240 research outputs found
The effects of selected therapeutic agents on cell cytotoxicity and Her-2 receptor expression using culturedbreast adenocarcinoma models
No full textDissertation (MSc)--University of Pretoria, 2013.Introduction: Epidemiological studies suggest that at least 1 in 29 South African women will be
diagnosed with breast cancer in their lifetime. Breast cancer is not a single disease. The
heterogeneity of breast cancer results in four distinct molecular subtypes including aggressive
human epidermal growth factor receptor-2 (Her-2) positive, where Her-2 receptors are overexpressed.
Trastuzumab (Herceptin®), is a recombinant, humanized, anti-Her-2 monoclonal antibody
that specifically targets subdomain IV of the extracellular domain of the Her-2 receptor and has
dramatically altered the prognosis of Her-2 positive breast cancer. Trastuzumab is, however,
associated with problems such as primary and acquired resistance, which has prompted
investigation into improving its efficacy.
Aim: To investigate the ability of selected therapeutic agents to alter in-vitro cell viability, cell cycling,
apoptosis and Her-2 expression in models of Her-2-positive and oestrogen receptor positive, Her-2
negative breast adenocarcinoma and bring about an alteration in the efficacy of trastuzumab.
Methods: MCF-7 cells which retain the ability to process oestrogen, and SK-Br-3 cells which overexpress
Her-2 gene products were used. Cells were exposed to trastuzumab, aspirin, calcipotriol,
doxorubicin, epidermal growth factor (EGF-human), geldanamycin, heregulin-β1 and β-oestradiol as
single agents and in combination with trastuzumab. Research methodologies included tetrazolium
conversion assay for cell viability, AMC-substrate cleavage and annexin-V for apoptosis, propidium
iodide staining for cell cycle analysis and anti-Her-2 affibody molecule for relative Her-2 receptor
density.
Results: Cell survival of 95.39% (±2.69) for MCF-7 cells and 74.17% (±1.60) for SK-Br-3 cells was
observed following trastuzumab (100 μg/ml) exposure. Trastuzumab resulted in statistically
significant G1 phase accumulation in MCF-7 cells at 72 hours and in SK-Br-3 cells from 24 hours.
Furthermore, trastuzumab decreased relative Her-2 receptor density in SK-Br-3 cells by
approximately 35% by 24 hours but had no effect in MCF-7 cells. The anti-proliferative effects of
trastuzumab were abrogated by EGF, a Her-1 ligand and heregulin-β1, a Her-3 and Her-4 ligand. Most agents altered distribution throughout the phases of cell cycle to a certain degree, with the G1
phase accumulation observed for trastuzumab being potentiated in some combinations. Most of the
agents, with the exception of doxorubicin and geldanamycin, did not promote apoptosis and
appeared instead to be anti-proliferative. Geldanamycin had the greatest effect on Her-2 receptor
density (approximately 80% by 24 hours) followed by EGF, heregulin and trastuzumab, with the
biological molecules in combination with trastuzumab producing a further significant reduction.
Conclusion: Endogenous Her-receptor ligands (EGF and heregulin) differentially altered the viability
parameters for trastuzumab which could play a role in the emergence of clinical resistance to
targeted therapy. Doxorubicin with concurrent trastuzumab significantly reduced cell viability
compared to each single agent in both cell lines. Furthermore, the cytostatic and cytotoxic abilities
of each of the other agents either mimicked trastuzumab alone or the selected agent alone when
exposed concurrently.gm2014PharmacologyUnrestricte
Giving Miss Marple a makeover : graduate recruitment, systems failure and the Scottish voluntary sector
No full textThe voluntary sector in Scotland, as across the globe, is becoming increasingly business like. Resultantly, there is an increasing demand for graduates to work in business and support functions. In Scotland, however, despite an oversupply of graduates in the labor market, the voluntary sector reports skills shortages for graduate-level positions; a leadership deficit was also reported in countries such as the United States. Through exploratory, mainly qualitative, case study and stakeholder research, this article proposes that one reason for this mismatch between the supply of and demand for graduates is a systems failure within the sector. Many graduates and university students remain unaware of potentially suitable paid job opportunities, in part because of the sector's voluntary label. To rectify this systems failure, thought needs to be given to the sector's nomenclature and the manner in which voluntary sector organizations attract graduate recruits, for example, through levering value congruence in potential recruits
Proteomic assessment of potential in vitro hepatotoxicity models
