Determination and characterization of genes involved in toxic mechanisms of the prymnesiophyte Prymnesium parvum

This thesis represents a study of the ecophysiology and toxicity of the prymnesiophyte Prymnesium parvum. The first aim was to investigate changes in the relative toxicity of P. parvum following a series of physiological shock treatments, meant to simulate environmental conditions under which harmful blooms of this species have been observed. As blooms of this haptophyte often occur in dynamic coastal brackish water systems, Prymnesium parvum is noted for its physiological flexibility, which may contribute to providing a competitive advantage over other coexisting species. Due to the unconfirmed nature of the compounds involved in toxigenic processes, two bioassays were employed to characterize changes in lytic capacity (extracellular vs. intracellular). These bioassays are considered physiologically relevant, as observed icthyotoxicity occurs through lysis of the gill cell membranes, rendering the fish unable to perform gas-exchange processes and obtain oxygen. Additionally, the gene expression of three polyketide synthase genes (PKS) were analyzed via quantitative PCR (qPCR), based on current chemical characterizations of toxic compounds produced by P. parvum. Low salinity and high irradiance were observed to alter the lytic effects of P. parvum on the sensitive cryptophyte Rhodomonas salina and erythrocytes. Furthermore, these two shock treatments were found to increase the transcript copy number in selected PKS genes, suggesting a possible correlation between toxicity and the PKS biosynthetic pathway. Allelochemical mediation has been suggested to affect competition and predatory relationships associated with formation of P. parvum blooms. As interactions between species are an integral part of understanding plankton ecology, interspecific interactions between P. parvum and three coexisting species were accordingly investigated. Combining bioassays with a functional genomic approach allowed differential characterization of cell-cell contact vs. waterborne cues depending on the organism with which incubated. A unique response on both the levels of toxicity, gene expression profile as well as PKS transcript copy number to the potential predator Oxhyrris marina suggest a fundamentally different type of interaction between the two species. Additionally, a dose-response time series experiment showed that changes in gene expression and toxicity did not occur immediately in P. parvum, rather after 60-90 minutes. Such a response by P. parvum may in fact signify a co-evolutionarily adaptive defense. Finally, examination of the effects of phosphorous limitation and low salinity stress on the gene expression profile and lytic capacity showed that the combination of these two stressors induces secretion or extracellular transport of toxic substances to a much higher degree than either stressor individually. Whether this observation is due to changes in membrane integrity due to homeostatic processes needs further research. The pattern of gene expression, however, revealed regulation of among others genes associated with active cellular transport processes, suggesting that maintenance of intracellular-extracellular homeostasis may play a role in the observed toxicity. In summary, these studies integrate the concepts of ecophysiology and functional genomics, providing a useful platform for further research regarding environmental factors associated with the toxicity of P. parvum. As functional genomic methods become more accessible, such approaches illustrate their potential application within the field of harmful algal research

auDeep: Unsupervised Learning of Representations from Audio with Deep Recurrent Neural Networks

auDeep is a Python toolkit for deep unsupervised representation learning from acoustic data. It is based on a recurrent sequence to sequence autoencoder approach which can learn representations of time series data by taking into account their temporal dynamics. We provide an extensive command line interface in addition to a Python API for users and developers, both of which are comprehensively documented and publicly available at https://github.com/auDeep/auDeep. Experimental results indicate that auDeep features are competitive with state-of-the art audio classification

A simple dot blot assay to measure hygromycin B phosphotransferase activity in whole cell extracts of Neurospora crassa

Hygromycin B (Hyg) is an aminocyclitol antibiotic with broad spectrum activity against prokaryotes and eukaryotes (Pettinger et al. 1953 Antibiot. Chemother. 3:1268- 1278). Hyg inhibits protein synthesis by blocking ribosomal translocation; it prevents polypeptide elongation by interfering with aminoacyl tRNA recognition and ribosomal A-site occupation (Cabanas et al. 1978 Euro. J. Biochem. 87:21-27, Hausner et al. 1988 J. Biol. Chem. 263:13103-13111). Hyg can lead to misreading during translation in vitro (Davies and Davies 1968 J. Biol. Chem. 243:3312-3316, Gonzales et al. 1978 Biochim. Biophys. Acta 521:459-469, Singh et al. 1979 Nature 277:146-148); however, this effect was not duplicated in vivo (Bakker 1992 J. Gen. Microbiol. 138:563-569)

Expression and Visualization of Red Fluorescent Protein (RFP) in Neurospora crassa

We report the expression of Discosoma red fluorescent protein (RFP) and RFP fusion proteins in Neurospora crassa. RFP was expressed under the control of the Neurospora ccg-1 promoter in transformants with single copies integrated at the his-3 locus by gene targeting. Because this RFP gene, tdimer2(12), contains a 677 bp direct tandem repeat of dsRed, RFP constructs underwent RIP at high frequency in rid+ strains. Fusion proteins of RFP to the amino terminus of Neurospora heterochromatin protein 1 (HP1) were localized to heterochromatic foci in Neurospora nuclei, consistent with prior findings with carboxy-terminal HP1-GFP fusion proteins

Expression and visualization of Green Fluorescent Protein (GFP) in Neurospora crassa

