107 research outputs found

    Accessible software frameworks for reproducible image analysis of host-pathogen interactions

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    Um die Mechanismen hinter lebensgefährlichen Krankheiten zu verstehen, müssen die zugrundeliegenden Interaktionen zwischen den Wirtszellen und krankheitserregenden Mikroorganismen bekannt sein. Die kontinuierlichen Verbesserungen in bildgebenden Verfahren und Computertechnologien ermöglichen die Anwendung von Methoden aus der bildbasierten Systembiologie, welche moderne Computeralgorithmen benutzt um das Verhalten von Zellen, Geweben oder ganzen Organen präzise zu messen. Um den Standards des digitalen Managements von Forschungsdaten zu genügen, müssen Algorithmen den FAIR-Prinzipien (Findability, Accessibility, Interoperability, and Reusability) entsprechen und zur Verbreitung ebenjener in der wissenschaftlichen Gemeinschaft beitragen. Dies ist insbesondere wichtig für interdisziplinäre Teams bestehend aus Experimentatoren und Informatikern, in denen Computerprogramme zur Verbesserung der Kommunikation und schnellerer Adaption von neuen Technologien beitragen können. In dieser Arbeit wurden daher Software-Frameworks entwickelt, welche dazu beitragen die FAIR-Prinzipien durch die Entwicklung von standardisierten, reproduzierbaren, hochperformanten, und leicht zugänglichen Softwarepaketen zur Quantifizierung von Interaktionen in biologischen System zu verbreiten. Zusammenfassend zeigt diese Arbeit wie Software-Frameworks zu der Charakterisierung von Interaktionen zwischen Wirtszellen und Pathogenen beitragen können, indem der Entwurf und die Anwendung von quantitativen und FAIR-kompatiblen Bildanalyseprogrammen vereinfacht werden. Diese Verbesserungen erleichtern zukünftige Kollaborationen mit Lebenswissenschaftlern und Medizinern, was nach dem Prinzip der bildbasierten Systembiologie zur Entwicklung von neuen Experimenten, Bildgebungsverfahren, Algorithmen, und Computermodellen führen wird

    Molecular approaches to increasing resistance of wheat (Triticum aestivum L.) towards two insect pests; Cereal aphid (Sitobion avenae F.) and Wheat bulb fly (Delia coarctata Fallen).

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    Cereal aphid (Sitobion avenae) and wheat bulb fly (Delia coarctata) are serious pests of wheat in the UK. At the present, chemical pesticides are used to control these insects, but they are limited in effectiveness, and have undersirable ecological impacts. There is a need to improve wheat genetically to be resistant to such inset pests. The objectives of this work were to investigate digestive biochemistry in the selected insect pests of wheat, and to determine effects of potential endogenous resistance factors in wheat on digestion, nutrition and other insect metabolic processes. The aim was to develop new strategies for crop protection. Digestive biochemistry in S. avenae and D. coarctata was studied to characterise gut proteases and their inhibition by host plant proteinase inhibitors (PIs). Investigation of proteolytic digestion in S. avenae gut showed that in spite of being a phloem-feeding insect, cereal aphid could digest ingested protein, using cysteine proteases. D. coarctata larvae contained mainly serine protease activity. A serine protease (DcSP) and a cysteine protease (DcCathL) from D. coarctata gut tissue were expressed as recombinant proteins. Only DcCathL was recovered in active form. DcCathL was insecticidal to Mamestra brassicae when injected into hemolymph, causing systemic and extensive melanisation. DcCathL selectively degraded recombinant serpins from M. brassicae in in vitro assays, and is suggested to interfere with regulation of the proteolytic cascade leading to phenoloxidase activation and melanin production in vivo. DcCathL has potential as a biopesticide if it could be made effective when orally delivered. A cationic amino acid transporter from D. coarctata gut (DcCAAT) was also cloned as a target for RNA interference. Potential resistance factors in wheat were characterised by expression as recombinant proteins. Two PIs from wheat (subtilisin/chymotrypsin inhibitor; WSCI, and cysteine proteinase inhibitor; WCPI) were expressed in the yeast Pichia pastoris, and purified. WSCI inhibited gut protease activity of both insects in in vitro and in vivo assays, whereas WCPI only inhibited S. avenae gut extract activity. On feeding, WSCI was antimetabolic to both insects, affecting both survival and growth, whereas WCPI was antimetabolic to S. avenae only. Wheat Hessian fly responsive (Hfr) genes are up-regulated in response to herbivory by Hessian fly (Mayetiola destructor). The protein product Hfr-3 was expressed and purified, and showed antimetabolic effects on survival and growth of both S. avenae and D. coarctata. Both accumulated and induced defence proteins, like WSCI, WCPI and Hfr-3, have the potential to act as endogenous resistance factors in wheat towards a range of insect pests. Developing a wheat variety constitutively expressing these defence proteins by using traditional breeding methods and/or modern biotechnological tools is discussed

