Noroviruses are the major cause of acute viral gastroenteritis worldwide. Thus, rapid and reliable pathogen detection and control are crucial to avoid epidemic outbreaks. Peptides which bind to these viruses with high specificity and affinity could serve as small and stable recognition elements in biosensing applications for a point-of-care diagnostic of noroviruses. They can be identified by screening large phage display libraries using the biopanning technique. In the present study, this method was applied to identify norovirus-binding peptide motifs. For this purpose, a biopanning based on column chromatography was established, and three rounds of selections were performed. After the second round, the cosmix-plexing recombination technique was implemented to enhance the chance of obtaining peptides with very high affinity. Biopanning data evaluation was based on next-generation sequencing (NGS), to show that this innovative method can enable a detailed analysis of the complete sequence spectrum obtained during and after biopanning. Highly enriched motifs could be characterized by their large proportion of the amino acids W, K, R, N, and F. Neighbourhood analysis was exemplarily performed for selected motifs, showing that the motifs FAT, RWN, and KWF possessed the fingerprints with the largest differences relative to the original library. This thesis thus presents next-generation sequencing-based analysis tools, which could now be transferred to any other biopanning project. The identified peptide motifs represent promising candidates for a future examination of their norovirus-specific binding. A new option for testing such phage-target interactions in the context of biopanning selections was studied in the second part of the thesis. For this purpose, a phage-based microarray was developed as a miniaturized binding assay. As a prerequisite, the different immobilization behaviour of phages on positively and negatively charged surfaces was studied, and a non-contact printing technique for bacteriophages was developed. Subsequently, the interaction of phages and antibodies directed against phage coat proteins was characterized in enzyme-linked immunosorbent assays, and the protocol was successfully transferred to the non-contact printed phage spots. At the proof-of-concept level, the phage array could finally be integrated into a microfluidic setup, showing a higher signal-to-background ratio relative to the static phage array. These results point the way towards a microfluidic phage array, allowing online monitoring, automation, and parallelisation of the phage array analysis. / Noroviren gelten als Hauptursache akuter viraler Magen-Darm-Erkrankungen. Nur eine zeitnahe und verlässliche Detektion und Kontrolle dieser Pathogene kann epidemische Ausbrüche vermeiden. Um dies zu ermöglichen, könnten Peptide, die an diese Viren mit hoher Spezifität und Affinität binden, als kleine und stabile Erkennungselemente in biosensorischen Anwendungen eingesetzt werden. Solche Peptide können mithilfe der Biopanning-Technik identifiziert werden, die auf dem Screening großer Phagen-Display-Bibliotheken beruht. In der vorliegenden Arbeit wurde diese Methode genutzt, um Norovirus-bindende Peptidmotive zu identifizieren. Dazu wurde ein auf Säulenchromatographie basierendes Biopanning entwickelt und drei Selektionsrunden durchgeführt. Die Cosmix-Plexing-Rekombinationstechnik wurde nach der zweiten Runde eingesetzt, um die Wahrscheinlichkeit der Gewinnung hochaffiner Binder zu erhöhen. Die Auswertung der Biopanningdaten erfolgte mittels Hochdurchsatzsequenzierung (Next-Generation Sequencing). Es konnte gezeigt werden, dass diese innovative Methode die detailierte Analyse des kompletten Sequenzspektrums während und nach dem Biopanning ermöglicht. Stark angereicherte Motive konnten durch ihren hohen Anteil an den Aminosäuren W, K, R, N und F charakterisiert werden. Eine Nachbarschaftsanalyse wurde exemplarisch für ausgewählte Motive durchgeführt. Dabei wurden die stärksten Unterschiede im Fingerprint im Vergleich zur Ausgangsbibliothek bei den Motiven FAT, RWN und KWF gefunden. Diese Dissertation stellt damit auf Next-Generation Sequencing basierende Analysetechniken vor, die für weitere Biopanningprojekte übernommen werden können. Die identifizierten Peptidmotive könnten