No full textThesis (PhD)--University of Pretoria, 2016.Scientifically credible and valid biological systems are essential in pharmaceutical research and development. Standardizing in vitro preclinical hepatotoxicity is confounded by the diversity of origin of cells and the ability to retain hepatocellular functions. Key determinants of valid hepatotoxicity models are resemblance to primary human hepatocytes (PHHs), adaptability to high-throughput screening and biological applicability. Numerous in vitro models, including immortalized cell lines and hepatocyte-like cells (HLCs) derived from induced pluripotent stem cells (iPSCs), attempt to reflect features of PHH. Additional influencing factors are the mechanical and geometric environment which dictate functionality and suggest a role for spatial organization as a requirement for mimicking PHH. As there is poor correlation between the cellular genome and proteome, assessing the hepatic phenotypes using proteomics is essential to capture functional cellular responses. The aim of this research was to determine proteomic differences between PHHs and differentially cultured and sourced human hepatocyte-derived cell lines or differentiated HLCs. Additionally, hepatocyte models were used to generate non-specific, proteome-wide information associated with exposure to selected known hepatotoxins to identify potential proteomic signatures of hepatotoxicity.PharmacologyPhDUnrestricte
The in vitro influences of epidermal growth factor and heregulin-β1 on the efficacy of trastuzumab used in Her-2 positive breast adenocarcinoma
No full textBACKGROUND: Human epidermal growth factor receptor-2 (Her-2) is over expressed in approximately 25-30% of all primary breast tumors resulting in a distinctive breast cancer subtype associated with a poor prognosis and a decrease in overall survival. Trastuzumab (Herceptin®), an anti-Her-2 monoclonal antibody, has dramatically altered
the prognosis of Her-2 positive breast cancer. Trastuzumab is, however, associated with primary and acquired resistance.
AIM AND METHODS: To investigate the in-vitro effects of trastuzumab on cell viability (tetrazolium conversion assay), cell cycling (propidium iodide staining), apoptosis (executioner caspases and annexin-V) and relative surface Her-2 receptor expression (anti-Her-2 affibody molecule) in Her-2-positive (SK-Br-3) and oestrogen receptor positive (MCF-7) breast adenocarcinoma cells and to determine potential augmentation of these effects by two endogenous ligands, epidermal growth factor (EGF) and heregulin-β1 (HRG- β1).
RESULTS: Cell viability was decreased in SK-Br-3 cells by exposure to trastuzumab. This was associated with G1 accumulation and decreased relative surface Her-2 receptor density, supporting the cytostatic nature of
trastuzumab in vitro. SK-Br-3 cells exposed to EGF and heregulin-β1 produced differential cell responses alone and in combination with trastuzumab, in some instances augmenting cell viability and cell cycling. Relative surface Her-2 receptor density was reduced substantially by trastuzumab, EGF and heregulin-β1. These reductions were
amplified when ligands were used in combination with trastuzumab.
CONCLUSION: Cell type specific interactions of endogenous ligands appear to be dependent on absolute Her-receptor expression and cross activation of signaling pathways. This supports the notion that receptor density of Her-family members and multiplicity of growth ligands are of mutual importance in breast cancer cell proliferation and therefore also in resistance associated with trastuzumab.The authors would like to acknowledge Roche Pharmaceuticals for the
donation of trastuzumab and the Cancer Association of South Africa
(CANSA) and the Research and Development Programme (RDP), University of Pretoria, for providing funding.http://www.cancerci.com/content/13/1/97am2014ay201
Human epidermal growth factor receptor 2-positive breast cancer : which cytotoxic agent best complements trastuzumab's efficacy in vitro?
No full textINTRODUCTION: Despite trastuzumab having enhanced selectivity for human epidermal growth factor receptor 2 (HER-2) overexpressing breast cancer cells, treatment is hampered by interindividual variation and tumors with high mitogenic potential. The lack of significant clinical benefit in certain patient cohorts suggests that HER-2 expression is ineffective as a sole prognostic indicator of response to therapy. Therefore, optimizing the clinical role of trastuzumab in drug combinations remains critical for clinical success.
AIM: To investigate the effects of trastuzumab in combination with either doxorubicin or geldanamycin on in vitro cell viability, cell cycling, apoptosis and relative HER-2 expression in HER-2-positive (SK-BR-3) and estrogen receptor-positive (MCF-7) breast adenocarcinoma models.