    A survey of the application of soft computing to investment and financial trading

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    Wheat Improvement

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    This open-access textbook provides a comprehensive, up-to-date guide for students and practitioners wishing to access in a single volume the key disciplines and principles of wheat breeding. Wheat is a cornerstone of food security: it is the most widely grown of any crop and provides 20% of all human calories and protein. The authorship of this book includes world class researchers and breeders whose expertise spans cutting-edge academic science all the way to impacts in farmers’ fields. The book’s themes and authors were selected to provide a didactic work that considers the background to wheat improvement, current mainstream breeding approaches, and translational research and avant garde technologies that enable new breakthroughs in science to impact productivity. While the volume provides an overview for professionals interested in wheat, many of the ideas and methods presented are equally relevant to small grain cereals and crop improvement in general. The book is affordable, and because it is open access, can be readily shared and translated -- in whole or in part -- to university classes, members of breeding teams (from directors to technicians), conference participants, extension agents and farmers. Given the challenges currently faced by academia, industry and national wheat programs to produce higher crop yields --- often with less inputs and under increasingly harsher climates -- this volume is a timely addition to their toolkit

    Statistical and image analysis methods and applications

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    STED Nanoscopy to Illuminate New Avenues in Cancer Research – From Live Cell Staining and Direct Imaging to Decisive Preclinical Insights for Diagnosis and Therapy

