notype, however LSCs expanded significantly faster on Ƴ-irradiated SDS decellularised HAM (p < 0.05) which was also corroborated by the highest expression of Ki67 and putative LSC marker, ABCG2. Colony forming efficiency assays showed a greater yield and proportion of holoclones in cells cultured on Ƴ-irradiated SDS decellularised HAM. Together our data indicate that SDS decellularised HAM may be a more efficacious substrate for the expansion of LSCs and the use of a c-irradiated HAM allows the user to start the manufacturing process with a sterile substrate, potentially making it safer.

Yes / Cytokines are extremely potent biomolecules that regulate cellular functions and play multiple roles in initiation and inhibition of disease. These highly specialised macromolecules are actively involved in control of cellular proliferation, apoptosis, cell migration and adhesion. This work, investigates the effect of transforming growth factor-beta2 (TGF-β2) on the biological regulation of chondrocyte and the repair of a created model wound on a multilayer culture system. Also the effect of this cytokine on cell length, proliferation, and cell adhesion has been investigated. Chondrocytes isolated from knee joint of rats and cultured at 4 layers. Each layer consisted of 2 × 105 cells/ml with and without TGF-β2. The expression of mRNA and protein levels of TGF-β receptors and Smad1, 3, 4, and 7 have been analysed by RT-PCR and western blot analysis. The effect of different supplementations in chondrocyte cell proliferation, cell length, adhesion, and wound repair was statistically analysed by One-way ANOVA test. Our results showed that the TGFβ2 regulates mRNA levels of its own receptors, and of Smad3 and Smad7. Also the TGF-β2 caused an increase in chondrocyte cell length, but decreased its proliferation rate and the wound healing process. TGF-β2 also decreased cell adhesion ability to the surface of the culture flask. Since, TGF-β2 increased the cell size, but showed negative effect on cell proliferation and adhesion of CHC, the effect of manipulated TGF-β2 with other growth factors and/or proteins needs to be investigated to finalize the utilization of this growth factor and design of scaffolding in treatment of different types of arthritis.

Ovine footrot is a complex multifactorial infectious disease, causing lameness in sheep with major welfare and economic consequences. Dichelobacter nodosus is the main causative bacterium; however, footrot is a polymicrobial disease with Fusobacterium necrophorum, Mycoplasma fermentans and Porphyromonas asaccharolytica also associated. There is limited understanding of the host response involved. The proinflammatory mediators, interleukin (IL)-1 and C-X-C Motif Chemokine Ligand 8 (CXCL8), have been shown to play a role in the early response to D. nodosus in dermal fibroblasts and interdigital skin explant models. To further understand the response of ovine skin to bacterial stimulation, and to build an understanding of the role of the cytokines and chemokines identified, primary ovine interdigital fibroblasts and keratinocytes were isolated, cultured and stimulated. The expression of mRNA and protein release of CXCL8 and IL-1 were measured after stimulation with LPS, D. nodosus or F. necrophorum, which resulted in increased transcript levels of IL-1 and CXCL8 in the M. fermentans-free cells. However, only an increase in the CXCL8 protein release was observed. No IL-1 protein release was detected, despite increases in IL-1 mRNA, suggesting the signal for intracellular pre-IL-1 processing may be lacking when culturing primary cells in isolation. The keratinocytes and fibroblasts naturally infected with M. fermentans showed little response to the LPS, a range of D. nodosus preparations or heat-inactivated F. necrophorum. Primary single cell culture models complement ex vivo organ culture models to study different aspects of the host response to D. nodosus. The ovine keratinocytes and fibroblasts infected with M. fermentans had a reduced response to the experimental bacterial stimulation. However, in the case of footrot where Mycoplasma spp. are associated with diseased feet, this natural infection gives important insights into the impact of multiple pathogens on the host response.