als vielversprechende Kandidaten zukünftig auf ihre Norovirus-spezifische Bindung hin getestet werden. Eine neue Möglichkeit, solche Phagen-Analyt-Interaktionen zu untersuchen, wurde im zweiten Teil der Dissertation untersucht. Dafür wurde als miniaturisierter Bindungsassay ein Phagen-basiertes Mikroarray entwickelt. Als Voraussetzung wurde zunächst das unterschiedliche Immobilisierungsverhalten von Bakteriophagen auf positiv und negativ geladenen Oberflächen untersucht und eine kontaktfreie Drucktechnik für Bakteriophagen etabliert. Anschließend wurde die Interaktion von Phagen und gegen sie gerichteten Antikörpern in Enzym-gekoppelten Immunadsorptionstests charakterisiert und das Protokoll erfolgreich auf die kontaktfrei gedruckten Phagenspots übertragen. Schließlich wurde erstmals die grundsätzliche Möglichkeit gezeigt, das Array in ein mikrofluidisches Setup zu integrieren, was zu einem höheren Signal-zu-Hintergrund-Verhältnis im Vergleich zum statischen Array führte. Diese Ergebnisse zeigen damit den Weg zu einem mikrofluidischen Phagen-Array auf, das sowohl die Möglichkeit des Online-Monitorings als auch der Automatisierung und Parallelisierung der Phagen-Array-Analyse bietet.

The evolution and adaptation of microorganisms is so rapid it can be seen in the time frame of days. The root cause for their evolution comes from selective environmental pressures that see organisms with beneficial mutations survive otherwise deadly encounters or outperform members of its population who fail to adapt. This does not always result in strict improvement of the individual as in the case of antibiotic resistant bacteria who often display fitness tradeoffs to avoid death (see Reviews [1-3]). For example, when an ampicillin resistance gene (ampC) containing plasmid that is occasionally found in the wild was transformed into S. typhimurium the bacteria had slower growth and impaired invasiveness [4]. In another example, capreomycin use with mycobacteria resulted in lower binding of the drug to the ribosome through mutations in rRNA methylase TlyA 16S rRNA, which decreases the overall fitness of the mycobacteria [5]. The evolutionary interactomes between bacteria and antibiotics do not end there, as antibiotic resistant bacteria often accumulate compensatory mechanisms to regain fitness. These range in effect with some altering individual cellular pathways and others having systemic affects [1]. My work has focused on the intersection of diabetes and related antibiotic resistant bacterial infections. Diabetes is one of the leading health issues in the United States, with over 10% of the adult population and over 26% of the elderly diagnosed (American Diabetes Association) [6]. Herein I further characterize the molecular pathways involved in diabetes, through the study of PAS kinase (PASK) function. PAS kinase is a serine-threonine protein kinase which regulates the pathways disrupted in diabetes, namely triglyceride accumulation, metabolic rate (respiration), adiposity and insulin production and sensitivity [7-9]. In this study I specifically focus on the effects of PAS kinase and its substrate, USF1/Cbf1p, and how their altered metabolic deficiencies can be suppressed using yeast cells. Through this study I further characterized the molecular function of USF1/Cbf1p through the identification of putative co-transcriptional regulators, identify novel genes involved in the regulation of respiration, and uncover a function or a previous uncharacterized protein, Pal1p. Part of the diabetes healthcare challenge results from the wide range of diseases that are associated with diabetes, including obesity [10, 11], renal failure [12, 13], neuropathies and neurodegeneration [14, 15], endocrine dysfunctions [16, 17], and cancers [18]. In addition, diabetes is a leading cause of lower limb amputations, due to poor circulation and the prevalence of ulcers [19-21], many of which are antibiotic resistant [22-25]. Phage therapy, based on the administration of bacterial viruses, is a viable option for the treatment of these diseases, with our lab recently isolating bacteriophages for several clinical cases. In the second half of my thesis, I present the study of the adaptation of bacteriophages to their hosts as well as report contributions of local ecology to their evolution.