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    Molecular imaging is established as an indispensable tool in various areas of cancer research, ranging from basic cancer biology and preclinical research to clinical trials and medical practice. In particular, the field of fluorescence imaging has experienced exceptional progress during the last three decades with the development of various in vivo technologies. Within this field, fluorescence microscopy is primarily of experimental use since it is especially qualified for addressing the fundamental questions of molecular oncology. As stimulated emission depletion (STED) nanoscopy combines the highest spatial and temporal resolutions with live specimen compatibility, it is best-suited for real-time investigations of the differences in the molecular machineries of malignant and normal cells to eventually translate the acquired knowledge into increased diagnostic and therapeutic efficacy. This thesis presents the application of STED nanoscopy to two acute topics in cancer research of direct or indirect clinical interest. The first project has investigated the structure of telomeres, the ends of the linear eukaryotic chromosomes, in intact human cells at the nanoscale. To protect genome integrity, a telomere can mask the chromosome end by folding back and sequestering its single-stranded 3’-overhang in an upstream part of the double-stranded DNA repeat region. The formed t-loop structure has so far only been visualized by electron microscopy and fluorescence nanoscopy with cross-linked mammalian telomeric DNA after disruption of cell nuclei and spreading. For the first time, this work demonstrates the existence of t-loops within their endogenous nuclear environment in intact human cells. The identification of further telomere conformations has laid the groundwork for distinguishing cancerous cells that use different telomere maintenance mechanisms based on their individual telomere populations by a combined STED nanoscopy and deep learning approach. The population difference was essentially attributed to the promyelocytic leukemia (PML) protein that significantly perturbs the organization of a subpopulation of telomeres towards an open conformation in cancer cells that employ a telomerase-independent, alternative telomere lengthening mechanism. Elucidating the nanoscale topology of telomeres and associated proteins within the nucleus has provided new insight into telomere structure-function relationships relevant for understanding the deregulation of telomere maintenance in cancer cells. After understanding the molecular foundations, this newly gained knowledge can be exploited to develop novel or refined diagnostic and treatment strategies. The second project has characterized the intracellular distribution of recently developed prostate cancer tracers. These novel prostate-specific membrane antigen (PSMA) inhibitors have revolutionized the treatment regimen of prostate cancer by enabling targeted imaging and therapy approaches. However, the exact internalization mechanism and the subcellular fate of these tracers have remained elusive. By combining STED nanoscopy with a newly developed non-standard live cell staining protocol, this work confirmed cell surface clustering of the targeted membrane antigen upon PSMA inhibitor binding, subsequent clathrin-dependent endocytosis and endosomal trafficking of the antigen-inhibitor complex. PSMA inhibitors accumulate in prostate cancer cells at clinically relevant time points, but strikingly and in contrast to the targeted antigen itself, they eventually distribute homogenously in the cytosol. This project has revealed the subcellular fate of PSMA/PSMA inhibitor complexes for the first time and provides crucial knowledge for the future application of these tracers including the development of new strategies in the field of prostate cancer diagnostics and therapeutics. Relying on the photostability and biocompatibility of the applied fluorophores, the performance of live cell STED nanoscopy in the field of cancer research is boosted by the development of improved fluorophores. The third project in this thesis introduces a biocompatible, small molecule near-infrared dye suitable for live cell STED imaging. By the application of a halogen dance rearrangement, a dihalogenated fluorinatable pyridinyl rhodamine could be synthesized at high yield. The option of subsequent radiolabeling combined with excellent optical properties and a non-toxic profile renders this dye an appropriate candidate for medical and bioimaging applications. Providing an intrinsic and highly specific mitochondrial targeting ability, the radiolabeled analogue is suggested as a vehicle for multimodal (positron emission tomography and optical imaging) medical imaging of mitochondria for cancer diagnosis and therapeutic approaches in patients and biopsy tissue. The absence of cytotoxicity is not only a crucial prerequisite for clinically used fluorophores. To guarantee the generation of meaningful data mirroring biological reality, the absence of cytotoxicity is likewise a decisive property of dyes applied in live cell STED nanoscopy. The fourth project in this thesis proposes a universal approach for cytotoxicity testing based on characterizing the influence of the compound of interest on the proliferation behavior of human cell lines using digital holographic cytometry. By applying this approach to recently developed live cell STED compatible dyes, pronounced cytotoxic effects could be excluded. Looking more closely, some of the tested dyes slightly altered cell proliferation, so this project provides guidance on the right choice of dye for the least invasive live cell STED experiments. Ultimately, live cell STED data should be exploited to extract as much biological information as possible. However, some information might be partially hidden by image degradation due the dynamics of living samples and the deliberate choice of rather conservative imaging parameters in order to preserve sample viability. The fifth project in this thesis presents a novel image restoration method in a Bayesian framework that simultaneously performs deconvolution, denoising as well as super-resolution, to restore images suffering from noise with mixed Poisson-Gaussian statistics. Established deconvolution or denoising methods that consider only one type of noise generally do not perform well on images degraded significantly by mixed noise. The newly introduced method was validated with live cell STED telomere data proving that the method can compete with state-of-the-art approaches. Taken together, this thesis demonstrates the value of an integrated approach for STED nanoscopy imaging studies. A coordinated workflow including sample preparation, image acquisition and data analysis provided a reliable platform for deriving meaningful conclusions for current questions in the field of cancer research. Moreover, this thesis emphasizes the strength of iteratively adapting the individual components in the operational chain and it particularly points towards those components that, if further improved, optimize the significance of the final results rendering live cell STED nanoscopy even more powerful

    SYNTHESIS AND EVALUATION OF ANTIMICROBIAL ACTIVITY OF PHENYL AND FURAN-2-YL[1,2,4] TRIAZOLO[4,3-a]QUINOXALIN-4(5H)-ONE AND THEIR HYDRAZONE PRECURSORS