<p dir="ltr">Cancer biological mechanisms are a vastly researched area in the field, yet they are not well understood in the various contexts in which cancer is found. Cancerous tumors often exist in harsh, stressful environments for normal cells, but cancer cells can thrive in these conditions. The tumor microenvironment (TME) typically has low oxygen levels (hypoxia), high acidity, and low nutrition. Exposure to the TME leads to many metabolic changes in the cells, enabling cancer to continue proliferating and migrating. However, these metabolic changes are not well understood, especially at the single-cell level. The ability to monitor cells in real time to determine the physical characteristics they undergo is critical to understanding the impact of these metabolic changes. Conventional methods focus on determining the genomic and proteomic changes in large numbers of cells, which may be overlooked if the changes are homogeneous across samples. In this work, we demonstrate the power of using multiple imaging techniques in combination with biochemical methods to visualize metabolic changes and determine the causes in various cancer cells under extreme hypoxia conditions.</p><p dir="ltr">The changes in the microtubule network that occur under hypoxia at the single-cell level are not widely researched. The use of confocal fluorescence microscopy can determine microtubule polymerization in conjunction with eGFP-transfected EB3, a protein that assists in microtubule polymerization. We have determined that hypoxic HeLa cells produce finger-like protrusions when exposed to hypoxia that help with cell migration and, ultimately, cancer cell metastasis. The formation of these protrusions is facilitated by localized mitochondria activities in the protrusions.</p><p dir="ltr">The metabolic changes in lipid droplets (LDs) under hypoxia at the single-cell level remain an elusive topic. The use of stimulated Raman spectroscopy (SRS) and coherent anti-Stokes Raman scattering (CARS) can determine the quantity and spatial-temporal distribution of LDs in cancer cells. We have found that LDs redistribute to the endoplasmic reticulum (ER) and increase in intensity in hypoxic MIA PaCa-2 and A549 cells. Time-lapse CARS microscopy revealed a release-accumulate process of these LDs on ER in hypoxia. We also studied the impact of carbon sources on LD formation and found that MIA PaCa2 cells prefer direct lipid uptake while glucose is also essential to reduce lipotoxicity. The use of hyperspectral stimulated Raman scattering (hSRS) also reveals that the content of the LDs changes to include less cholesteryl ester and a decrease in lipid saturation level.</p><p dir="ltr">Collectively, these findings shed new light on the understanding of cytoskeleton dynamics and lipid metabolism in hypoxic conditions. The discoveries made within this research would lead to better treatment strategies for effective treatment of hypoxia-resistant cancer cells.</p>

Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Introduction – Due to spatial separation from the native vascular bed, solid tumours develop regions with limited access to nutrients essential for growth and survival. The promotion of a process known as macroautophagy may facilitate in the maintenance of intracellular amino acid levels, through breakdown of cytoplasmic proteins, so that they remain available for macromolecular biosynthesis and ATP production. Several studies point to the potential ability of some cancers to temporarily increase autophagy and thereby prolong cell survival during metabolic stress. The validity of these claims is assessed when a commonly used breast cancer cell line and an epithelial breast cell line are starved of amino acids in this study. Furthermore, we go on to hypothesize that acute amino acid deprivation during treatment will result in an elevated sensitivity of MDAMB231 cells to doxorubicin toxicity but limit its cytotoxic side-effects in MCF12A cells. Methods and study design- Human breast cancer cells (MDAMB231) and breast epithelial cells (MCF12A) cultured in complete growth medium were compared to those incubated in medium containing no amino acids. Steady state autophagy levels were