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Coming dissertations at Uppsala university

  • 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.

  • LL-37-derived cyclic antimicrobial drug leads : Design, synthesis, activity and different ways of creating them  Author: Taj Muhammad Link: Publication date: 2019-11-27 12:17

    In an era where last-line antibiotics are failing, one of the powerful approaches to develop novel therapeutic agents is to turn back to nature in order to identify possible drug candidates. Among the potential candidates, antimicrobial peptides (AMPs) have garnered much attention as an antimicrobial. These are broad spectrum host defense molecules produced by all living organisms. LL-37 is such a multitask human defense peptide that mediates various host immune responses and also exerts antimicrobial activity. However, the direct use of this 37-amino acid long α-helical peptide is hampered by protease susceptibility, in particular for antimicrobial applications. A small 12-residues peptide, referred as KR-12, derived from LL-37, has been reported to have selective toxic effect on bacteria. 

    Analogues of KR-12 were generated in the form of Alanine and Lysine scans to find out the positions important for improved activity and selectivity. Backbone-cyclised dimers based on KR-12 and KR-12 analogues, tethered by linkers of two to four amino acid residues, were synthesised to explore the concept of cyclisation, dimerisation and cross-linking as means to enhance peptide stability and activity. Antimicrobial activities of the linear peptides and cyclic dimers were assayed against human pathogens, in buffer and/or physiological conditions. Proteolytic stability, permeabilisation efficacy on microbial membranes and, their structures were also characterised.  

    From Ala and Lys scans, it was possible to identify two key positions for the enhanced broad-spectrum antibacterial activity: replacement of Gln5 with Lys, and Asp9 with either Ala or Lys. In serum stability assay, KR-12 and analogues were found to be unstable. The backbone-cyclised KR-12 dimers showed improved antimicrobial activity and increased stability compared to monomeric KR-12. KR-12 monomers adopt a well-defined α-helical structure in membrane-mimicking environment, while cyclised dimers were unstructured in solution judged by NMR. The KR-12 (Q5K, D9A) cyclised dimers retained antimicrobial activity in physiological conditions. Circular dichroism showed that the cyclic dimer, cd4-PP, had 77% helical content when bound to lyso-phosphatidylglycerol micelles.

    Moreover, the limits of cyanobactin-macrocyclase PatGmac were explored to cyclise peptides larger than their natural substrates, namely the PawS derived peptide Sunflower Trypsin Inhibitor-1 (SFTI-1) and the cyclotide kalata B1. PatGmac was used very efficiently to cyclise SFTI-1. In addition, semi-pure butelase 1, isolated from Clitoria ternatea seeds, was immobilised on NHS column. The immobilised column was then used to produce substrates ranging from 16 to 34 varying length.

  • 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.