Aspergillus niger is an important industrial producer of organic acids and enzymes producing large amounts of spent fungal biomass. In the European Research Area Industrial Biotechnology (ERA-IB)... Show moreAspergillus niger is an important industrial producer of organic acids and enzymes producing large amounts of spent fungal biomass. In the European Research Area Industrial Biotechnology (ERA-IB) funded project, we effectively aimed to improve the composition of post-fermentation fungal biomass for extraction of the value-added product chitosan as a derivative of cell wall chitin (FunChi). As chitin/chitosan is not encountered in plant or human tissue, it often acts as an elicitor to plant and animal immune responses in order to fight off possible impending fungal infections. The application of both chitin and chitosan oligomers have been shown to prime plants against infection. This thesis discusses the identification of genes that are important for chitin deposition in the cell wall of A. niger. In addition, the work described here also investigates the genes that facilitate chitin cross-linking to the cell wall. The relevance of all findings are discussed in relation to both the improvement of chitin extraction from post-fermentation biomass and to the integrity of the fungal cell wall. Show less
Natural vanilla flavor is one of the most important in the world. However, the cost of this flavor is expensive. Production of this flavor by alternative methods, could reduce the cost. Towards... Show moreNatural vanilla flavor is one of the most important in the world. However, the cost of this flavor is expensive. Production of this flavor by alternative methods, could reduce the cost. Towards this end, using microbiological methods, fungal endophytes were isolated and identified, by morphological characters and PCR-based methods, from vanilla pods, the site of vanilla flavor. The fungal endophytes were tested for their involvement in the synthesis of vanilla flavor. In this way, three vanilla pod endophytes Hypoxylon investiens, Pestalotiopsis microspora, Diaporthe phaseolorum were found to modify the amounts of vanilla flavor and aroma metabolites vanillin, vanillyl alcohol, p-hydroxybenzoic acid, p-coumaric acid, p-xylene, α-phellandrene, 3-carene, α-terpineol, p-hydroxybenzaldehyde, α-cubebene, β-caryophyllene in Vanilla material containing media (in vitro) as well as in living Vanilla plant material (in vivo - acclimatized plants and calli). These metabolites were synthesized either de novo or through biotransformation reactions of precursors. That the latter describes the involvement of several metabolites related to vanilla flavor, implies complex flavor notes, typical of natural vanilla flavor, as opposed to the synthetic version. The analytical methods used include GC-MS, HPLC, 1H and 2D NMR. The results support the hypothesis that endophytes play a role in vanilla flavor metabolite biosynthesis. Show less
This thesis focuses on the role of chemokine receptors CXCR3 and CCR2 in the inflammatory process and infection control using the zebrafish model. It describes the regulatory interplay between an... Show moreThis thesis focuses on the role of chemokine receptors CXCR3 and CCR2 in the inflammatory process and infection control using the zebrafish model. It describes the regulatory interplay between an atypical and a conventional chemokine receptor during chemotaxis in macrophages, the role of chemotactic signaling in cell polarization and explores an in vivo screening workflow for human anti-inflammatory drugs using zebrafish. Show less
In this thesis, we have studied the potential of the zebrafish larval model in studying the ECS, as a complementary model to the existing rodent models. More specifically, we have looked at the... Show moreIn this thesis, we have studied the potential of the zebrafish larval model in studying the ECS, as a complementary model to the existing rodent models. More specifically, we have looked at the role of the ECS in regulating locomotion and anxiety, and its interaction with the hypothalamic-pituitary-interrenal (HPI) axis, or stress axis. This study has provided us with an interesting animal model which allows for pharmacological screening of Cnr1 agonists, and their involvement in the CNS, as shown by a change in locomotion, anxiety-like behavior and HPI axis activity. The zebrafish larval model can be used as a complementary model to the existing rodent animal models, to study the ECS. The zebrafish larval model brings several interesting features, such as optical transparency and possibilities for high-throughput screening. Furthermore, a complete ECS is present, there is lack of endogenous activity, allowing for exogenous compound screening, and zebrafish data is generally in line with rodent literature. Since the ECS is involved in many diseases, more research of this system may result in the discovery of novel drugs and drug targets. Show less