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    A variety of 1-(s-phenyl)-[1,2,4]triazolo[4,3-a]quinoxalin-4(5H)-one (3a-3h) and 1-(s-furan-2-yl)-[1,2,4]triazolo[4,3- a]quinoxalin-4(5H)-one (5a-d) were synthesized from thermal annelation of corresponding hydrazones (2a-h) and (4a-d) respectively in the presence of ethylene glycol which is a high boiling solvent. The structures of the compounds prepared were confirmed by analytical and spectral data. Also, the newly synthesized compounds were evaluated for possible antimicrobial activity. 3-(2-(4-hydroxylbenzylidene)hydrazinyl)quinoxalin-2(1H)-one (2e) was the most active antibacterial agent while 1-(5-Chlorofuran-2-yl)-[1,2,4]triazolo[4,3-a]quinoxalin-4(5H)-one (5c) stood out as the most potent antifungal agent

    Exploitation of small cysteine-rich spider protein toxins as bio-insecticides

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    Recombinant fusion protein technology allows specific insecticidal protein and peptide toxins to display activity in orally-delivered bio-pesticides. Here, some small cysteine-rich protein toxins were evaluated as insecticides, including δ-amaurobitoxin-Pl1a (Pl1a) from tangled nest spider (Pireneitega luctuosa), ω-atracotoxin-Hv1a (Hv1a) from funnel web spider (Hadronyche versuta) and κ-theraphotoxin-Ec2a (Ec2a) from Eucratoscelus constrictus, which target insect voltage-gated sodium channels, calcium activated potassium channels and voltage-regulated potassium channels, respectively. Recombinant proteins were produced using the yeast Pichia pastoris as expression host, by combining the coding sequences of the toxin with that of snowdrop lectin ("carrier"), that can deliver these toxins to the central nervous system of the target pest. Experimental results showed the toxins alone had limited or even no activities without being fused to the N-terminal of snowdrop lectin "carrier". Further, fusion of toxins to proteins other than snowdrop lectin also gave products with low or no biological activity. The absence of biological activity suggested that the toxin protein was not folding properly when expressed without fusion to the snowdrop lectin carrier, which meant GNA could not only direct transport of the toxins across the insect gut as a carrier, but also can help toxins to achieve correct folding. For example, the toxin Pl1a and a Pl1a/GNA fusion protein both caused mortality when injected into cabbage moth (Mamestra brassicae) larvae, but the Pl1a/GNA fusion protein was approximately 6 times as effective as recombinant Pl1a on a molar basis. Pl1a alone was not orally active against cabbage moth larvae, but a single 30 μg dose of the Pl1a/GNA fusion protein caused 100% larval mortality within 6 days when fed to 3rd instar larvae, and caused significant reductions in survival, growth and feeding in 4th - 6th instar larvae. To attempt to further improve the folding of recombinant fusion proteins, the predicted Pro-regions of toxins, between the signal peptide and the final mature sequence of the protein were examined. Inclusion of the Pro-region in the expression construct was hypothesised to result in improved folding of the toxin when expressed in P. pastoris. The results proved that the new type fusion protein (Pro-region/toxin/GNA) had much higher biological activity than toxins alone and higher activity than toxin/GNA fusion proteins. In addition, the Pro-region was successfully removed from the Pro-region/toxin/GNA proteins after expression. For example, the LD50 of Pro-Hv1a/GNA was decreased by 12 fold compared to Hv1a/GNA when injected into Mamestra brassicae larvae of different stages of development. Increased biological activity of Pro-Hv1a/GNA when compared to Hv1a/GNA was also observed when the proteins were injected into slugs. The increased biological activity of Pro-Hv1a/GNA on injection was also observed as increased oral toxicity of the fusion protein to insects. A single dose (20 μg) of fusion protein Hv1a/GNA caused no mortality to 5th instar larvae of M. brassicae, or 30% mortality to 3rd instar larvae; in contrast, 20 μg Pro-Hv1a/GNA caused 30% mortality to 5th instar larvae, and 90% mortality to 3rd instar larvae. Fusion proteins have the potential to be a new class of bio-pesticides for commercial application and have potential uses in complementing or replacing existing pesticides. Insecticide-resistant strains of peach potato aphid (Myzus persicae), designated "kdr", "super-kdr" and "kdr+super-kdr" contain mutations in the voltage-gated sodium channel (NaCh). Pl1a/GNA and Pro-Hv1a/GNA fusion proteins have the LC50 values of 0.35 and 0.19 mg ml-1 when fed to wild-type M. persicae. For insecticide-resistant aphids, the LC50 for the Pl1a/GNA fusion protein, which targets NaCh, was increased by 2-6 fold correlating with pyrethroid resistance (wild-type < kdr < super-kdr < kdr+super-kdr strains). In contrast, the LC50 for the Pro-Hv1a/GNA, which targets calcium channels, showed limited correlation with pyrethroid resistance. Therefore, mutations in the sodium channel in pyrethroid-resistant aphids also protect against a fusion protein containing a sodium channel-specific toxin, despite differences in ligard-channel interactions. This may be because changes to the spatial structure of domain II as a result of these mutations presumably also disturb the binding of Pl1a to receptor site 4, in domain II of sodium ion channel. However, mutations in the sodium channel do not confer resistance to a fusion protein targeting calcium channels. The use of fusion proteins with differing targets could delay resistance development in M. persicae