monitored using classical protein markers of autophagy (LC3-II and beclin-1) and the acidic compartmentalization in cells (Lysotracker™ red dye) in conjunction with autophagy inhibition (bafilomycin A1 and ATG5 siRNA). Cell viability was monitored using several techniques, including caspase 3/7 activity. ATP levels were assessed using a bioluminescent assay, while mass spectrometry based proteomics was used to quantify cellular amino acid levels. Similar techniques were used to monitor autophagy during doxorubicin treatment, while cellular doxorubicin localization was monitored using immunofluorescence microscopy. Finally, a completely novel GFP-LC3 mouse tumour model was designed to assess autophagy and caspase activity within tumours in vivo, during protein limitation and doxorubicin treatment. Results - Amino acid deprivation resulted in a transient increase in autophagy at approximately 6 hours of amino acid starvation in MDAMB231 cells. The amino acid content was preserved within these cells in an autophagy-dependent manner, a phenomenon that correlated with the maintenance of ATP levels. Inhibition of autophagy during these conditions resulted in decreased amino acid and ATP levels and increased signs of cell death. MCF12A cells displayed a greater tolerance to amino acid starvation during 24 hours of amino acid starvation. Evidence indicated that autophagy was important for the maintenance of amino acid and ATP levels in these cells and helped prevent starvation-induced cell death. Furthermore, data showed that concomitant amino acid withdrawal resulted in decreased cellular acidity in MDAMB231 cells, and increased acidity in MCF12A cells, during doxorubicin treatment. These changes correlated with evidence of increased cell death in MDAMB231 cells, but a relative protection in MCF12A cells. A novel model was used to apply these techniques in vivo, and although mice fed on a low protein diet during high dose doxorubicin treatment had increased mean survival and smaller tumour sizes, evidence suggested that autophagy is protecting a population of cells within these tumours. Conclusions - This novel approach to tumour sensitization could have several implications in the context of cancer therapy, and given the delicate relationship that autophagy has with the cancer microenvironment, efforts to determine the mechanisms involved in autophagy and sensitization could lead to new and innovative treatment opportunities for cancer management. / AFRIKAANSE OPSOMMING: Inleiding – As gevolg van hul skeiding van die oorpronklike vaskulêre netwerk, ontwikkel soliede gewasse areas met beperkte toegang tot noodsaaklike voedingstowwe. Die bevordering van 'n proses wat as makro-autofagie bekend staan, kan die handhawing van intrasellulêre aminosuur vlakke fasiliteer. Voorafgenoemde proses word waarskynlik deur die afbreek van sitoplasmiese proteïene teweegebring om sodoende vir makro-molekulêre biosintese en ATP produksie beskikbaar te kan wees. Verskeie studies dui daarop dat sommige kankersoorte die vermoë het om autofagie tydelik te verhoog, en daarby sel oorlewing gedurende metaboliese stress te verleng. Die geldigheid van hierdie eise word evalueer wanneer 'n algemeen beskikbare borskanker sellyn, en 'n borsepiteelsellyn in hierdie studie van aminosure verhonger word. Verder, veronderstel ons dat akute aminosuur ontneming gedurende behandeling 'n verhoogde sensitiwiteit van MDAMB231 selle tot doxorubicin toksisiteit tot gevolg sal hê, maar terselfdetyd die middel se sitotoksiese newe-effekte in MCF12A selle sal beperk. Metodes en studie ontwerp – Menslike borskanker- (MDAMB231) en bors epiteel selle (MCF12A) wat in volledige groeimedium gekweek is, is vergelyk met selle wat in aminosuur vrye medium gekweek is. Basislyn autofagie-vlakke is gemonitor deur die gebruik van klassieke autofagie proteïen merkers (LC3-II en beclin-1) en die asidiese kompartementalisering in selle (Lysotracker™ rooi kleurstof) saam met autofagie inhibisie (bafilomycin A1 and ATG5 siRNA). Sellewensvatbaarheid is deur die gebruik van verskeie tegnieke, insluitend caspase 3/7 aktiwiteit, gemonitor. ATP-vlakke is deur die gebruik van 'n bioluminiserende tegniek gemeet, terwyl