Prostate cancer (PCa) is one of the most prevalent cancer in males. Although the majority of the patients can benefit from the present clinical treatments, 20%-30% of the patients who originally... Show moreProstate cancer (PCa) is one of the most prevalent cancer in males. Although the majority of the patients can benefit from the present clinical treatments, 20%-30% of the patients who originally respond to the therapy still develop incurable, castration-resistance bone metastases, which is a main cause of death in PCa . In this thesis, I combined an advanced zebrafish xenograft model with in vitro cellular approaches and mice xenografts to study the early stage of PCa metastasis. Using this comprehensive esearch platform, I identified multiple key signaling pathways that play essential roles in promoting the onset of PCa metastatis. The pathways I discovered include Cripto-associated EMT plasticity, CDC-42-N-Wasp-Cortactin associated mechanosensing and mechanotransduction, microenvironment dependent NF-ĸB-Activin A signaling pathway, and AMPK-Autophagy dependent metabolic stress coping pathway. Show less
Plants are colonized by an astounding number of microorganisms that can provide different life-support functions, including nutrient acquisition and protection against (a)biotic stresses like... Show morePlants are colonized by an astounding number of microorganisms that can provide different life-support functions, including nutrient acquisition and protection against (a)biotic stresses like drought or pathogen attack Here, the diversity of bacteria living inside plant root tissue was explored with a focus on Actinobacteria, and in particular Streptomyces. Streptomycetes are filamentous bacteria that are commonly found in soil. They were brought into the laboratory for their ability to produce a large diversity of natural products, including many different antibiotics. In plant-associated environments, Streptomyces can be found in the rhizosphere, the endosphere and the phyllosphere. In these niches, they receive nutrients from the plants, feasting on various biopolymers and exudates. In return, the plant may benefit from their presence by enhanced nutrient acquisition, pathogen antagonism and induced systemic resistance To date, however, plant-Streptomyces interactions are not well understood and the mechanisms underlying plant colonization and invasion by Streptomyces remain largely elusive. Also, the chemistry of plant-Streptomyces interactions is yet underexplored, leaving us with a reservoir of untapped natural products that may contribute to solving the problem of emerging antibiotic resistance. Therefore, this research was focused on the aforementioned topics. Show less
The recently introduced nature-originated deep eutectic solvents, so-called natural deep eutectic solvents (NADES) are considered as truly green solvents, which composed of natural ingredients... Show moreThe recently introduced nature-originated deep eutectic solvents, so-called natural deep eutectic solvents (NADES) are considered as truly green solvents, which composed of natural ingredients found abundantly in organisms. NADES has been used in diverse applications, including extraction of bioactive molecules, enzymatic processes, agricultural and pharmaceutical fields, and protein stabilization. Despite the various applications, their physiological roles in organisms still remain unclear. In the thesis, the possible roles of NADES were investigated using a model plant (Drosera species) and for macromolecules. The chemical analysis of Drosera exudates indicated that the exudates contained sugars, amino acids, and organic acids which might be NADES components, and they could be involved in the biological roles of exudates. Several NADES are also proven to be optimum media to improve the stability and activity of enzymes. Additionally, NADES protected the enzymes from microorganism contamination. Besides enzymes, DNA and RNA were well protected from degradation by NADES. Based on the results of the thesis, a new biological role of NADES associated to macromolecules were deduced. Show less
In this thesis, we will utilize embryonic zebrafish tumour models to understand the interaction between engrafted human cancer cells and macrophages from the host, test drug administration... Show moreIn this thesis, we will utilize embryonic zebrafish tumour models to understand the interaction between engrafted human cancer cells and macrophages from the host, test drug administration modalities and anti-cancer efficacies of newly-developed PDT and PACT compounds, and test a light-triggered liposomal system for targeted drug delivery specifically to cancer cells in vivo. In chapter 2, we investigate the role of macrophages in tumour-induced angiogenesis. We show that macrophage-dependent angiogenesis is driven by macrophage recruitment to lactic acid secreted by glycolytic B16 melanoma cells. Chemical inhibition of macrophages and glycolysis blocks the initiation of angiogenesis in these models, suggesting that macrophages attracted to glycolytic melanoma cells contribute to the tumour-induced angiogenesis process.In chapters 3 and 4, we explore novel PDT and PACT compounds, respectively, for treatment of conjunctival melanoma in zebrafish. We inject conjunctival melanoma cells into the retro-orbital site to establish an orthotopic model and into the Duct of Cuvier to generate an ectopic model. Our results prove that zebrafish provides a fast vertebrate cancer model to test the optimal administration regimen of drugs, conditions of light irradiation, host toxicity and anti-cancer efficacy of PDT and PACT drugs against conjunctival melanoma.In chapter 5, we focus on modifying liposomes to be light triggered in order to deliver drugs specifically to cancer cells. We inject MDA231 breast cancer cells into the Duct of Cuvier at 2 days post fertilization (dpf) to initiate metastasis to the CHT. We successfully demonstrate that light-triggered, cell-specific delivery of liposome-encapsulated doxorubicin reduces the xenograft cancer cell burden without enhanced cytotoxicity of the zebrafish embryos. In chapter 6, we summarize the novel anti-cancer strategies, which we have developed using zebrafish xenograft models. In the same chapter, we frame our findings in the current scientific landscape and discuss future perspectives. Show less
