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Coming theses from other universities

  • Arterial stiffness and risk factors for cardiovascular disease in young adults Author: Ulrika Fernberg Link: Publication date: 2019-08-30 10:18
  • Studies on cysteine-rich peptides from Nemertea and Violaceae : Proteomic and transcriptomic discovery and characterization Author: Erik Jacobsson Link: Publication date: 2019-08-30 09:06

    The overall aims of the projects included in this thesis were to discover, synthesize and characterize disulphide-stabilized peptides from marine worms (Nemertea sp.) and plants (Viola sp.). 

    One of the main outcomes of this thesis is the discovery of a new family of highly active cysteine-rich toxins, alpha nemertides, from nemertean worms (paper II). Functional characterization and production routes of nemertides were further explored (papers II-III). In addition, 12 new cyclotides from the bog violet were discovered (paper I). Finally, transcriptomes and mucus of the Antarctic nemertean Parborlasia corrugatus were investigated for toxin content (paper IV).

     In paper I wild-type leaf and callus tissue of the endangered bog violet, V. uliginosa, were analyzed using transcriptomics and LC-MS, resulting in the discovery of 12 new cyclotides (i.e. cysteine-rich cyclic peptides). In addition, cyclotide expression under different cell-growth conditions was monitored.

    In paper II  the discovery and initial characterization of a new family of highly active peptides, the alpha nemertides, from the epidermal mucus of the world’s longest animal; Lineus longissimus is described. The most abundant alpha nemertide, alpha-1, was extracted in minute amounts, prompting the use solid phase peptide synthesis (SPPS) for further characterization. The tertiary structure of alpha-1 was elucidated and revealed an inhibitory cystine knot (ICK) framework. The knotted core-structure is similar to the cyclic cystine knot (CCK) motif, found in the cyclotides described in paper I.

    In manuscript III, the production route established in paper II was used to produce nemertides alpha 1-7. These were tested in vivo in an Artemia microwell assay as well as on an extended panel of voltage-gated sodium channels (NaV1.1 – 1.8 and BgNaV1). All seven alpha nemertides were highly active in the in vivo Artemia assay with EC50 values in the sub to low µM range. The alpha nemertides were also active in the NaVs tested. However, differences in the activity profiles were observed, indicating an opportunity for future optimization of alpha nemertides to reach higher specificity to certain NaV subtypes.

    In manuscript IV, the exploration of nemertide toxins was extended to include the Antarctic P. corrugatus. Resulting findings include a set of cysteine-rich peptides, some similar to the nemertides previously discovered in paper II. Two purified peptides and one fraction were evaluated for their membranolytic activity.

  • The Glycobiology of Human Adenovirus Infections : implications for tropism and treatment Author: Naresh Chandra Link: Publication date: 2019-08-30 06:00

    Human adenoviruses (HAdVs) are common human pathogens, causing gastrointestinal, ocular, and respiratory infections on a regular basis. Epidemic keratoconjunctivitis (EKC) is a severe ocular infection for which no approved antivirals are available. HAdV-D37 is one of the causative agents of EKC and uses sialic acid (SA)-containing glycans as cellular receptors. HAdV-D37 interacts with SA via the knob domain of the trimeric virus fiber protein, containing three SA-binding sites. HAdV-D37 also bind to glycosaminoglycans (GAGs), but the outcome of this interaction remains unknown. Here, using biochemical and cell-based assays, the impact of GAGs on HAdV-D37 infection (paper I) was investigated. We found that HAdV-D37 interacts with both soluble and cell-surface sulfated GAGs via the knob domain of the viral fiber protein. Remarkably, removal of heparan sulfate (HS; a type of GAG) from human corneal epithelial (HCE) cells by heparinase III enhanced HAdV-D37 infection. We propose that sulfated GAGs in bodily secretions and on plasma membranes function as decoy receptors that prevent the virus from binding to SA-containing receptors and inhibit subsequent virus infection. We also found abundant HS in the basement membrane of the human corneal epithelium. We suggest that this layer of HS functions as a barrier to sub-epithelial infection of HAdV-D37. Based on this finding, we hypothesized that GAG-mimetics may act as artificial decoy receptors and inhibit HAdV-D37 infection. Here, the antiviral effect of suramin (a known GAG-mimetic) and its analogs against HAdV-D37 (paper II) was evaluated. Interestingly, all compounds displayed antiviral effects by inhibiting the binding of HAdV-D37 to HCE cells. The antiviral effect of suramin was HAdV species-specific. We report for the first time that virus binding to cell-surface decoy receptor constitutes a potential target for antiviral drug development.