massa-spektrometrie-gebaseerde “proteomics” gebruik is om sel aminosuur vlakke te kwantifiseer. Soortgelyke tegnieke is gebruik om autofagie gedurende doxorubicin behandeling waar te neem, terwyl sellulêre doxorubicin lokalisasie deur die gebruik van immunofluoresensie mikroskopie gemonitor is. Ten slotte, is 'n unieke GFP-LC3 muismodel in hierdie studie ontwikkel. Hierdie model is gebruik om autofagie en caspase aktiwiteit in gewasse in vivo te bestudeer tydens proteïen beperking en doxorubicin behandeling. Resultate – Aminosuur ontneming het tot 'n tydelike verhoging in autofagie na ongeveer 6 ure van aminosuur verhongering in MDAMB231 selle gelei. Die aminosuur inhoud van hierdie selle het op 'n autofagie-afhanklike manier behoue gebly. Hierdie verskynsel het met die handhawing van ATP-vlakke gekorreleer. Autofagie inhibisie gedurende hierdie kondisies het 'n verlaging in aminosuur en ATP-vlakke teweeggebring, sowel as vermeerderde tekens van seldood tot gevolg gehad. MCF12A selle het 'n groter toleransie tot aminosuur verhongering tydens die 24 uur aminosuur verhongeringsperiode getoon. Getuienis het aangedui dat autofagie belangrik vir die handhawing van aminosuur en ATP-vlakke in hierdie selle was, en gehelp het om verhongerings-geïnduseerde seldood te voorkom. Verder het data gewys dat aminosuur ontrekking tot verminderde sellulêre asiditeit in MDAMB231 selle, en verhoogde asiditeit in MCF12A selle gedurende doxorubicin behandeling gelei het. Hierdie veranderinge stem ooreen met getuienis van toenemende seldood in MDAMB231 selle, maar 'n relatiewe beskerming in MCF12A selle. 'n Unieke model was gebruik om hierdie tegnieke in vivo toe te pas. Alhoewel verhoogde oorlewing en kleiner gewasse in muise op 'n lae proteïen dieet gedurende hoë dosis doxorubicin behandeling opgemerk is, het bewyse voorgestel dat autofagie 'n populasie selle binne die gewasse beskerm. Gevolgtrekkings – Hierdie unieke benadering tot tumor sensitisering kan verskeie implikasies in die konteks van kanker behandeling hê. Gegewe die delikate verhouding van autofagie met die kanker mikro-omgewing, kan pogings om die meganismes betrokke in autofagie en sensitisering te bepaal, tot nuwe en innoverende behandelings vir kanker lei.

CD2AP is a member of the CD2AP/CIN85 family of adaptors and involved in several cellular processes, such as kidney podocyte development, actin mediated membrane trafficking and T cell activation. It contains three SH3 domains whose binding properties and interaction partners remain largely unexplored. The CD2AP SH3 interaction with the novel partner Rab5-activating GEF RIN3 was studied extensively by isothermal titration calorimetry (ITC), peptide scanning arrays, mutagenesis and X-ray crystallography. Mapping of the interaction regions showed that human RIN3 contains two binding sites for the CD2AP SH3 domains. From these studies, the CD2AP SH3 recognition motif P-x-P/A-x-x-R emerged. Two crystal structures (1.65 &Aring; and 1.2 &Aring;) of the SH3 1 and SH3-2 domains in complex with RIN3 epitopes 1 and 2 respectively revealed that these residues serve as anchoring points. With the aid of bioinformatics tools, this motif was used to conduct a peptide array-based screen for additional signalling partner candidates. One of the hits was the Arf-GAP ARAP1. ITC data indicate that the three SH3 domains differentially recognise three ARAP1 epitopes, with the first ARAP1 epitope binding to SH3-2 in the nanomolar range. A crystal structure (1.6 &Aring;) of the SH3-2 domain in complex with the first ARAP1 epitope implicates two additional anchoring residues that extend beyond the PPII helical region of the canonical motif. The CD2AP/ARAP1 interaction was confirmed in podocytes and cancer cells at the endogenous protein level. Even though RIN3 and ARAP1 are involved in membrane trafficking, a direct link to CD2AP had not been reported before. Other candidates from the peptide array analyses were also investigated by ITC. In conclusion, this study led to the elucidation of the CD2AP SH3-1 and SH3-2 domain binding signatures and the identification of putative novel binding partners for all three SH3 domains. Lastly, insight was gained into the binding preferences of CD2AP SH3-3.