RESULTS: HER-2-rich SK-BR-3 cells demonstrated a greater sensitivity to the effects of doxorubicin than MCF-7 cells. Concurrent trastuzumab exposure resulted in a further reduction in cell viability. This decreased cell viability induced by doxorubicin was associated with activation of executioner caspases as well as with alterations in cell-cycle kinetics, primarily promoting S-phase accumulation. Doxorubicin had no effect on surface HER-2 density expression. Geldanamycin reduced cell viability significantly greater in SK-BR-3 than MCF-7 cells, and was associated with G2 cell-cycle accumulation. The addition of trastuzumab did not augment these effects. Geldanamycin promoted substantial reductions in relative surface HER-2 density in SK-BR-3 cells.
CONCLUSION: The in vitro data supported the rationale for using doxorubicin in trastuzumab-based therapies. Therefore, despite the incidence of cardiotoxicity, doxorubicin could retain a fundamental role in treating HER-2-positive breast cancer. While geldanamycin is a potent cytotoxic agent, its concurrent use with trastuzumab requires further research into the transient or permanent nature of alterations in HER-2 status in cell progeny.The authors would like to acknowledge Roche Pharmaceuticals for the kind donation of trastuzumab and the Cancer Association of South Africa (CANSA) as well as the Research and Development Program (RDP), University of Pretoria, for providing their generous funding, and to Dr AD Cromarty and Dr JJ van Tonder without which this project would not have been possible.http://www.dovepress.comam201
Characterisation of inducible proteins in the HepG2 cell line proteome
No full textThesis (PhD (Pharmacology))--University of Pretoria, 2022.Rationale: Attaining scientifically robust, clinically representative, and functionally translatable preclinical models for pharmaceutical research has become increasingly desirable. Relevant hepatic models are pivotal for the screening and accurate identification of safe new drug candidates. Currently, primary human hepatocytes are considered the gold standard for hepatic modelling However, their limited accessibility, high variability and terminal nature hamper their widespread adoption. Immortalised cell cultures, for example the human hepatoma cell line (HepG2), have served as widespread substitutes. However, the representativeness of HepG2 cells is of concern due to the relatively large phenotypic disparity when compared to primary human hepatocytes. Recent studies have postulated that the adoption of HepG2 cells into three dimensional (3D) conformations may promote a more closely correlated phenotype. Little is known regarding the extent of proteomic changes which occur when cells as cultured as spheroids, nor is there sufficient insight into the temporal dynamics which may drive these changes. The aim of this study was to investigate whether proteomic changes occur in HepG2 cells when cultured as spheroids compared to traditional monolayer culture techniques and if so whether these changes were culture time dependent. Additionally, HepG2 spheroid cultures were assessed for improved metabolic adaption following extended-time spheroid-based culture in the presence of specific drugs, a key characteristic of human hepatocytes that would be critical for hepatotoxicity testing models.
Methods: HepG2 cells were cultured as spheroids using two established 3D culture models; cells were seeded into hanging drop plates (20 000 cells/spheroid) and into 81 well, 3D micro moulds (1000 cells/spheroid), then maintained under untreated control or drug exposed groups, using a 7-drug cocktail representing substrates of phase I metabolizing enzymes. Spheroid cultures were characterized for protein content, size, viability, and immunohistochemistry. Cultures from different groups and culture times were compared by means of quantitative proteomic methods using isobaric labelling proteomics. Sample proteins were reduced, alkylated, precipitated, digested with trypsin and labelled using 6-plex tandem mass tag (TMT) isobaric labels. Proteomic analysis was performed using a Dionex Ultimate 3000 RSLCnano liquid chromatography system coupled to a Thermo Scientific Fusion Lumos Orbitrap Mass Spectrometer. Protein identification was conducted using an alternating SequestHT/MS Amanda schema. Relative protein quantitation for the different spheroid groups were expressed relative to original monolayer cultured HepG2 control cells. Relative metabolic competences of spheroid cultures were assessed using a defined hepatic metabolism phenotyping cocktail using an in-house developed liquid chromatography tandem mass spectrometry method.
