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Coming dissertations at MedFak

  • Towards time-resolved molecular interaction assays in living bacteria Author: João Crispim Encarnação Link: Publication date: 2019-12-18 10:48

    Rare and neglected diseases such as multidrug resistant (MDR) tuberculosis, malaria and trypanosomiasis are re-emerging in Europe. New strategies are needed to accelerate drug discovery to fight these pathogens. AEGIS is a Pan-European project that combines different technologies to accelerate the discovery of molecules suitable for drug development in selected neglected diseases. This thesis is part of the AEGIS research area that considers time in a multidisciplinary approach, combining biology, physics and mathematics to provide tools to characterize biological events for improving drug development and information about the target diseases and lead compounds.

    Real-time cell binding assays (RT-CBA) of receptor-ligand interactions are fundamental in basic research and drug discovery. However, this kind of assays are still rare on living cells, especially in the microbiology field. In this project, we apply the same high-precision assay type on bacterial systems and explored the interior of the cell with a time resolved assay.

    The effect of temperature was evaluated in the RT-CBA using LigandTracer to ensure that it was possible to use the technology in a range of temperatures suitable for bacteria. A method for attaching Gram positive and negative bacteria on the surface of a normal Petri dish, showing a high reproducibly and a high cellular viability after 16 h. With these two key steps, an RT-CBA fit for microbiology is available.

    Next, to answer biological questions, intracellular interactions were explored by expression and validation of intracellular proteins with fluorescent tags suitable for RT-CBAs. First, we used the subunit B from the Shiga toxin (STxB) as a model to understand different aspects about the internalization processes. RT-CBAs allowed to discovery new features of STxB binding and mechanism to deliver small molecules or small proteins into cancer cells. Then, for exploring intracellular interactions, insect cells were bioengineered for evaluating the ability of small molecules to internalize and bind to its target. Using Carbonic anhydrase II – sulfonamides as a model system, the molecular interaction in the cytoplasm could be measured using a quencher label approach. The development of this kind of novel RT-CBA tools provide new information about drug candidates for targets that are not properly expressed in bacterial cells.

    The assays in this project can make drug design more efficient. Furthermore, the evaluation of binding activity of the new compounds developed by AEGIS, focusing on rare/neglected diseases, in a biological environment has the potential to accelerate drug discovery for the targeted emerging diseases.

  • Dried blood sampling and digital readout to advance molecular diagnostics Author: Johan Björkesten Link: Publication date: 2019-11-28 13:23

    A drastically increased capacity to measure large sets of molecular features in numerous patient samples in great detail will be required to fulfill the vision of precision medicine and wellness, which may characterize molecular diagnostics in the 21st century. Also sampling procedures need a renaissance to permit continuous sampling at population levels at reasonable cost.

    Blood sampling is typically performed via venipuncture to draw several milliliters of blood for plasma isolation. This is inconvenient, time-consuming and costly, as well as hard to standardize. The effect on plasma protein profiles by pre-centrifugation delay was investigated in Paper II, demonstrating time- and temperature-dependent release of proteins from blood cells upon delayed plasma isolation, but almost no protein degradation as analyzed by two 92-plex protein panels (Olink® Proteomics). An alternative sampling method, where blood drops from a finger stick are collected dried on paper, is relatively non-invasive, potentially home-based and cheap. Dried blood spots can also be shipped via regular mail and compactly stored. The effect of drying and long term storage stability of a large set of proteins from dried blood spots was investigated in Paper I using Olink® technology. The main findings were that drying slightly but consistently influenced the recorded levels of blood proteins, and that long-term storage decreased the detected levels of some of the proteins with half-lives of decades.

    Some molecular diagnostic investigations require great accuracy to be useful, arguing for digital enumeration of individual molecules. Digital PCR is the gold standard but Paper III presents an alternative approach based on rolling circle amplification of single molecules. Another instance where extreme assay performance is required is for rare mutation detection from liquid biopsies. Paper V presents a new method offering essentially error-free genotyping of individual molecules by majority-vote decisions for counting rare mutant DNA in blood. Yet other diagnostic investigations require very simple assays. Paper IV presents a novel one-step method to detect nucleic acid sequences by combining the power of rolling circle amplification and the specificity of DNA strand displacement in a format simple enough to be used at the point of care.   

    Altogether, the thesis spans technologies for advanced molecular diagnostics, from sample collection over assay techniques to an improved readout.

  • Morphometry of the Optic Nerve Head as a Diagnostic Tool for Glaucoma Author: Camilla Sandberg Melin Link: Publication date: 2019-11-27 09:37

    Glaucoma is a chronic optic nerve head (ONH) disease. Gradual retinal ganglion cell and nerve fiber loss lead to morphological ONH change and visual field defects. Initial loss is often focal. Rate of progression and life expectancy guide treatment. Currently, confocal scanning laser tomoghraphy (HRT) and optic coherence tomography (OCT) are available for ONH imaging. However, there is no consensus for which morphometric measurement of ONH nerve fiber content to use for glaucoma follow-up.

    Purpose: To measure ONH nerve fiber content as neuroretinal rim area (NRA) with HRT, estimate NRA measurement variation and its impact on designing a follow-up strategy. To develop a custom algorithm, Pigment epithelium central limit-Inner limit of the retina Minimal Distance (PIMD), for measuring ONH nerve fiber content in OCT data cubes. To measure PIMD in glaucomatous eyes, estimate the variance sources for PIMD and their impact on designing strategies for glaucoma follow-up.

    Methods: NRA was measured with HRT in non-glaucomatous and glaucomatous eyes. Sources of variance for NRA were estimated. An OCT data cube of a non-glaucomatous eye was used in developing the PIMD algorithm. PIMD was measured in 500 radii along the ONH circumference. PIMD averaged over the circumference is PIMD-2π. Sources of variance for PIMD-2π were estimated for glaucomatous eyes. Strategies for following PIMD-2π and segments of PIMD-2π within subject over time were proposed.

    Results: Variation among subjects was substantial for NRA and PIMD-2π. Contrarily, within subject variation was small for NRA and PIMD-2π. When within subject variation, a previously reported loss rate for progressing glaucoma, and measuring NRA 3 times every 4 months were applied, a significant loss was detected after 54 months. When within subject variation and a PIMD-2π loss rate resulting in blindness after 20 years were applied, a significant PIMD-2π loss was detected in 16 months with visits every 4 months. Within subject segmental PIMD-2π loss can be detected from the 3rd visit. Loss rate of each PIMD can be estimated with linear regression from the 4th visit. Change in segmental PIMD-2π loss rate can be detected at a later visit.

    Conclusions: Small within subject variation allows for within subject NRA and PIMD follow-up over time. Segmental PIMD-2π has potential to detect focal glaucomatous defects and worsening of existing defects. There is potential to detect a change in segmental PIMD-2π loss rate. Segmental PIMD-2π has potential as a tool for within subject follow-up of glaucoma.