Micropollutant bioremoval in wastewater treatment systems: from microbial population structure to function

Dissertation presented to obtain the Ph.D degree in BiologyThe continuous release of micropollutants into receiving waters due to insufficient elimination from wastewater treatment plants (WWTP) raises global concerns regarding their potential risks to the environment and human health.(...

Antimicrobial activity and mode of action - Examples from natural products, peptides, and peptidomimetics

Infections caused by bacteria are the third leading cause of death worldwide. Antimicrobials are used to treat and prevent those infections and enabled the development of the modern healthcare system as we know it. However, those achievements are threatened by the global emergence of antimicrobial-resistant bacteria. There is an obvious need for developing new antimicrobials. Natural environments, like the Arctic Ocean, offer largely underexplored biodiversity with promising natural antimicrobial products. This thesis investigates marine and marine-inspired molecules for their antibacterial properties and potential applications

Genetic characterization of Rhodococcus rhodochrous ATCC BAA-870 with emphasis on nitrile hydrolysing enzymes

Includes abstract.Includes bibliographical references.Rhodococcus rhodochrous ATCC BAA-870 (BAA-870) had previously been isolated on selective media for enrichment of nitrile hydrolysing bacteria. The organism was found to have a wide substrate range, with activity against aliphatics, aromatics, and aryl aliphatics, and enantioselectivity towards beta substituted nitriles and beta amino nitriles, compounds that have potential applications in the pharmaceutical industry. This makes R. rhodochrous ATCC BAA-870 potentially a versatile biocatalyst for the synthesis of a broad range of compounds with amide and carboxylic acid groups that can be derived from structurally related nitrile precursors. The selectivity of biocatalysts allows for high product yields and better atom economy than nonselective chemical methods of performing this reaction, such as acid or base hydrolysis. In order to apply BAA-870 as a nitrile biocatalyst and to mine the organism for biotechnological uses, the genome was sequenced using Solexa technology and an Illumina Genome Analyzer. The Solexa sequencing output data was analysed using the Solexa Data Analysis Pipeline and a total of 5,643,967 reads, 36-bp in length, were obtained providing 4,273,289 unique sequences. The genome sequence data was assembled using the software Edena, Velvet, and Staden. The best assembly data set was then annotated automatically using dCAS and BASys. Further matepaired sequencing, contracted to the company BaseClear® BV in Leiden, the Netherlands, was performed in order to improve the completeness of the data. The scaffolded Illumina and mate-paired sequences were further assembled and annotated using BASys. BAA-870 has a GC content of 65% and contains 6997 predicted protein-coding sequences (CDS). Of this, 54% encodes previously identified proteins of unknown function. The completed 5.83 Mb genome (with a sequencing coverage of 135 X) was submitted to the NCBI Genome data bank with accession number PRJNA78009. The genome sequence of R. rhodochrous ATCC BAA-870 is the seventh rhodococcal genome to be submitted to the NCBI and the first R. rhodochrous subtype to be sequenced. An analysis of the genome for nitril

Laboratory directed research and development annual report. Fiscal year 1994

The Department of Energy Order DOE 5000.4A establishes DOE`s policy and guidelines regarding Laboratory Directed Research and Development (LDRD) at its multiprogram laboratories. This report represents Pacific Northwest Laboratory`s (PNL`s) LDRD report for FY 1994. During FY 1994, 161 LDRD projects were selected for support through PNL`s LDRD project selection process. Total funding allocated to these projects was $13.7 million. Consistent with the Mission Statement and Strategic Plan provided in PNL`s Institutional Plan, the LDRD investments are focused on developing new and innovative approaches in research related to our {open_quotes}core competencies.{close_quotes} Currently, PNL`s core competencies have been identified as integrated environmental research; process science and engineering; energy systems development. In this report, the individual summaries of LDRD projects (presented in Section 1.0) are organized according to these core competencies. The largest proportion of Laboratory-level LDRD funds is allocated to the core competency of integrated environmental research. Projects within the three core competency areas were approximately 91.4$35K or less. The projects described in this report represent PNL`s investment in its future and are vital to maintaining the ability to develop creative solutions for the scientific and technical challenges faced by DOE and the nation. The report provides an overview of PNL`s LDRD program, the management process used for the program, and project summaries for each LDRD project

Biomimetic Based Applications

The interaction between cells, tissues and biomaterial surfaces are the highlights of the book "Biomimetic Based Applications". In this regard the effect of nanostructures and nanotopographies and their effect on the development of a new generation of biomaterials including advanced multifunctional scaffolds for tissue engineering are discussed. The 2 volumes contain articles that cover a wide spectrum of subject matter such as different aspects of the development of scaffolds and coatings with enhanced performance and bioactivity, including investigations of material surface-cell interactions