No description available.

Small stable protein domains are attractive scaffolds for engineering affinity proteins due to their high tolerance to mutagenesis without loosing structural integrity. The albuminbinding domain is a 5 kDa three-helix bundle derived from the bacterial receptor Protein G with low-nanomolar affinity to albumin. In this thesis, the albumin-binding domain is explored as a scaffold for engineering novel affinity proteins with the possible benefit of combining a prolonged serum half-life with specific targeting in a single small scaffold protein. Previously, a library was created by randomizing surface-exposed residues in order to engineer affinity to a new target antigen in addition to the inherent albumin affinity. Here, phage display selections were separately performed against the tumor antigens ERBB2 and ERBB3. The ERBB3 selection resulted in a panel of candidates that were found to have varying affinities to ERBB3 in the nanomolar range, while still retaining a high affinity to albumin. Further characterization concluded that the clones also competed for binding to ERBB3 with the natural activating ligand Heregulin. The selections against ERBB2 resulted in sub-nanomolar affinities to ERBB2 where the binding site was found to overlap with the antibody Trastuzumab. The binding sites on ABD to albumin and either target were found in both selections to be mutually exclusive, as increased concentrations of albumin reduced the level of binding to ERBB2 or ERBB3. An affinity-matured ERBB2 binder, denoted ADAPT6, which lacked affinity to albumin was evaluated as a radionuclide-labeled imaging tracer for diagnosing ERBB2-positive tumors. Biodistribution studies in mice showed a high renal uptake consistent with affinity proteins in the same size range and the injected ADAPT quickly localized to the implanted tumor. High contrast images could be generated and ERBB2-expressing tissue could be distinguished from normal tissue with high contrast, demonstrating the feasibility of the scaffold for use as diagnostic tool. In a fourth study, affinity maturation strategies using staphylococcal cell-surface display were evaluated by comparing two replicate selections and varying the stringency. A sub-nanomolar target concentration was concluded to be inappropriate for equilibrium selection as the resulting output was highly variable between replicates. In contrast, equilibrium sorting at higher concentrations followed by kinetic-focused off-rate selection resulted in high output overlap between attempts and a clear correlation between affinity and enrichment. / <p>QC 20160510</p>

The study of social evolution and virulence in parasites is concerned with fitness consequences of trade-offs between parasite life history traits and interactions between parasite species and/or genotypes with their hosts. I develop our understanding of social evolution and virulence in parasites in several ways. (1) I review empirical evidence for the fundamental predictions of virulence-transmission trade-off theory and demonstrate that the fit between theory and data is primarily qualitative rather than quantitative; that parasites differ in their degree of host generalism, and this is likely to impact virulence in four ways. (2) I take a comparative approach to examine the underlying causes of an observed statistical variation in the size of parasite infectious doses across taxa, revealing that mechanisms used by parasites to infect hosts are able to explain variation in both infectious dose and virulence. (3) I formally compare data on human pathogens to explain variation in virulence across taxa, revealing that immune subversion and not growth rate, explains variation in virulence. This allows me to predict that immune subverters and not fast growing parasites are likely to cause the most virulent clinical infections. (4) Using bacteria and their naturally infecting viruses (bacteriophage), I take an experimental approach to investigate the consequences of coinfection for parasite life history traits, and find that viruses cultured under a mix of single infections and coinfections evolved plasticity; they killed hosts more rapidly when coinfecting, and this resulted in high fitness under both single infection and coinfection conditions. (5) I experimentally investigate how selection within and between hosts and patches of hosts affects the fitness and virulence of populations of these viruses. I find that limited host availability favours virulent, faster killing parasites with reduced transmission; suggesting high, rather than low, virulence may be common in spatially structured host-parasite communities.

