| 1. |
- Dahlin, Paul, 1983-
(författare)
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Analysis of sterol metabolism in the pathogenic oomycetes Saprolegnia parasitica and Phytophthora infestans
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The primary objective of this thesis was to investigate the sterol metabolism of two pathogenic oomycetes, specifically the processes of sterol synthesis and sterol acquisition in the fish pathogen Saprolegnia parasitica (Saprolegniales) and the plant pathogen Phytophthora infestans (Peronosporales). Furthermore, the effects of steroidal glycoalkaloids from Solanaceous plants, on P. infestans, were examined. The improved understanding of these processes should help to identify approaches for the identification of new oomycete inhibitors targeting sterol metabolism in agriculture and aquaculture farming systems, and to guide plant-breeding strategies to defend solanaceous plants against oomycetes.For these reasons, the molecular basis of the metabolic pathways of sterol synthesis and/or sterol acquisition was investigated. Sterols are derived from isoprenoids and indispensable in various biological processes. Our biochemical investigation of an oxidosqualene cyclase revealed that sterol synthesis in S. parasitica begins with the formation of lanosterol (Paper I), and a reconstruction of the complete sterol synthesis pathway to the final compound, fucosterol, in S. parasitica was performed using bioinformatics (Paper II). Complementary to this work, the extent to which P. infestans, which is incapable of de novo sterol synthesis, is able to modify exogenously provided sterols was investigated by determining the growth impact of various sterol supplements in the growth media (Paper II). Building on the sterol investigations, the solanaceous sterol derivatives from the glycoalkaloid family were analysed. These compounds contain both a steroidal and a carbohydrate (glycan) moiety. Data obtained by feeding various deuterium-labeled sterols to potato shoots, supported the theory that steroidal glycoalkaloids in Solanum tuberosum are produced from cholesterol (Paper III). Since these steroidal glycoalkaloids are thought to play a role in plant defense, their physiological effects on P. infestans were investigated (Paper IV). Unexpectedly we found that non-glycosylated steroidal alkaloids had a greater inhibitory effect than steroidal glycoalkaloids. Steroidal glycoalkaloids derived from other Solanaceous species exhibited different physiological effects on the growth of P. infestans. This research was conducted on two oomycete species belonging to the Saprolegniales and Peronosporales orders, hence the results presented are likely to be representative of each of these two oomycete orders.
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| 2. |
- Larsbrink, Johan, 1982-
(författare)
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Strategies for the Discovery of Carbohydrate-Active Enzymes from Environmental Bacteria
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The focus of this thesis is a comparative study of approaches in discovery of carbohydrate-active enzymes (CAZymes). CAZymes synthesise, bind to, and degrade all the multitude of carbohydrates found in nature. As such, when aiming for sustainable methods to degrade plant biomass for the generation of biofuels, for which there is a strong drive in society, CAZymes are a natural source of environmentally friendly molecular tools.In nature, microorganisms are the principal degraders of carbohydrates. Not only do they degrade plant matter in forests and aquatic habitats, but also break down the majority of carbohydrates ingested by animals. These symbiotic microorganisms, known as the microbiota, reside in animal digestive tracts in immense quantities, where one of the key nutrient sources is complex carbohydrates. Thus, microorganisms are a plentiful source of CAZymes, and strategies in the discovery of new enzymes from bacterial sources have been the basis for the work presented here, combined with biochemical characterisation of several enzymes.Novel enzymatic activities for the glycoside hydrolase family 31 have been described as a result of the initial projects of the thesis. These later evolved into projects studying bacterial multi-gene systems for the partial or complete degradation of the heterogeneous plant polysaccharide xyloglucan. These systems contain, in addition to various hydrolytic CAZymes, necessary binding-, transport-, and regulatory proteins. The results presented here show, in detail, how very complex carbohydrates can efficiently be degraded by bacterial enzymes of industrial relevance.