Results: HepG2 spheroid cultures were shown to progressively upregulate expression of hepatic marker proteins over the 28-day culture time course. Quantitative proteomic methods initially identified over 5000 proteins which when filtered to include only proteins with at least three identified peptides and presence in all replicates, reduced to approximately 4800 proteins. Identified proteins showed differential expression between spheroid culture groups compared to monolayer cultures. Additionally, progressive differential proteome expression was evident between spheroid cultures according to time spent in culture and based on presence or absence of drug exposure. Thorough bioinformatics analyses revealed insights into the potential mechanisms responsible for the proteomic differences and identified that while spheroids exposed to drug appear to adapt metabolically to their culture environments, the nature of these changes may lack impact for clinical relevance. Metabolomic investigations corroborated the proteomic findings showing that, while maintaining some capacity for phase I metabolism, overall, clinically relevant metabolism for cytochrome P450 enzymes was absent.
Conclusion: Quantitation of large protein cohorts demonstrated that there are adaptive changes occurring during long-term culture of HepG2 cell spheroids. While the proteomic changes observed, under all culture conditions, do not support adoption of these cultures for modelling drug metabolism, use in other bespoke model systems which concern other aspects of liver function is feasible. The high-quality proteomic data generated shows temporal dynamics in spheroid cultures which have implications for their utility. It necessitates the requirement for more comprehensive investigations into the relevance of the time-points used when adopting spheroid cultures.NRF fundingPharmacologyPhD (Pharmacology)Unrestricte
A “Choose Your Own Adventure” Recipe for Teaching Scholarly Communications Concepts
No full textThis recipe was developed as a way to walk graduate students and faculty through the complex topic of scholarly communications, including publishing models, assessing journal quality, funder open access policies, and author rights. The recipe takes the form of an interactive online story using the open source tool Twine and can be customized
or different disciplinary groups with ease
The generation of human induced pluripotent stem cell lines from individuals of Black African ancestry in South Africa
No full textThe lack of equitable representation of African diversity in scientific resources, such as genome-wide association studies and human induced pluripotent stem cell (hiPSC) repositories, has perpetuated inequalities in the advancement of health research. HiPSCs could be transformative in regenerative and precision medicine, therefore, the generation of diverse lines is critical in the establishment of African-relevant preclinical cellular models. HiPSC lines were derived from two healthy donors of Black African ancestry using Sendai virus reprogramming of dermal fibroblasts, and characterised to confirm stemness markers, trilineage differentiation, and genetic integrity. These hiPSCs represent a valuable resource for modelling African relevant disease biology
Learner identity and resettlement in Australia : experiences of adults in the Sudanese community
No full textThis thesis was scanned from the print manuscript for digital preservation and is copyright the author.
Researchers can access this thesis by asking their local university, institution or public library to
make a request on their behalf. Monash staff and postgraduate students can use the link in the References field
Is Switzerland doing enough regarding the prevention of syphilis, gonorrhea and chlamydia infections ?
No full textSexually transmitted infections are a major problem for medicine and for public health services worldwide. More than 30 sexually transmittable pathogenic micro-organisms are known, including bacteria, viruses, fungi, protozoa and ectoparasites. According to estimates from the World Health Organisation more than 333 million of bacterial sexually transmitted infections occur worldwide per year. Sexually transmitted infections, by their nature, affect individuals, within partnerships and larger sexual networks, and in turn populations.
This report focuses on three bacterial sexually transmitted infections in Switzerland that are Chlamydia trachomatis, Neisseria gonorrhea and Treponema pallidum (syphilis) in Switzerland. The prevalence of these infections has been increasing alarmingly for a decade. All three infections can be asymptomatic and their diagnosis and treatment can therefore occur too late or worse not at all, even though treatments are available. This is an important problem as untreated sexually transmitted infections may cause complications such as ascending infections, infertility, ectopic pregnancies and serious long-term neurological sequels. The consequences of these infections should not be underestimated. They constitute a significant public health burden as well as serious financial burden.
The increases in chlamydia, syphilis and gonorrhea infections have also been observed in many European countries. Countries, where rising numbers of sexually transmitted infections have been observed, have reacted in different ways. Some have developed clinical guidelines or implemented screening programs, while others are still in their observational phase.
The aim of this mémoire is to assess whether Switzerland is doing enough regarding the prevention of chlamydial, syphilis and gonorrheal infections. After first describing the infections, surveillance systems of sexually transmitted infections are assessed, then the epidemiological trends of these three infections are described, and finally the prevention measures implemented in Switzerland to respond to the increasing number of infections are described. The reaction of the United Kingdom to the same problem is reported for comparison. [Author, p. 7
- …