Plant architecture has distinct forms in different plant species, but also within a species the finalarchitecture of a plant is determined by its gradual development and changes therein induced by... Show morePlant architecture has distinct forms in different plant species, but also within a species the finalarchitecture of a plant is determined by its gradual development and changes therein induced by environmental conditions during the plant’s life cycle. Higher plants have the ability tocontinually produce new organs and tissues during their lifetime. This unique ability is provided by pools of stem cells organized in so-called meristems. Primary meristems located at apices, of the plant body are involved in elongation of the main plant axes, whereas secondary meristems in lateral positions play a role in branching and axis thickening. First chapter is reviewing the current advances in our understanding of the molecular mechanisms that regulate plant meristems and their developmental phase transitions in Arabidopsis thaliana. AT-HOOK MOTIF NUCLEAR LOCALIZED (AHL) gene family is introduced in chapter 1,and that of family member AHL15 described and studied more in detail in chapter 2. In chapter 3 we identified a new branch of the ageing pathway in Arabidopsis, in which the longevity-promoting AHL15 protein represses ageing independent of miR156/157. Chapter 4 of this thesis focuses on AHL15's effect on Arabidopsis vascular cambium activity and secondary growth. Analysis showed that in an ahl15 loss-of-function mutant, interfascicular secondary growth is significantly reduced. Show less
Acidobacteria is a widely distributed phylum but their functional roles in ecosystem processes are still largely elusive. The Granulicella genus belongs to the class Acidobacteriia and is known to... Show moreAcidobacteria is a widely distributed phylum but their functional roles in ecosystem processes are still largely elusive. The Granulicella genus belongs to the class Acidobacteriia and is known to produce copious amount of Extracellular Polymeric Substances (EPS), which are fundamental for microbial life. The major goal of my thesis was to investigate and understand the metabolism of Granulicella sp. strains WH15 and 5B5, and the functions and environmental fate of EPS of Granulicella sp. WH15 (WH15EPS). Optimization of carbon concentration and manganese in culture medium allowed our strains, especially WH15, to grow faster in laboratory conditions, producing extractable amounts of EPS. Using the Stable Isotope Probing technique, we observed the incorporation of WH15EPS by Singulisphaera and its connections to other Planctomycetes and Acidobacteria, which were not reported before. In addition, our results showed, in the genomes of the microbes which incorporated WH15EPS, a large diversity of glycoside hydrolase with biotechnological potential and a high number of unclassified microorganisms that could be targeted for future studies. The data presented in this thesis establish a solid fundamental basis for more mechanistic studies of Acidobacteria and other uncultivated microbes. Show less
Plant-microbe interaction resulted in different physio/chemical responses by host plant and interacting rhizobacteria. This thesis focuses on how different plants and rhizobacteria combinations... Show morePlant-microbe interaction resulted in different physio/chemical responses by host plant and interacting rhizobacteria. This thesis focuses on how different plants and rhizobacteria combinations modulate plant metabolism. Factorial combinations of different plant species, including Arabidopsis thaliana (model plant), Brassica oleracea var. italica (crop) and Artemisia annua (medicinal plant), and phylogenetically distinct rhizobacterial species, including Pseudomonas fluorescens SS101 (Pf SS101), Microbacterium and three Paraburkholderia species, were used as study model systems in this thesis. Untargeted metabolomics was used to assess the impact of these rhizobacteria on the shoot chemistry of the host plant species. This study revealed that root treatment of different plant species with rhizobacteria altered 18-78% of the detected plant secondary metabolites in the shoot. We also assessed the impact of a known bacterial trait on plant phenotype and chemistry. cysH mutation of Pf SS101 affected the chain elongation of aliphatic glucosinolate biosynthesis in Arabidopsis whereas it led to an accumulation of indolic glucosinolates and flavonoids in Broccoli. To further discover the bacterial traits affected during the interaction with Broccoli roots, genome wide transcriptome analysis was carried out, resulting in upregulation of genes involved in flagellar assembly, chemotaxis, and motility together with nutrient uptake and (an)ion transporter in Paraburkholderia species. Show less
