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Yes / Metastatic melanoma, a deadly form of skin cancer, often develops resistance to the BRAF inhibitor drug vemurafenib, highlighting the need for understanding the underlying mechanisms of resistance and exploring potential therapeutic strategies targeting integrins and TGF-β signalling. In this study, the role of integrins and TGF-β signalling in vemurafenib resistance in melanoma was investigated, and the potential of combining vemurafenib with cilengitide as a therapeutic strategy was investigated. In this study, it was found that the transcription of PAI1 and p21 was induced by acquired vemurafenib resistance, and ITGA5 levels were increased as a result of this resistance. The transcription of ITGA5 was mediated by the TGF-β pathway in the development of vemurafenib resistance. A synergistic effect on the proliferation of vemurafenib-resistant melanoma cells was observed with the combination therapy of vemurafenib and cilengitide. Additionally, this combination therapy significantly decreased invasion and colony formation in these resistant cells. In conclusion, it is suggested that targeting integrins and TGF-β signalling, specifically ITGA5, ITGB3, PAI1, and p21, may offer promising approaches to overcoming vemurafenib resistance, thereby improving outcomes for metastatic melanoma patients.

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Thesis (MScMedSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Mycobacterium tuberculosis is the etiological agent for tuberculosis, an infectious disease which is one of the leading causes of morbidity and mortality in developing countries. The emergence of drug resistant tuberculosis has negatively impacted the efficacy of current treatment regimens and threatens to undermine tuberculosis control programs worldwide. Rifampicin forms the backbone of the World Health Organization’s recommended treatment regimen for the treatment of drug susceptible tuberculosis. Resistance to rifampicin is caused by mutations in the 81 bp core region of the rpoB gene which encodes the β subunit of RNA polymerase. Numerous studies have shown that mutations at codons 531 and 526 are the most frequent in clinical isolates, yet little is known concerning the mechanistic effect of these mutations on the fidelity of RNA polymerase. In the present study, we aimed to determine the influence of rpoB mutations on the gene expression profile of M. tuberculosis cultured in vitro. To accomplish this, rifampicin resistant clinical isolates and spontaneous mutants (selected in vitro from H37Rv and a drug-sensitive clinical strain) harbouring rpoB H526Y and S531L mutations were subjected to whole genome sequencing and genome-wide transcriptional profiling. When comparing the transcription profile of H37Rv to the in vitro rpoB mutants, a large proportion of the differentially expressed genes were found to encode for proteins involved in intermediary metabolism and respiration; and cell wall and cell processes. The majority of these differentially expressed genes were downregulated. Prominent differential expression in the same functional categories was also evident when comparing the clinical isolates with these mutations; however, a greater number of genes were differentially expressed in this case. Furthermore, expression of genes that are part of the WhiB7 regulon were found to be upregulated in the rpoB526 mutants, and downregulated in the rpoB531 mutants. These findings indicate that both the position of the rpoB mutation, as well as the genetic background of the strain, play an important role in the gene expression profile of rpoB mutants. Surprisingly, transcriptional profiling of cultures that were exposed to the critical concentration of rifampicin for 24 hours did not exhibit significant differential gene expression. Whole genome sequencing, followed by bioinformatic analysis, revealed that the in vitro mutants harbour synonymous and non-synonymous single nucleotide polymorphisms in addition to the respective rpoB mutations. This suggests that the mycobacterial genome is constantly evolving, challenging previous assumptions of relatively static mycobacterial genomes. The findings from this research have provided novel insight into understanding the influence of resistance-conferring mutations on the biology of M. tuberculosis and have shown that further studies are urgently needed to better understand the complex physiology of this pathogen. This knowledge will be critical for the success of future drug development endeavours. / AFRIKAANSE OPSOMMING: Mycobacterium tuberculosis is die etiologiese agent vir tuberkulose, een van die grootste oorsake van morbiditeit en sterftes in ontwikkelende lande. Die verskyning van middelweerstandige tuberkulose het 'n negatiewe impak op die effektiwiteit van die huidige behandeling van tuberkulose en dreig om tuberkulose beheerprogramme wêreldwyd te ondermyn. Weerstandigheid teen rifampisien, een van die eerste-lyn anti-tuberkulose middels, word veroorsaak deur mutasies in die 81 bp kerngedeelte van die rpoB geen. Die mees algemene mutasies in kliniese isolate word gevind in kodons 531 en 526; alhoewel daar min inligting beskikbaar is oor die effek van hierdie mutasies op die funksie van RNS polimerase. Die doel van hierdie studie was om die effek van verskillende rpoB mutasies op die geen-uitdrukkingsprofiel van M. tuberculosis te bepaal. Rifampisien-weerstandige kliniese isolate en in vitro mutante (geselekteer vanaf H37Rv en 'n rifampisien-sensitiewe kliniese isolaat) met rpoB H526Y en S531L mutasies was vir hierdie doel geselekteer en gebruik om die heel genoom volgorde te bepaal en geenuitdrukking te kwantifiseer. Vergelyking van die H37Rv transkripsieprofiel met in vitro geselekteerde rpoB mutante het getoon dat 'n groot aantal gene wat differensieel uitgedruk is, vir proteïene kodeer wat betrokke is in selwand-prosesse en intermediêre metabolisme en respirasie. Die meerderheid van hierdie differensieel uitgedrukte gene was afgereguleer. ’n Soortgelyke verskynsel is ook waargeneem in kliniese isolate, met die verskil dat 'n groter aantal gene in hierdie geval differensieel-uitgedruk was. Gene wat deel vorm van die WhiB7 regulon is opgereguleer in die rpoB526 mutante, terwyl dit afgereguleer was in die rpoB531 mutante. Hierdie resultate is ’n baie sterk aanduiding dat beide die posisie van die rpoB mutasie, asook die genetiese agtergrond van die organisme, ’n belangrike rol speel in die uitdrukking van gene in rifampisin weerstandige M. tuberculosis. Kulture wat aan die kritiese konsentrasie van rifampisien vir 24 uur blootgestel is, het teenverwagting geen differensiële geen-uitdrukking getoon nie. Verder het die heel genoom volgorde bepaling van die in vitro mutante getoon dat sinonieme en nie-sinonieme enkel-nukleotied polimorfismes teenwoordig is in die onderskeie rpoB mutante. Dit dui daarop dat die mikobakteriële genoom voortdurend verander, moontlik nie so stadig as wat voorheen vermoed is nie. Die bevindinge van hierdie navorsing bied nuwe insigte om die invloed van mutasies wat middel-weerstandigheid veroorsaak op die biologie van M. tuberculosis te verstaan. Dit het ook getoon dat verdere studies dringend nodig is om die komplekse fisiologie van hierdie patogeen te verstaan. Hierdie kennis sal van kardinale belang wees vir die sukses van toekomstige ondernemings om nuwe anti-tuberkulose middels te ontwikkel.

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Microbiology / Master / Master of Philosophy

published_or_final_version / abstract / Clinical Oncology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Microbiology / Master / Master of Philosophy

published_or_final_version / abstract / Microbiology / Doctoral / Doctor of Philosophy