<p>This thesis describes recombinant proteins that have been generated by rational and combinatorial protein engineering strategies for use in subunit vaccine delivery and tumor targeting.</p><p>In a first series of studies, recombinant methods for incorporating immunogens into an adjuvant formulation, e.g. immunostimulating complexes (iscoms), were evaluated. Protein immunogens, which are not typically immunogenic in themselves, are normally administered with an adjuvant to improve their immunogenicity. To accomplish iscom incorporation of a <i>Toxoplasma gondii</i> surface antigen through hydrophobic interaction, lipids were added either <i>in vivo</i> via <i>E. coli</i> expression, or <i>in vitro</i> via interaction of an introduced hexahistidyl (His6) peptide and a chelating lipid. The possibility of exploiting the strong interaction between biotin and streptavidin was also explored, in order to couple a<i> Neospora caninum</i> surface antigen to iscom matrix, i.e. iscom particles without any antigen. Subsequent analyses confirmed that the immunogens were successfully incorporated into iscoms by the investigated strategies. In addition, immunization of mice with the recombinant Neospora antigen NcSRS2, associated with iscoms through the biotin-streptavidin interaction, induced specific antibodies to native NcSRS2 and reduced clinical symptoms following challenge infection. The systems described in this thesis might offer convenient and efficient methods for incorporating recombinant immunogens into adjuvant formulations that might be considered for the generation of future recombinant subunit vaccines.</p><p>In a second series of studies, Affibody® (affibody) ligands directed to the extracellular domain of human epidermal growth factor receptor 2 (HER2/neu), which is known to be overexpressed in ∼ 20-30% of breast cancers, were isolated by phage display <i>in vitro</i> selection from a combinatorial protein library based on the 58 amino acid residue staphylococcal protein A-derived Z domain. Biosensor analyses demonstrated that one of the variants from the phage selection, denoted His₆-Z_HER2/neu:4, selectively bound with nanomolar affinity (KD ≈ 50 nM) to the extracellular domain of HER2/neu (HER2-ECD) at a different site than the monoclonal antibody trastuzumab. In order to exploit avidity effects, a bivalent affibody ligand was constructed by head-to-tail dimerization, resulting in a 15.6 kDa affibody ligand, termed His₆-(Z_HER2/neu:4)₂, that was shown to have an improved apparent affinity to HER2-ECD (KD ≈ 3 nM) compared to the monovalent affibody. Moreover, radiolabeled monovalent and bivalent affibody ligands showed specific binding in vitro to native HER2/neu molecules expressed in human cancer cells. Biodistribution studies in mice carrying SKOV-3 xenografted tumors revealed that significant amounts of radioactivity were specifically targeted to the tumors <i>in vivo</i>, and the tumors could easily be visualized with a gamma camera. These results suggest that affibody ligands would be interesting candidates for specific tumor targeting in clinical applications, such as <i>in vivo</i> imaging and radiotherapy.</p>

<p>Mast cells (MC) are potent inflammatory cells that are known primarily for their prominent role in IgE mediated allergies. However, they also provide beneficial functions to the host, e.g. in bacterial and parasitic defence. MCs react rapidly upon stimulation by releasing potent granule-stored mediators, and serine proteases of the chymase or tryptase families are such major granule constituents. </p><p>As a first step towards a better understanding of the biological function of these proteases, we have determined the extended cleavage specificities of four mammalian mast cell chymases, by utilizing a substrate phage display approach. The specificities of these enzymes have then been used to compare their functional characteristics.