Gold nanoprobes for the detection of mutations associated with antibiotic resistance in Mycobacterium tuberculosis complex

Dissertação para obtenção do Grau de Mestre em Genética Molecular e BiomedicinaTuberculosis is still one of the leading human infectious diseases, with 8.7 million new cases reported in 2011 alone. Also, the increasing rate of multidrug-resistant tuberculosis (MDR-TB) and its treatment difficulties pose a serious public health threat especially in developing countries. Resistance to isoniazid and rifampicin, first line antibiotics, is commonly associated with point mutations in katG, inhA and rpoB genes of Mycobacterium tuberculosis complex (MTBC). Therefore, the development of a cheap, fast and simple molecular method to assess susceptibility profiles would have a huge impact in the capacity of early diagnosis and treatment of MDR-TB patients. Gold nanoparticles functionalised with thiol-modified oligonucleotides (Au-nanoprobes) have shown the potential to provide a rapid and sensitive detection method for MTBC and single base mutations associated with antibiotic resistance, namely the characterisation of the three most relevant codons in rpoB gene associated to rifampicin resistance. In this work the Au-nanoprobe approach is extended towards the discrimination of specific mutations within inhA gene which is associated with resistance to isoniazid. Using a multiplex PCR reaction for rpoB and inhA genes, it was possible to assess both loci in parallel, and extend the potential of the Au-nanoprobe method to MDR-TB molecular characterisation with special application in the most frequent genotypes circulating in the Lisbon Health Region

Gold Nanoparticles to Tackle Drug Resistance in Cancer

The most common type of cancer treatment, chemotherapy, often fails due to the acquisition of resistance. It is therefore of the utmost importance to better understand the cellular mechanisms of resistance, while developing new strategies to tackle drug resistance. Nanomedicine promises to develop new tools for diagnosing and treating of diseases, improving human health. This thesis explored five main strategies to overcome drug resistance in cancer cells: i) new anti-cancer compounds, ii) nanovectorisation and targeting of compounds, iii) gold nanoparticles (AuNP) for hyperthermia and combination with chemotherapy iv) AuNP assisted angiogenesis arrest with laser ablation and chemotherapy v) AuNP mediated gene silencing of efflux pumps. We started by characterizing the mechanism of action of a new anti-tumour compound (ZnD), that was able to reduce the viability of a colon cancer cell line (HCT116 DR) resistant to doxorubicin (DOX), a first-line chemotherapeutic. We vectorized our compound with AuNP, resulting in increased toxicity to HCT116 DR mouse xenografts. Taking advantage of the photothermal properties of AuNP, we combined chemotherapy with photo hyperthermia by irradiating AuNP with a green laser. Hyperthermia was especially effective against HCT116 DR. Since angiogenesis is a milestone in cancer development, we aimed to prevent it, using an anti-angiogenic peptide vectorized with AuNP and laser irradiation, resulting in a vascularization reduction of 91% in vivo. We have observed that ABCB1 efflux pump was the major cause of resistance of DOX resist cell line, therefore we silenced its mRNA with an anti-sense oligonucleotide AuNP. Our results showed that although the silencing was effective, cells did not return to a sensitive phenotype, requiring further experiments. Altogether this thesis shows the potential of nanotechnology for cancer treatment, both in chemotherapy and in surgery, where green lasers are already used. This work can be applied to drug resistant tumours, increasing the efficacy of treatment

Characterisation of microbial attack on archaeological bone

As part of an EU funded project to investigate the factors influencing bone preservation in the archaeological record, more than 250 bones from 41 archaeological sites in five countries spanning four climatic regions were studied for diagenetic alteration. Sites were selected to cover a range of environmental conditions and archaeological contexts. Microscopic and physical (mercury intrusion porosimetry) analyses of these bones revealed that the majority (68%) had suffered microbial attack. Furthermore, significant differences were found between animal and human bone in both the state of preservation and the type of microbial attack present. These differences in preservation might result from differences in early taphonomy of the bones. © 2003 Elsevier Science Ltd. All rights reserved