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| 3. |
- Brown, Christian, 1976-
(författare)
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Characterization of specific domains of the cellulose and chitin synthases from pathogenic oomycetes
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Some oomycetes species are severe pathogens of fish or crops. As such, they are responsible for important losses in the aquaculture industry as well as in agriculture. Saprolegnia parasitica is a major concern in aquaculture as there is currently no method available for controlling the diseases caused by this microorganism. The cell wall is an extracellular matrix composed essentially of polysaccharides, whose integrity is required for oomycete viability. Thus, the enzymes involved in the biosynthesis of cell wall components, such as cellulose and chitin synthases, represent ideal targets for disease control. However, the biochemical properties of these enzymes are poorly understood, which limits our capacity to develop specific inhibitors that can be used for blocking the growth of pathogenic oomycetes.In our work, we have used Saprolegnia monoica as a model species for oomycetes to characterize two types of domains that occur specifically in oomycete carbohydrate synthases: the Pleckstrin Homology (PH) domain of a cellulose synthase and the so-called ‘Microtubule Interacting and Trafficking’ (MIT) domain of chitin synthases. In addition, the chitin synthase activity of the oomycete phytopathogen Aphanomyces euteiches was characterized in vitro using biochemical approaches.The results from our in vitro investigations revealed that the PH domain of the oomycete cellulose synthase binds to phosphoinositides, microtubules and F-actin. In addition, cell biology approaches were used to demonstrate that the PH domain co-localize with F-actin in vivo. The structure of the MIT domain of chitin synthase (CHS) 1 was solved by NMR. In vitro binding assays performed on recombinant MIT domains from CHS 1 and CHS 2 demonstrated that both proteins strongly interact with phosphatidic acid in vitro. These results were further supported by in silico data where biomimetic membranes composed of different phospholipids were designed for interaction studies. The use of a yeast-two-hybrid approach suggested that the MIT domain of CHS 2 interacts with the delta subunit of Adaptor Protein 3, which is involved in protein trafficking. These data support a role of the MIT domains in the cellular targeting of CHS proteins. Our biochemical data on the characterization of the chitin synthase activity of A. euteiches suggest the existence of two distinct enzymes responsible for the formation of water soluble and insoluble chitosaccharides, which is consistent with the existence of two putative CHS genes in the genome of this species.Altogether our data support a role of the PH domain of cellulose synthase and MIT domains of CHS in membrane trafficking and cellular location.
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| 4. |
- Leijon, Felicia, 1983-
(författare)
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Understanding and manipulating primary cell walls in plant cell suspension cultures
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The cell wall is required for many aspects of plant function and development. It is also an accessible and renewable resource utilized both in unrefined forms and as raw material for further development. Increased knowledge regarding cell wall structure and components will contribute to better utilization of plants and the resources they provide. In this thesis aspects of the primary cell wall of Populus trichocarpa and Nicotiana tabacum are explored.In Publication I a method for isolation and biochemical characterization of plant glycosyltransferases using a spectrophotometric or a radiometric assay was optimized. The radiometric assay was applied in Publication II where the proteome of the plasmodesmata isolated from P. trichocarpa was analyzed. Proteins identified belonged to functional classes such as “transport”, “signalling” and “stress responses”. Plasmodesmata-enriched fractions had high levels of callose synthase activity under ion depleted conditions as well as with calcium present.The second part of the thesis comprises the alteration of the cell wall of N. tabacum cells and A. thaliana plants through in vivo expression of a carbohydrate binding module (CBM) (Publication III). In tobacco this resulted in cell walls with loose ultrastructure containing an increased proportion of 1,4-β-glucans. The cell walls were more susceptible to saccharification, possibly due to changes in the structure of cellulose or xyloglucan. Arabidopsis plants showed increased saccharification after mild pretreatment, suggesting that heterologous expression of CBMs is a promising method for cell wall engineering. In Publication IV cellulose microfibrils (CMFs) and nanocrystals (CNCs) were extracted from the transgenic cells. CNC preparation resulted in higher yields and longer CNCs. Nanopapers prepared from the CMFs of the CBM line demonstrated enhanced strength and toughness. Thus, changes to the ordered regions of cellulose were suggested to take place due to CBM expression.