</p><p>The major mucosal MC chymase in mice, mMCP-1, was found to possess a strict preference in four amino acid positions of the peptide substrate. Using this sequence to search the mouse proteome for potential <i>in vivo</i> substrates led to the identification of several very interesting potential novel substrates. Some of them may explain the increased epithelial permeability provided by this enzyme.</p><p>Human MCs, express only one single α-chymase, and the rodent α-chymases have secondarily gained elastase-like primary cleavage specificity. However, rodents express additional chymases, the β-chymases, and rodent β-chymases may have adopted the function of the α-chymases. The cleavage specificities of the human chymase and two rodent β-chymases were therefore determined (rat rMCP-1 and mouse mMCP-4). N-terminal of the cleaved bond the three chymases showed similar preferences, but C-terminal the human chymase and mMCP-4 shared a high preference for acidic amino acids in the P2´ position and therefore seem to be functional homologues. The molecular interactions mediating the preference for acidic amino acids in position P2´ were further investigated. By site-directed mutagenesis of the human chymase, amino acids Arg143 and Lys192 were concluded to synergistically mediate this preference.</p><p>Our data show that chymases, of different MC subpopulations, display quite different extended cleavage specificities. However mouse do possess a MC chymase with almost identical cleavage specificity as the human MC chymase indicating a strong evolutionary pressure to maintain this enzyme specificity.</p>

Viruses are the most abundant biological entity on Earth and outnumber their hosts ten-to-one. Ocean viruses (phages) impact bacterial-driven global biogeochemical cycles through lysis, manipulating host metabolism, and horizontal gene transfer. However, knowledge of virus-host interactions and viral roles in ecosystems remains limited due to few cultured marine phage genomes and non-quantitative culture-independent metagenomes. Here, I develop and apply novel and well-tested bioinformatic techniques to explore Pacific Ocean viral communities using quantitative datasets derived from rigorously-tested preparation methods. To evaluate concentration and purification methods, I examined triplicate metagenomes from a single ocean sample using four protocols. Concentration protocols showed statistical differences in taxonomy whereas purification protocols did not. Specifically, TFF-concentrated metagenomes contained trace bacterial contamination and had fewer abundant taxa as compared to FeCl₃-precipitated metagenomes. K-mer analysis using the complete dataset revealed polymerase choice defined access to "rare" sequences.To explore unknown viral sequences, I organized known and unknown sequence space into 27K high-confidence protein clusters (PCs) from 32 diverse Pacific Ocean Virus (POV) metagenomes, which doubled available PCs and included the first pelagic deep-sea viral metagenomes. Using PCs as a whole-viral-community diversity metric revealed decreases from coastal to open ocean, winter to summer, and deep to surface, that correlate with data from microbial genetic diversity markers (no parallel viral markers exist).Biologically, POV metagenomes showed that viruses likely reprogram central metabolic pathways in microbial communities far beyond the "photosynthesis viruses" paradigm. Gene distribution patterns from 35 viral gene families (31 new) revealed niche-specific (photic vs aphotic zone) altered pathway carbon flux presumably optimized to best locally generate energy and drive viral replication. Further, these PCs define the first "core" (180 genes) and "flexible" (423K genes total) viral community genome. Functionally, core genes again suggest niche-differentation with extensive Fe-S cluster-related genes for electron transport and metabolic enzyme catalysis in photic samples, and manipulation of host pressure-sensitive genes in aphotic samples. Taxonomically, these data deconstruct the culture-based paradigm that tailed viruses dominate in the wild - instead they appear ubiquitous, but not abundant.