Plant exudates appeared long time ago in the history line of life in the early Devonian. They are typical saps produced by specialized cells. They can be naturally excreted outside or right after... Show morePlant exudates appeared long time ago in the history line of life in the early Devonian. They are typical saps produced by specialized cells. They can be naturally excreted outside or right after plants are damaged or infected. Through time, diverse cultures recognized them as valuable sources of natural medicines. Of various types of plant exudates, the most outstanding are gums, mucilage, essential oils, oleoresins, resins, phloem like saps, and latexes. Regardless of their commercial importance, in the aspect of biology, plant exudates are believed to be involved in plant defense mechanisms, but their roles still remain unclear. In this thesis, based on diverse experiments, many features of plant exudates were unveiled. Interestingly, even if plant exudates possess a broad chemical diversity, their specialized biological functions resulted in less variation of their metabolomes as compared with their bearing tissues. This lower variation indicates that plant exudates might partake of a general-broad defense against herbivores and pathogens in their bearing tissues. This defense involves both mechanical and chemical traits which might complement and modulate each other, eventually resulting in a chemo-mechanical defense layer. Of the diverse chemicals in plant exudates, terpenes are one of the most common metabolites in these saps. Show less
In this Ph.D. thesis, we characterized two novel upstream regulators of JUB1, HB40, and HB22, in Arabidopsis thaliana. In Chapter 2, we described functions of HB40 in modulating growth and... Show moreIn this Ph.D. thesis, we characterized two novel upstream regulators of JUB1, HB40, and HB22, in Arabidopsis thaliana. In Chapter 2, we described functions of HB40 in modulating growth and development via the regulation of JUB1 and GA inactivation genes GA2OXs. In Chapter 3, we studied whether the HB40-JUB1 regulatory unit plays a role in drought tolerance. Our results suggest that HB40-JUB1 is involved in the modulation of growth, development and drought response. In Chapter 4, we showed that HB40 and its homologues HB21 and HB53 are functionally redundant in repressing hypocotyl elongation under dark conditions. Like HB40, HB21 and HB53 positively and directly regulate the expression of JUB1. In Chapter 5, we investigated the potential gene regulatory network (GRN) controlled by HB22. Show less
The effects of anthropogenic noise on interactions between predators and their prey are still little understood. This thesis aims to fill pressing knowledge gaps on this topic by studying how... Show moreThe effects of anthropogenic noise on interactions between predators and their prey are still little understood. This thesis aims to fill pressing knowledge gaps on this topic by studying how anthropogenic noise affected various stages in the prey-hunting of predators and the predator-avoidance of prey. For predators, I investigated whether an¬thropogenic noise could influence habitat choice (chapters 2 and 3), foraging efficiency (chapter 3) and communication between foraging group members (chapter 4). For prey, I studied how anthropogenic noise affected prey behaviour outside of predation con¬texts (chapter 5) and if current and previous vessel noise exposure affected anti-predator behaviour when a simulated predator attacked (chapter 6). The multi-disciplinary studies of this thesis combine to unravel more insight into the influence of noise on predator-prey relationships in the marine environment. Further investigations should focus on revealing the mechanistic underpinning of noise effects on behaviour of both predator and prey. Only then will we be able to reduce the impact of noise on marine ecosystems. Show less
As a contribution to the changing legislation and evolving societal attitudes concerning environmental issues, this project aims to enhance and manipulate the plants’ own natural defense mechanisms... Show moreAs a contribution to the changing legislation and evolving societal attitudes concerning environmental issues, this project aims to enhance and manipulate the plants’ own natural defense mechanisms against western flower thrips (WFT). Accordingly, an approach based on treatments of seeds (Solanum lycopersicum) and cuttings (Chrysanthemum morifolium) was undertaken to protect plants from their early critical young stage onwards. Known putative defense secondary metabolites were exogenously applied whereas, external application of plant hormones was explored as a means to trigger innate defense responses. Natural Deep Eutectic Solvents (NADES), as environmentally benign solvents, significantly improved the solubilizing properties of poorly-soluble insecticidal metabolites but did not enhance resistance against WFT. On the contrary, seed treatments with the ubiquitous plant hormone jasmonic acid (JA) appeared to induce cultivar dependent defenses as it only reduced silver damage in tomato cultivar Carousel. Sulfuric acid scarification, prior to JA seed incubation, significantly augmented the embryonic receptivity of a non-responsive cultivar thus, highlighting the importance of seed coat permeability. Moreover, we demonstrate that water dipping of indole-3-butyric acid (IBA)-coated chrysanthemum cuttings repeatedly reduced herbivory, both by thrips as well as by leaf miner. Show less