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| 5. |
- Malm, Erik, 1981-
(författare)
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Analyzing the properties and biosynthesis of β-glucans from Gluconacetobacter and poplar
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Glucans are polysaccharides integral to many materials and biological functions. Under the umbrella of Biomime, the Swedish Center for Biomimetic Fiber Engineering, this work has aimed to improve basic understanding of the biosynthesis of such glucans. This has been achieved through direct investigation of cellulose structure, and by developing the tools to analyze glucan biosynthesis. Notably we have identified a novel chemical effector of glucan synthesis processes and developed a proteomic toolkit useful for analyzing membrane-bound glycosyltransferases, the enzyme group responsible for glucan biosynthesis. During this work, glucan synthesis has been studied using both Gluconacetobacter and Populus cell suspension cultures.Publication I. Gluconacetobacter cellulose (BC) was used as a base to create a novel and well characterized nano-material with improved mechanical properties. This novel composite of BC and hydroxyethylcellulose (HEC) had improved tensile strength compared to pure BC. Through thorough study utilizing dispersion measurements, electron microscopy, nuclear magnetic resonance and X-ray diffraction it was shown that the improved properties derived from a layer of HEC coating each fibril.Publication II. Bacterial cellulose was labeled in specific positions with 13C (C4 and C6). These samples were analyzed by CP/MAS NMR along with cellulose samples from cotton and Halocynthia sp. For each sample spectral fitting was performed and general properties of crystal allomorph composition and fibril widths were determined. Calculations were also made for water accessible surfaces of the fibrils. The results showed that water accessible C4 surface signals are reflective of the allomorph composition of the sample, along with a distorted signal that derives due to fibril imperfections. Water accessible surface signals from the C6 region are instead derived from rotamer conformations of the C6 hydroxymethyl groupsfrom glucose residues.In Publication III, a high-throughput screen was used to identify an inhibitor of Golgi-derived glycosyltransferase activity, termed chemical A. The structural basis for inhibition was determined and in vitro assays of callose synthesis were performed. The in vitro assays revealed chemical A to also be an activator of callose synthesis. To understand this activation kinetic studies were performed, showing that chemical A is a mixed type of activator, which can bind either the free enzyme or the enzyme-substrate complex. Chemical A has uses in chemical genetics for dissecting processes involving callose synthesis, such as stress response and cell-plate formation.In publication IV, we present an in-house developed platform for proteomics with a distributed processing model. This in-house system has been central to many proteomics tasks, including for those presented in publication V, and is being distributed as the Automated Proteomics Pipeline (APP).In publication V, conditions for enrichment of Detergent-Resistant Microdomains (DRM) have been optimized for Populus trichocarpa cell cultures. The proteins enriched in DRM were identified using mass spectrometry based proteomics, and a functional model for DRM was proposed. This model involves proteins specialized in stress response, including callose synthase, and cell signaling. This further strengthens the arguments for DRMs as sites of specific cellular functions and confirms they play a role in glucan synthesis.
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| 6. |
- Rzeszutek, Elzbieta
(författare)
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Cell wall biosynthesis in the pathogenic oomycete Saprolegnia parasitica
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The oomycete Saprolegnia parasitica is a fungus-like microorganism responsible for the fish disease saprolegniosis, which leads to important economic losses in aquaculture. Currently, there is no efficient method to control the infection and therefore methods for disease management are urgently needed. One of the promising approaches to tackle the pathogen is the inhibition of cell wall biosynthesis, specifically the enzymes involved in carbohydrate biosynthesis. The cell wall of S. parasitica consists mainly of cellulose, β-1,3 and β-1,6-glucans, whereas chitin is present in minute amounts only. The available genome sequence allowed the identification of six putative chitin (Chs) and cellulose (CesA) synthase genes. The main objective of this work was to characterize CHSs and CesAs from S. parasitica and test the effect of cell wall related inhibitors on pathogen growth. The tested inhibitors included nikkomycin Z, a competitive inhibitor of CHS as well as inhibitors of cellulose biosynthesis, namely flupoxam, CGA325'615 and compound I (CI). All drugs strongly reduced the growth of S. parasitica and inhibited the in vitro formation of chitin or cellulose, demonstrated by the use of a radiometric assay. The chemicals also affected the expression of some of the corresponding Chs and CesA genes.One of the CHSs, namely SpCHS5, was successfully expressed in yeast and purified to homogeneity as a full length protein. The recombinant enzyme was biochemically characterized and demonstrated to form chitin crystallites in vitro. Moreover, our data indicate that SpCHS5 most likely occurs as a homodimer which can further assemble into larger multi-subunit complexes. Point mutations of conserved amino acids allowed us to identify the essential residues for activity and processivity of the enzyme.In addition to the cell wall related inhibitors, a biosurfactant naturally produced by Pseudomonas species, massetolide A, was tested, showing strong inhibition of S. parasitica growth.Altogether, our data provide key information on the fundamental mechanisms of chitin and cellulose biosynthesis in oomycetes and the biochemical properties of the enzymes involved. They also demonstrate that the enzymes involved in cell wall biosynthesis represent promising targets for anti-oomycete drugs, even when the corresponding polysaccharides, such as chitin, occur in small amounts in the cell wall.