Unlike thin tissues (e.g., skin) which has been successfully grown, growing thick tissues (e.g., bone and muscle) still exhibit certain limitations due to lack of nutrients (e.g., glucose and oxygen) feeding on cells in extracapillary space (ECS) region, or also known as scaffold in an in vitro static culture. The transport of glucose and oxygen into the cells is depended solely on diffusion process which results in a condition where the cells are deprived of adequate glucose and oxygen supply. This condition is termed as hypoxia and leads to premature cell death. Hollow fibre membrane bioreactors (HFMBs) which operate under perfusive cell culture conditions, have been attempted to reduce the diffusion limitation problem. However, direct sampling of glucose and oxygen is almost impossible; hence noninvasive methods (e.g., mathematical models) have been developed in the past. These models have defined that the glucose diffusivity in cell culture medium (CCM) is similar to the diffusivity in water; thus, they do not represent precisely the nutrient transport processes occurring inside the HFMB. In this research, we define glucose as our nutrient specie due to its limited published information with regard to its diffusivity values, especially one that corresponds to cell/tissue engineering (TE) experiments. A series of well-defined diffusion experiments are carried out with TE materials of varying pore size and shapes imbibed in water and CCM, namely, cellulose nitrate (CN) membrane, polyvinylidene fluoride (PVDF) membrane, poly(L-lactide) (PLLA) scaffold, poly(caprolactone) (PCL) scaffold and collagen scaffold. A diffusion cell is constructed to study the diffusion of glucose across these materials. The glucose diffusion across cell-free membranes and scaffolds is investigated first where pore size distribution, porosity and tortuosity are determined and correlated to the effective diffusivity. As expected, the effective diffusivity increases correspondingly with the pore size of the materials. We also observe that the effective glucose diffusivity through the pores of these materials in CCM is smaller than in water. Next, we seeded human osteoblast cells (HOSTE85) on the scaffolds for a culture period of up to 3 weeks. Similar to the first series of the diffusion experiments, we have attempted to determine the effective glucose diffusivity through the pores of the scaffolds where cells have grown at 37°C. The results show that cell growth changes the morphological structure of the scaffolds, reducing the effective pore space which leads to reduced effective diffusivity. In addition, the self-diffusion of glucose in CCM and water has also been determined using a diaphragm cell method (DCM). The results have shown that the glucose diffusivity in CCM has significantly reduced in comparison to the water diffusivity which is due to the larger dynamic viscosity of CCM. The presence of other components and difference in fluid properties of CCM may also contribute to the decrease. We finally employ our experimentally deduced effective diffusivity and self-diffusivity values into a mathematical model based on the Krogh cylinder assumption. The glucose concentration is predicted to be the lowest near the bioreactor outlet, or in the scaffold region, hence this region becomes a location of interest. The governing transport equations are non-dimensionalised and solved numerically. The results shown offer an insight into pointing out the important parameters that should be considered when one wishes to develop and optimise the HFMB design.

The use of chemotherapy in the treatment of cancer has stimulated the demand for better chemotherapeutic agents that are more potent at destroying tumor cell populations and more selective for the specific tumor versus normal host tissues. This project is directed at discovering new anti-tumor agents that are effective against breast cancer based on structures derived from marine organisms, specifically brominated pyrroles. We utilized an in vitro breast cancer model to study the effects of pyrroles on tumor proliferation and survival, as well as growth arrest and cell death. Our findings indicate that the substituted pyrrole JG-03-14 induces time dependent cell death in breast tumor cells where the cell death involves apoptosis and autophagy. Residual growth arrest in p53 wild type cells is characteristic of senescence. JG-03-14 also demonstrated substantial anti-proliferative effects in multi-drug resistant cells. These findings indicate JG-03-14 would potentially be developed for the treatment of breast cancer.