    HAdVs are the major cause of infectious conjunctivitis, constituting up to 75% of all conjunctivitis cases worldwide. Species B HAdV type 3 (HAdV-B3) causes pharyngoconjunctival fever (PCF), whereas HAdV-D8, -D37, and -D64 cause EKC. Recently, HAdV-D53, -D54, and -D56 have emerged as new EKC-causing agents. HAdV-E4 causes both PCF and EKC. SA-containing glycans have been established as cellular receptors for HAdV-D37. By means of cell-based assays, we investigated if ocular HAdVs other than HAdV-D37 also use SA-containing glycans as receptors on HCE cells (paper III). It was found that SA-containing glycans function as cellular receptors for five (HAdV-D8, -D37, -D53, -D54, and -D64) out of six EKC-causing species D HAdVs. We showed that these viruses interact with SAs via the knob domain of the viral fiber protein. HAdV-E4 and -D56 infection of cells was independent of SAs. Surprisingly, HCE cells were completely refractory to HAdV-B3 infection. A trivalent sialic acid (TSA) derivative ME0462 (compound 17a in paper II), designed to bind to SA-binding sites on HAdV-D37 fiber knob, also showed potent antiviral activity against several EKC-causing HAdVs. This suggests that ME0462 can be used as a broad-spectrum antiviral against known and emerging EKC-causing HAdVs. Surface plasmon resonance (SPR) analysis confirmed a direct interaction between ME0462 and fiber knobs of EKC-causing HAdVs.

    Recently, a TSA derivative (ME0322; designed to bind to SA-binding sites on HAdV-D37 fiber knob) was shown potent antiviral against HAdV-D37 in vitro. To improve the antiviral potency of this compound, six new TSA derivatives were synthesized and their inhibitory effects were evaluated against HAdV-D37 (paper IV). Interestingly, the best compound 17a was found approximately three orders of magnitude more potent (IC50 (binding) = 1.4 nM, IC50 (infection) = 2.9 nM) than ME0322 (IC50 in µM range). SPR data showed that HAdV-D37 fiber knob binds to TSA compounds with high affinities. Structural data revealed the trivalent binding mode of all newly synthesized TSA compounds to HAdV-D37 fiber knob. Ophthalmic toxicity of compound 17a (best compound) was also investigated in rabbits without any sign of toxicity.

    HAdV-D36 is a member of species D HAdV and has the ability to infect a broad range of animals, which is unusual for HAdVs. Another remarkable feature of HAdV-D36 is that this virus induces obesity in experimental animals. Several epidemiological studies highlighted a link between HAdV-D36 and human obesity. There is no information about the cellular receptor usage by HAdV-D36. Using structural biology and cell-based approaches, we investigated the cellular receptor(s) for HAdV-D36 (paper V).  We show that HAdV-D36 attaches to host cells (via the fiber knob) using the coxsackie and adenovirus receptor (CAR), SA-containing glycans, and one or more unknown proteins or glycoproteins. Using glycan microarray, we found that HAdV-D36 displays binding preference to a rare SA-variant: 4-O,5-N-diacetylneuraminic acid (Neu4,5Ac2), over the more common SA (in humans) i.e. 5-N-acetylneuraminic acid (Neu5Ac). Structural analysis of HAdV-D36 fiber knob:Neu4,5Ac2 complex explained this preference. To date, Neu4,5Ac2 has not been detected in humans, although it is synthesized by many domestic and livestock animals. Our results indicate that HAdV-D36 has evolved to utilize a specialized set of cellular receptors that coincide with a unique host range and pathogenicity profile.

    These studies provide insights into multiple roles of glycans in HAdV infection cycle and highlight the therapeutic potential of glycans/glycan-mimetics in HAdV-D37 infection.