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| 7. |
- Fugelstad, Johanna, 1981-
(författare)
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Functional characterization of cellulose and chitin synthase genes in Oomycetes
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Some species of Oomycetes are well studied pathogens that cause considerable economical losses in the agriculture and aquaculture industries. Currently, there are no chemicals available that are environmentally friendly and at the same time efficient Oomycete inhibitors. The cell wall of Oomycetes consists of b-(1à3) and b-(1à6)-glucans, cellulose and in some species minute amounts of chitin. The biosynthesis of cellulose and chitin in Oomycetes is poorly understood. However, cell wall synthesis represents a potential target for new Oomycete inhibitors. In this work, cellulose and chitin synthase genes and gene products were analyzed in the plant pathogen Phytophthora infestans and in the fish pathogen Saprolegnia monoica. A new Oomycete CesA gene family was identified, containing four subclasses of genes designated as CesA1 to 4. The gene products of CesA1, 2 and 4 contain pleckstrin homology (PH) domains located at the N-terminus, which is unique to the Oomycete CesAs. Our results show that the SmCesA2 PH domain binds to phosphoinositides, F-actin and microtubules in vitro and can co-localize with F-actin in vivo. Functional characterization of the CesA genes by gene silencing in P. infestans led to decreased cellulose content in the cell wall. The cellulose synthase inhibitors DCB and Congo Red inhibited the growth of the mycelium of S. monoica and had an up-regulating effect on SmCesA gene expression. Zoospores from P. infestans treated with DCB were unable to infect potato leaves. In addition, two full-length chitin synthase genes (Chs) were analyzed from S. monoica. Expression of SmChs2 in yeast yielded an active recombinant protein. The biochemical characterization of the in vitro product of SmChs2 confirmed that the protein is responsible for chitin formation. The chitin synthase inhibitor nikkomycin Z inhibited the SmChs2 both in vivo and in vitro. Altogether these results show that at least some of the CesA1-4 genes are involved in cellulose biosynthesis and that synthesis of cellulose is crucial for infection of potato by P. infestans. The PH domain is involved in the interaction of CesA with the cytoskeleton. In addition, we firmly demonstrate that the SmChs2 gene encodes a catalytically active chitin synthase.
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| 8. |
- Guanglin, Kuang, 1987-
(författare)
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Theoretical Studies of Protein-Ligand Interactions
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The protein-ligand interaction is an important issue in rational drug design and protein function research. This thesis focuses on the study of protein-ligand interactions using various molecular modeling methods, which are used in combination to predict the binding modes and calculate the binding free energies of several important protein-ligand systems, as summarized below.In Paper I, we investigated the binding profile of a type I positive allosteric modulator (PAM) NS-1738 with the α7-nicotinic acetylcholine receptor (α7-nAChR). NS-1738 is found to have three different binding sites on α7-nAChR and has moderate binding affinities to the receptor.In Paper II, we revealed the binding mechanism of a PET radio-ligand [18F]ASEM with α7-nAChR. Using metadynamics simulations, we managed to find a stable state which is not observed in molecular docking and unbiased molecular dynamics simulations. Free energy analysis further confirmed that this stable state is the global minimum with respect to the selected collective variables.In Paper III, we studied the binding modes and binding affinities of two probes (AZD2184 and thioflavin T) for the detection of amyloid β(1-42) fibrils in clinical studies. We found that AZD2184 and thioflavin T are able to bind to several sites of the Aβ(1-42) fibril. Due to the small size, planarity and neutrality of AZD2184, it binds more strongly with Aβ(1-42) fibril at all sites. By contrast, thioflavin T has more significant conformational changes after binding, which is the reason that thioflavin T can be used as a fluorescent probe in in vitro studies.In Paper IV, we studied the binding profile of PtdIns(3,4,5)P3 with the plecsktrin homology (PH) domain of Saprolegnia monoica cellulose synthase. We first studied the binding modes of the inositol groups with the PH domain in solution, the results of which were then used to guide the modeling of the binding mode of PtdIns(3,4,5)P3 in a membrane with the PH domain.