Platinum-based drugs are an important group of alkylating-like agents which are used in cancer chemotherapy treatment. Cisplatin and oxaliplatin in particular are still commonly used today and are the focus of this thesis. As with most chemotherapy drugs, the efficacy of these drugs are limited by toxicity as well as tumour resistance, and therefore by increasing our understanding of these areas it is hoped to one day achieve personalised chemotherapy. The use of ICP-MS in the study of bio-sciences is still relatively new, however it has the ability to provide robust, fast and accurate methods for the quantification of platinum in biological samples. The research presented here utilised mass spectrometry in the study of the formation of Pt-DNA adducts in the clinical samples, the binding of oxaliplatin to short peptides and the effect of selenium supplementation on oxaliplatin in colorectal cancer cell lines. A comparison in the number of Pt-DNA adducts in saliva and leukocyte samples obtained from patients undergoing Pt-based chemotherapy demonstrated a lack of correlation between the two sample types. Samples were taken pre- and post-treatment and analysed via SF-ICP-MS and significant inter-patient variability was observed as expected. In both leukocyte and saliva samples, not only was Pt from previous chemotherapy cycles observed, but Pt was detected in the DNA in both sample types 1 hour after treatment. However a lack of correlation between platinum levels seen in the blood and saliva, combined with unexpected difficulties obtaining patient adherence to the saliva sampling protocol, indicated that saliva does not at present offer a reliable alternative to leukocytes for this assay. The binding of oxaliplatin to short nitrogen and sulfur rich peptides was investigated. Platinum binding to the peptides was observed and no significant differences in the level of binding were observed between the range of N and S rich peptides studied in this investigation. Partly due to the inability to reproduce biological conditions in this study, oxaliplatin was observed as a whole molecule, and furthermore dimers and multimers were also observed. The effect of selenium supplementation on the total cellular uptake of platinum was investigated in cultured cells via ICP-MS and LA-ICP-MS. It was observed that selenium decreased the amount of Pt taken up by the cancer cells. This was seen in analysis of populations of cells as well as by single cell analysis. Furthermore, while problems were encountered measuring selenium in subcellular experiments, the effect of selenium on the subcellular distribution of platinum as well as the number of Pt-DNA adducts could be determined.

Comparou-se a eficácia da baciloscopia in natura e pós concentração (Coloração Ziehl Neelsen) e cultura tradicional e automatizada MA de amostras de escarro ou saliva/muco para a detecção do Complexo M. tuberculosis (MTB) em Centro de Referencia Ambulatorial para Tuberculose na Cidade de São Paulo. A identificação do grupo MTB baseou-se no crescimento em meio com Ácido para nitrobenzóico, e visualização do fator corda. Do total de 374 amostras, 228 eram de pacientes sob diagnóstico inicial e 146 em tratamento. 83 amostras eram saliva/muco. Destas, sete foram positivas à baciloscopia, 03 (3,6%) no material concentrado e uma (1,2%) in natura (p=0.5 McNemar Test). Cinco amostras (6%) positivas na cultura pelo método tradicional e 07 (8,4%) p=0.5 pelo MA. As amostras de escarro eram 74 foram positivas na baciloscopia, sendo 34 (11,7%) in natura e 45 (15,5%) no material concentrado, p=0.001. No método tradicional 56 (19,2%) e 67 (23%) pelo MA p=0.001. Independente da característica da amostra a baciloscopia pós concentração e a cultura pelo MA foram mais sensíveis. / Effectiveness of bacilloscopy were compared in natura and pos-concentrated (Ziehl Neelsen staining), and tradicional and automated culture-AM of sputum or saliva/mucus samples for detection of M. tuberculosis complex (MTB) in Ambulatory Reference Center in the city of São Paulo. Identification of MTB group was based on growth in medium with para nitrobenzoic acid, and cord factor visualization. From the total 374 samples, 228 were from patients under initial diagnostic and 146 in treatment. 83 samples were saliva/mucus. Seven of these were bacilloscopy positive, 03 (3.6%) in concentrated material and one (1.2%) in natura (p=.500ª McNemar Test). Five samples (6%) positive for culture by traditional method and 07 (8.4%) p=0.5 for AM. For sputum 74 were bacilloscopy positive, being 34 (11.7%) in in natura and 45 in concentrated material, p=0.001. In traditional method, 56 (19.2%) and 67 (23%) for AM, p=0.001. Independently of sample characteristics, pos-concentrated bacilloscopy and culture by AM were more effectiveness.