We report the first successful imaging of GFP expression in Neurospora crassa. GFP was expressed under the control of the heterologous ToxA promoter from Pyrenophora tritici-repentis in transformants carrying multiple or single copies of the GFP construct. GFP was also detected in ascospores but not during earlier stages of the sexual cycle

Preliminary specification and design documentation for software components to achieve catallaxy in computational systems

This Report is about the preliminary specifications and design documentation for software components to achieve Catallaxy in computational systems. -- Die Arbeit beschreibt die Spezifikation und das Design von Softwarekomponenten, um das Konzept der Katallaxie in Grid Systemen umzusetzen. Eine Einführung ordnet das Konzept der Katallaxie in bestehende Grid Taxonomien ein und stellt grundlegende Komponenten vor. Anschließend werden diese Komponenten auf ihre Anwendbarkeit in bestehenden Application Layer Netzwerken untersucht.Grid Computing

Measuring extracellular enzymes in pure and mixed cultures of Trametes versicolor (L.:Fr.) Pilat and Trichoderma harzianum Rifai

The biocontrol potential of Trichoderma harzianum Rifai, Trichoderma polysporum (Link.:Pers.) Rifai, Scytalidium aurantiacum Klingstr. et Beyer or a Penicillium sp. against Trametes versicolor (L. :Fr.) Pilat, Neolentinus lepideus (Fr.) Redh. et Ginns, Postia placenta (Fr.) M.Lars. et Lomb. and Irpex lacteus Fr. was evaluated using agar plate, soil bottle and small size wood wafer tests. Although T. harzianum arrested growth of several wood decay fungi, it never killed them on wood samples. Of the tested microfungi, S. aurantiacum performed best in the wood-based assays. At the same time, the utility of small size wood assays for evaluation of biocontrol potential was demonstrated. Such assays are more rapid and versatile than full-scale soil bottle or sawdust tube tests, and provide more information than agar plate screenings. Microscopic examination of hyphal interactions between T. versicolor and T. harzianum on microscope slide cultures yielded little additional information, partly due to the experimental system chosen. No hyphal interference or lysis of cell walls was observed, possibly due to the high nutrient levels in the slide cultures. The activities of enzymes related to wood decay were studied in pure and mixed liquid cultures of T. versicolor and T. harzianum at low (0.4 mM) and high (4 mM) nitrogen concentrations. In high nitrogen medium, total filter paper cellulase, specific and total cellobiase, and specific and total laccase increased when compared to activities obtained in low nitrogen concentrations. Conversely, specific filter paper cellulase and peroxidase activities were enhanced under nitrogen limiting conditions. Influences of T. harzianum on extracellular enzyme production of T. versicolor were observed. Total and specific laccase activities were induced in media containing both low and high nitrogen levels, whilst filter paper cellulase activities decreased. Peroxidase and cellobiase activities remained at approximately the same level or were decreased in mixed cultures. The experimental system chosen allowed no separation of mycelia or culture liquids of the two fungi incubated in mixed culture. Therefore, few conclusions with respect to induction, inhibition or regulation of the monitored enzymes could be reached. None of the enzyme activities was correlated with biomass production. Laccases and Poly R-478 peroxidase activity indicated survival of the T. versicolor, since T. harzianum did not produce these two enzymes

The kinetochore interaction network (KIN) of ascomycetes

Chromosome segregation relies on coordinated activity of a large assembly of proteins, the kinetochore interaction network (KIN). How conserved the underlying mechanisms driving the epigenetic phenomenon of centromere and kinetochore assembly and maintenance are remains unclear, even though various eukaryotic models have been studied. More than 50 different proteins, many in multiple copies, comprise the KIN or are associated with fungal centromeres and kinetochores. Proteins isolated from immune sera recognized centromeric regions on chromosomes and thus were named centromere proteins (CENPs). CENP-A, sometimes called centromere-specific H3 (CenH3), is incorporated into nucleosomes within or near centromeres. The constitutive centromere-associated network (CCAN) assembles on this specialized chromatin, likely based on specific interactions with and requiring presence of CENP-C. The outer kinetochore comprises the Knl1-Mis12-Ndc80 (KMN) protein complexes that connect CCAN to spindles, accomplished by binding and stabilizing microtubules (MTs) and in the process generating load-bearing assemblies for chromatid segregation. In most fungi the Dam1/DASH complex connects the KMN complexes to MTs. Fungi present a rich resource to investigate mechanistic commonalities but also differences in kinetochore architecture. While ascomycetes have sets of CCAN and KMN proteins that are conserved with those of budding yeast or metazoans, searching other major branches of the fungal kingdom revealed that CCAN proteins are poorly conserved at the primary sequence level. Several conserved binding motifs or domains within KMN complexes have been described recently, and these features of ascomycete KIN proteins are shared with most metazoan proteins. In addition, several ascomycete-specific domains have been identified here

A theoretical and computational basis for CATNETS

The main content of this report is the identification and definition of market mechanisms for Application Layer Networks (ALNs). On basis of the structured Market Engineering process, the work comprises the identification of requirements which adequate market mechanisms for ALNs have to fulfill. Subsequently, two mechanisms for each, the centralized and the decentralized case are described in this document. These build the theoretical foundation for the work within the following two years of the CATNETS project. --Grid Computing