Browsing by Author "Naran, Krupa"

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    Development of potential immunodiagnostic & therapeutic techniques using SNAP-fusion proteins as tools for the validation of Triple-negative Breast Cancer
    (2020) Magugu, Freddy-Junior Siybaulela; Barth, Stefan; Naran, Krupa
    Globally, breast cancer is the leading cause of death in the female population aged 45 and below with a breast cancer incidence reaching 18.1 million in the year 2018. Triple negative breast cancer (TNBC) is part of a group of cancers that lack the expression of Progesterone receptor (PR), Estrogen receptor (ER) and Human epidermal growth factor receptor 2 (HER2). TNBC is commonly associated with early stage metastasis with low survival rates as well as a high frequency of recurrence and proves to be problematic in both the young and elderly female populations. Conventional diagnostic methods for TNBCs include mammography, magnetic resonance imaging (MRI) and ultrasound while therapeutic methods include mastectomy and breast conserving surgery (coupled with radiation therapy). The lack of effective therapeutic options, poor prognostic value and high rates of metastasis, has made treatment of TNBC difficult. The major focus of this work was on the following tumour associated antigens (TAAs): CSPG4 (a transmembrane protein found in 50% of TNBC cases), EGFR (which is overexpressed in 13-76% of TNBCs), and MSLN (which is overexpressed in 67% of TNBCs) as potential targets for monospecific therapy. The evolution of antibody-based immunotherapy strategies has led to applications of single chain variable fragment (scFv) & single domain/nanobody (VHH) antibody formats for diagnostic and therapeutic purposes. In this work, these recombinant antibody fragments have been combined with SNAP-tag, a modified version of the human DNA repair enzyme O6-alkylguanine-DNA-alkyltransferase (AGT), which autocatalytically binds benzyl-guanine modified substrates such as fluorophores or small molecule toxins covalently in a 1:1 stoichiometry. In this study, the primary aim was the comparison of different antibody formats fused to SNAPtag and the potential of these biopharmaceuticals towards immunodiagnosis and therapy of TNBCs. First functionalities of two scFv SNAP fusion proteins and one VHH SNAP fusion protein previously not having been described are provided through binding analyses on receptor positive tumour cell lines. This was achieved by in-silico design and molecular cloning of genetically fused antiCSPG4(scFv), -MSLN(scFv), -MSLN(VHH), -EGFR(scFv) & -EGFR(VHH) to SNAP-tag. The final constructs were confirmed by Sanger sequencing and subsequently transfected into a mammalian vector system (HEK293T) for transient expression of the engineered fusion proteins. Full length protein purified from cell culture supernatant was analysed for diagnostic/therapeutic activities dependant on the substrate attached in the form of a fluorophore or small molecule toxin resulting in recombinant antibody-drug conjugates (ADCs). The study shows promise in providing new immunodiagnostic and therapeutic agents that are specific and less harmful than the current state of the art procedure
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    Elucidation of mechanisms of antibiotic subversion in mycobacteria
    (2015) Naran, Krupa; Warner, Digby F; Mizrahi, Valerie
    The intrinsic resistance of Mycobacterium tuberculosis ( Mtb ) to antibiotics is generally attributed to multiple factors, most significantly the low permeability of the mycobacterial cell wall, the operation of various drug inactivating systems, and the activity of efflux pumps. This study aimed to investigate the role of various components of the "intrinsic resistome" that limit the efficacy of antitubercular agents. The DNA damage response: the SOS response was hypothesized to play a role in antibiotic- mediated cellular death, and that disabling the mycobacterial SOS response, by generating non-cleavable LexA mutants (lexA Ind-), could be used as a tool to validate antibiotic-mediated cell death. To this end, the M. smegmatis (Msm) cleavable LexA was shown to be essential for induced mutagenesis and damage tolerance and that an intact DNA damage repair system is required to respond to antibiotic-mediated DNA damage. In contrast, Mtb cleavable LexA was required for induced mutagenesis but not necessarily damage sensitivity. In addition, the Mtb SOS response does not contribute significantly to remediation of antibiotic-mediated DNA damage. Collectively, these data suggest that DNA repair mechanisms differ between the mycobacterial species and despite effectively inactivating the LexA-dependent DNA repair mechanism(s) in Msm and Mtb, these organisms are able to circumvent this pathway and successfully remediate damaged DNA sustained under various conditions. Furthermore, Mtb auto-bioluminescent reporter strains were generated by introducing the lux operon downstream of the recA or radA promoters. Analysis of a panel of antimicrobials against these strains allowed for the identification of true DNA-damaging agents and the evaluation of the kinetics of the DNA-damage response, in a concentration- and time-dependent manner. Efflux-mediated drug resistance: This study aimed to evaluate the interactions between pairwise combinations of selected antimicrobials and efflux pump inhibitors (EPIs), in vitro and ex vivo, and to identify a novel verapamil (VER)-analogue with improved efficacy against Mtb. Subsequently, a candidate EPI was identified, with equivalent in vitro synergistic effects to VER when used in combination with various antibiotics but with reduced cytotoxic effects, ex vivo, when compared to VER. Mycothiol-mediated protection : It was hypothesized that undetectable levels of mycothiol ( MSH ) in Mtb would potentiate the use of current antibiotics. To investigate the contribution of the cellular antioxidant, MSH, to the mitigation of antimicrobial efficacy, this study aimed to disrupt MSH production by conditionally knocking-down expression of the essential gene, mshC. The mshC knock-down mutants (in all configurations) were not anhydrotetracycline (ATC)-regulatable in liquid or on solid medium, which was subsequently validate d with quantitative gene expression analysis. These data suggest that a tetracycline (Tet)-based conditional expression system may not be applicable to mshC. In conclusion, Mtb has a multitude of inherent mechanisms to subvert the effects of antimicrobial treatment. This study has contributed to the understanding of certain aspects of the intrinsic resistome and in doing so, established tools that can be used in future drug discovery programmes.