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| 9. |
- Klinter, Stefan, 1985-
(författare)
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Identification and characterisation of chitin and cellulose synthases in oomycetes : New tools for biochemical studies and structure determination
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Despite resembling ‘true’ fungi in terms of morphological features, oomycetes form a distinct eukaryotic lineage of filamentous microorganisms that belongs to the stramenopiles, a group of protists also comprising the closely-related brown algae and diatoms. Many oomycetes are devastating pathogens of plants and animals, globally causing significant economic losses in the agriculture and aquaculture industries, and posing considerable environmental damage to natural ecosystems. Although the cell wall (CW) is critical for the viability and morphogenesis of the organism it surrounds, our knowledge of oomycete CW architecture and biosynthetic enzymes is limited. Given the vast threat that pathogenic oomycetes pose, uncovering the details of CW biosynthesis and regulation in these pathogens may reveal new opportunities for disease control.To this end, we aimed to elucidate the role of putative membrane-bound glycosyltransferase family 2 enzymes implicated in the biosynthesis of oomycete CW polysaccharides. Suitable gene candidates were identified, and their products analysed, as illustrated by the oomycete-wide discovery and phylogenetic analysis of the chitin synthase gene family (paper I), and the identification of the cellulose synthase genes in Saprolegnia parasitica (paper II) and Phytophthora capsici (paper III). Expression of promising candidate genes was verified using different techniques, including gene expression analysis (papers II and III), and the effect of inhibitors on hyphal growth (papers I and II) and enzymatic activity in in vitro assays (paper II). Single enzymes representing putative chitin synthases from various organisms (unpublished data) and cellulose synthases from S. parasitica (extended data for paper II), and P. capsici cellulose synthase 1 (paper III) were produced, and partly enriched or even purified, in yeast strains specifically engineered to facilitate the biochemical characterisation of the recombinant proteins in in vitro enzyme assays. To advance functional investigations and structure determination of integral membrane proteins, we developed DirectMX, a method that allows the reconstitution of target proteins with their surrounding lipids directly from crude cell membranes into Salipro nanoparticles (paper IV).
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| 10. |
- Zhang, Ye, 1984-
(författare)
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High cell density perfusion process development for antibody producing Chinese Hamster Ovary cells
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Perfusion operation mode is currently under fast expansion in mammalian cell based manufacturing of biopharmaceuticals, not only for labile drug protein but also for stable proteins such as monoclonal antibodies (mAbs). Perfusion mode can advantageously offer a stable cell environment, long-term production with high productivity and consistent product quality. Intensified high cell density culture (HCDC) is certainly one of the most attractive features of a perfusion process due to the high volumetric productivity in a small footprint that it can provide. Advancements in single-use technology have alleviated the intrinsic complexity of perfusion processes while the maturing in cell retention devices has improved process robustness. The knowledge for perfusion process has been gradually built and the “continuous” concept is getting more and more acceptance in the field.This thesis presents the development of robust perfusion process at very high cell densities in various culture systems. Four HCDC perfusion systems were developed with industrial collaborators with three different mAb producing Chinese Hamster Ovary (CHO) cell lines: 1-2) WAVE Bioreactor™ Cellbag prototype equipped with cell separation by hollow fiber filter utilizing Alternating Tangential Flow (ATF) and Tangential Flow Filtration (TFF) techniques; 3) Fiber matrix based CellTank™ prototype; 4) Glass stirred tank bioreactor equipped with ATF. In all the systems, extremely high viable cell densities above 130 million viable cells per milliliter (MVC/mL) up to 214 MVC/mL were achieved. Steady states were maintained and studied at 20-30 MVC/mL and 100-130 MVC/mL for process development. Perfusion rate selection based on cell specific perfusion rate (CSPR) was systematically investigated and exometabolome study was performed to explore the metabolic footprint of HCDC perfusion process.
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