The complete genome sequence of bacteriophage VR7 has been determined. The genome sequence is 169,285 nt, with an overall G+C content of 40,3%, compared with 35.3 % of T4. VR7 encodes 293 putative protein-encoding open reading frames (ORFs) and tRNAMet. In total, 211 of the 293 VR7 open reading frames encode putative proteins that share 30% ‒ 97% amino acid sequence identity with those found in T4; 46 ORFs resemble genes from other T4-like phages, 9 show similarities to genes of non T4 type phages and 27 ORFs lack any database matches. Homologs to the T4 α-gt, β-gt, SegA, SegB, SegC, SegD, SegE, I-TevI, I-TevII, I-TevIII, gp42, Ac, NrdG, NrdD, Arn, IPI, IPII, IPIII, Mrh as well as the T4-specific tRNAs are all absent in VR7. The amino acid sequences of the three major structural proteins gp23, gp18 and gp19 of VR7 show 84.9%, 71.3% and 69.9% aa identity respectively with adequate proteins of T4. In total, 43 PE, 43 PM and 44 PL have been identified in VR7. Moreover, phage VR7 encodes homologues of all transcription-associated proteins of T4. The functional complementation experiments of VR7 MotA, sharing only 34% amino acid sequence identity with MotA of T4, have been performed. It has been demonstrated, that the presence of plasmid encoded VR7 MotA complements the T4motAΔ mutant for growth in E. coli, and activates middle-mode transcription during the growth of T4motA-. Bacteriophages VR5, VR7 and VR20 have been characterized. It has been demonstrated that these phages... [to full text] / Nustačius 169,285 b.p. bakteriofago VR7 genomo nukleotidų seką aptikta viena tRNRMET ir 293 hipotetiniai ASR. Du šimtai vienuolika šio fago genų koduoja baltymus, kurie yra 30% ‒ 97% homologiški atitinkamiems fago T4 baltymams. Keturiasdešimt šeši fago VR7 baltymai neturi analogų T4, bet yra homologiški įvairių kitų T4 giminingų fagų baltymams, 9 baltymai nėra artimi T4-giminingų fagų koduojamiems baltymams, o 27 bakteriofago VR7 ASR koduoja baltymus, kuriems homologų NCBI duomenų bazėje nėra. Fago VR7 genome nėra genų, koduojančių bakteriofago T4 : α ir β gliukoziltransferazes (α-gt , β-gt) , DNR endonukleazes SegA, SegB, SegC, SegD, DNR metilazę Dam, dCMP hidroksimetilazę gp42, atsparumą akriflavinui sąlygojantį baltymą Ac bei ląstelės šeimininkės σ32 fosforilinime dalyvaujančio mrh geno produkto. Nustatyta, kad GC sudaro 40,3% fago VR7 genominės DNR, kai tuo tarpu fago T4 - 35%. Taipogi nustatyta, kad VR7 gp18, gp19 ir gp23 yra tik 71.3% , 69.9% ir 84.9% homologiški atitinkamiems fago T4 baltymams. Tiriant bakteriofago VR7 transkripcijos reguliaciją buvo aptikti 43 ankstyvieji, 43 vidurinieji bei 44 vėlyvieji promotoriai. Šio fago genominėje DNR taip pat buvo identifikuoti visų fago T4 transkripcijos reguliacijoje dalyvaujančių baltymų homologai. Klonavus bakteriofago VR7 geną motA buvo atliktas funkcinės komplementacijos tyrimas fago T4motA- sistemoje in vivo. Nustatyta, kad plazmidėje koduojamas fago VR7 viduriniosios transkripcijos aktyvatorius MotA, kurio... [toliau žr. visą tekstą]

The Life Marker Chip instrument, which has been selected to fly as part of the 2018 ExoMars rover mission payload, aims to detect up to 25 organic molecules in martian rocks and regolith, as markers of extant life, extinct life, meteoritic in-fall and spacecraft contamination. Martian samples will be extracted with a solvent and the resulting liquid extracts will be analysed using multiplexed microarray-format immunoassays. The LMC is under development by an international consortium led by the University of Leicester and the work described within this thesis was carried out at Cranfield University as part of the consortium’s broader program of work preparing the LMC instrument for flight in 2018. Within this thesis four specific areas of LMC instrument development are addressed: the investigation of immunoassay compatible liquid extraction solvents, the study of likely interactions of martian sample matrix with immunoassays, the development of antibodies for the detection of markers of extinct life and demonstration of solvent extraction and immunoassay detection in a flight representative format. Cont/d.