To gain more insight into the evolutionary development of orchid flowers and fruits, the orchid species Erycina pusilla was studied. The evolutionary origin of the median petaloid sepal, the callus... Show moreTo gain more insight into the evolutionary development of orchid flowers and fruits, the orchid species Erycina pusilla was studied. The evolutionary origin of the median petaloid sepal, the callus on the labellum, and the stelidia was studied. These organs were found to be derived from a sepal, a stamen that gained petal identity, and stamens that became staminodes, respectively. The “Oncidiinae” model was proposed, explaining the duplications, diversifying selection and changes in spatial expression of different MADS-box genes that shaped the perianth, enabling the rewardless flowers of E. pusilla to mimic an unrelated rewarding flower for pollinator attraction.After pollination the inferior orchid ovary develops into a fruit. This process is described for E. pusilla up to fruit dehiscence. The fruit associated MADS-box genes and proteins together with other dehiscence-related genes were analyzed in order to propose a first “orchid fruit developmental protein and gene network” model. Fruit development was further studied by transcriptome analyses presenting data obtained from different developmental phases.By analyzing the anatomy of ripe fruits of different orchid species, possible correlations were found between fruit valve lignification patterns, life form, growth strategy, ecology, fruit orientation, dehiscence type, number of valves and slits, and phylogenetic relationships. Show less
Plants produce an astonishing variety of secondary metabolites (SMs) which are thought to play vital roles in the fitness of plants through ecological interactions. The most characteristic features... Show morePlants produce an astonishing variety of secondary metabolites (SMs) which are thought to play vital roles in the fitness of plants through ecological interactions. The most characteristic features of SMs are their striking chemical diversity and inter- or intraspecific variation. Due to the large number, high structural diversity and multifunctionality of SMs, it is still an ongoing challenge to understand how this SM diversity comes about, and why such a large diversity is maintained in nature. In this thesis this question was studied using the pyrrolizidine alkaloids (PAs) of Jacobaea species as the study system from an evolutionary and biosynthetic perspective. PA variations were studied among and within Jacobaea species, and species-specific PA profiles were observed. In order to understand how PA diversity is related to species phylogeny, the evolutionary histories and phylogenetic signals of individual PAs were investigated under the phylogenetic context of Jacobaea species and no strong phylogenetic signals were found. To shed light on the mechanisms underlying PA diversity, a gene-to-metabolite approach targeting cytochrome P450 monooxygenases which play an important role in the evolution of chemical diversity was applied to study their involvement in PA biosynthesis and PA diversity. Show less
The PID-directed shift in PIN polarity has been broadly accepted as one of the essential mechanisms for the regulation of auxin transport polarity. We verified that both PID functionality and its... Show moreThe PID-directed shift in PIN polarity has been broadly accepted as one of the essential mechanisms for the regulation of auxin transport polarity. We verified that both PID functionality and its subcellular localization do not depend on PDK1 function. However, by detailed analysis on these phenotypes and the expression of the auxin response reporter, we found the pdk1 pdk2 double mutant to be impaired in auxin transport in vascular tissues. Together with other mutant phenotypes, we suspect that PDK1 may be the master regulator of AGC1 kinases. The pdk1 pdk2 short root phenotype caused by phloem differentiation defects phenocopied the pax mutant. Complementation results of wild type and phosphomimic PAX in the pdk1 pdk2 background suggest that PDK1-dependent PAX phosphorylation and activation are essential for its full biological function. We also explain the molecular basis of PDK1 basal localization and the unnecessity of this polarity for vascular development. In addition, we investigated downstream action after PID phosphorylation. Several conserved tyrosine residues close to serine phosphorylation sites in the PIN1 and PIN2 HLs are mutated. Two of these tyrosines redundantly affected PIN polarity. However, PID mediated phosphorylation and tyrosine-based PIN trafficking are independent processes. Show less
In this thesis, the role of plant-mediated soil legacy effects in shaping aboveground plant-insect interactions was investigated. This work shows that soil legacy effects on plant-insect... Show moreIn this thesis, the role of plant-mediated soil legacy effects in shaping aboveground plant-insect interactions was investigated. This work shows that soil legacy effects on plant-insect interactions are a common phenomenon in nature, both in individual responding plant species and their associated herbivores, as well as in responding plant communities and associated herbivores. This thesis elucidates two potential mechanisms how soils can influence aboveground insects. First, soils can alter the way plants defend them selves in terms of secondary chemistry and phytohormonal defenses. Second, soils can also directly impact aboveground insects by changing the insect microbiome. This work shows that a large part of the insect microbiome is taken up directly from the soil microbiome, and leads to exciting new research directions on the longterm and evolutionary implications of soils for aboveground insects. Show less