    Image Registration Workshop Proceedings

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    Automatic image registration has often been considered as a preliminary step for higher-level processing, such as object recognition or data fusion. But with the unprecedented amounts of data which are being and will continue to be generated by newly developed sensors, the very topic of automatic image registration has become and important research topic. This workshop presents a collection of very high quality work which has been grouped in four main areas: (1) theoretical aspects of image registration; (2) applications to satellite imagery; (3) applications to medical imagery; and (4) image registration for computer vision research

    Characterisation of aphid proteins as targets for aphid control

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    There have been extensive investigations of allozymes in aphid species, but only a relatively small amount of variation has been found between and, especially, within species. While modern molecular methods have shown that there is a large amount of variation amongst and within aphid species at the DNA level, there has been no concomitant detailed investigation of protein variation. 2DE was used as a powerful method to analyse the protein complement of aphid tissues. When the protein profiles of remnant and gut tissues were compared within and between several aphid species, higher levels of conservation were found in remnant tissue proteins than in the gut tissue proteins. These conservation levels may indicate different evolutionary processes in the two tissue types. The remnant proteins may have specific functions across all aphid species, which restrict the chances of accumulating mutations. The gut proteins do not appear to be similarly constrained, with the wide variation in gut protein profiles observed amongst aphid species possibly related to differences in their host ranges. The presence of protein homologues or common precursor molecules was indicated where some protein appeared to have slight, but distinct, differences between the species. The protein data from both tissues was qualitatively analysed to produce parsimonious comparisons between the aphid species. The gut protein data gave strong relationships between the species, which were in agreement with a classification based on aphid morphometries. However, the high level of conservation in the remnant proteins appeared to have obscured any separation of the species using this data. The effects of changing diet on the proteins of the aphid gut were also explored using 2DE. Within each clone, and therefore within each species, a small subset of proteins varied with host plant. On both host plant species, an analysis of this variation found that the changes included both additions and absences within the aphid gut protein profile. A polyclonal antiserum was raised against total proteins from M. persicae, fed on Chinese cabbage. The cross-reactivity of anti- whole M. persicae antiserum with large numbers of Western blotted proteins from other aphid species confirmed the protein conservation observed after 2DE protein analysis. A second polyclonal antiserum, raised against gut proteins from M persicae fed on Chinese cabbage, also showed cross-reactivity with Western blotted proteins from other aphid species. Probing with lectins, which specifically bind to secondary carbohydrate structures, showed that many of these cross-reacting gut proteins were glycosylated. As has been found with some antisera raised against proteins from other insects, the secondary carbohydrate structure of the proteins may account for some of the cross-reactivity seen with proteins from other species. The cross-reactivity of the anti-gut antibody may also indicate the presence of homologous proteins occurring in the guts of aphid species, previously indicated after 2DE separation and silver staining of aphid proteins. After establishing a suitable artificial diet for the long term culture of M. persicae, the effects of including the polyclonal antisera raised against aphid proteins in the diet were assessed. Inclusion of anti- M. persicae gut antiserum in artificial diet had a detrimental effect on the longevity of feeding aphids. The findings of the thesis are discussed in context of aphid control and the current trend towards in planta methods
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