Characterization of antimicrobial peptides deriving from insects and their application in the biomedical field

Antibiotics are the current drugs used to treat pathogenic bacteria, but their prolonged use contributes to the development and spread of drug-resistant microorganisms. The antibiotic resistance issue led to the need to find new alternative molecules, which should be less prone to bacterial resistance. Antimicrobial peptides (AMPs) aroused great interest as potential next-generation antibiotics. AMPs are involved in several defence-related processes such as the binding and neutralization of endotoxins, the modulation of the immune responses to infection and the killing of pathogens. Antimicrobial peptides are small molecules with an amino acid composition ranging from 10 to 100 residues and are biosynthesized by all living organisms but it is known that the class of insects represents the largest source of these molecules. This aspect is related to insect’s biodiversity and their ability to live in hostile environments rich of pathogens. Most insect AMPs are cationic molecules due to the presence of basic residues and according to their amino acid sequences and structures, they can be classified in four different groups: cysteine-rich peptides (e.g., defensins), the α-helical peptides (e.g., cecropins), glycine-rich proteins (e.g., attacins) and proline-rich peptides (e.g., drosocins). Insect AMPs have demonstrated to be useful in several applications concerning the pharmaceutical as well as the agricultural fields. Moreover, insect AMPs aroused great interest for their biomedical application thanks to the increasing number of peptides that can inhibit human pathogens. For this reason, this Ph.D. project aimed to the identification of antimicrobial peptides deriving from insects, particularly from the Black Soldier Fly Hermetia illucens (L.) (Diptera: Stratiomyidae). Through a combination of transcriptomics and bioinformatics analysis, 57 antimicrobial peptides have been identified from H. illucens insect. Through an in silico analysis, the biological activity have been predicted and the physio-chemical properties have been calculated for all the identified peptides. Based on the bioinformatics results, the in vitro production of the most promising sequences has been performed through molecular cloning strategies in order to evaluate the antibacterial activity in vitro. Particularly, some of the identified peptides (C16571, C46948, C16634, and C7985) showed the ability to inhibit E. coli growth at a concentration value of 3 μM. For the C15867 peptide, recombinantly produced and expressed, a MIC (Minimum Inhibitory Concentration) value of 18 μM has been determined. Moreover, an in vivo approach was carried out for the identification of antimicrobial peptides by extracting the hemolymph from the H. illucens larvae, recovering then the peptides fraction from the larvae’s plasma and its antibacterial activity has been evaluated against both Gram-positive and Gram-negative bacteria. The performed analysis showed that a small amount (7.5/15 μL) of the peptide fraction recovered from the larvae’s plasma was able to inhibit the cell growth of different bacterial strains

Threonyl tRNA synthetases as antibiotic targets and resistance mechanisms

Threonyl tRNA synthetases (ThrRSs) catalyse the attachment of L-threonine to its cognate tRNAThr. These enzymes are essential for the translation of proteins. There are currently two known natural product ThrRS inhibitors, borrelidin and obafluorin, produced by Streptomyces parvulus Tü 4055 and Pseudomonas fluorescens ATCC 39502 respectively. Both of their biosynthetic gene clusters (BGCs) encode secondary ThrRSs which provide self-resistance mechanisms. In the borrelidin BGC, this is BorO and in the obafluorin BGC, this is ObaO. While the biosynthesis of both compounds is well understood, the mechanisms of self-resistance are not, and the mechanism of action of obafluorin remains elusive. Here the structure of BorO was solved and it was found that both ThrRSs in the producer, S. parvulus (Sp), are resistant to borrelidin. Mutagenesis of the Escherichia coli target, EcThrRS, identified a L489T mutation which is sufficient to confer resistance by preventing borrelidin binding, explaining resistance by BorO. In SpThrRS, this residue is a glutamate and an EcThrRS, L489Q mutation is not sufficient to confer resistance, meaning that SpThrRS has a distinct borrelidin resistance mechanism. It was unexpectedly found that ObaO can confer resistance to borrelidin due to the presence of a methionine in this same position and the EcThrRS L489M mutant is resistant to borrelidin. Introduction of this mutation allowed the first structure of ObaO to be solved by X-ray crystallography. Cryogenic electron microscopy (Cryo-EM) structures of obafluorin bound to both EcThrRS and ObaO were solved, showing that EcThrRS covalently links to obafluorin through Y462, while the interaction is non-covalent for ObaO. Spontaneous resistant mutagenesis identified the serine (S463) immediately adjacent to Y462as an essential component of the ObaO resistance to obafluorin. Finally, a survey of published genomes for additional copies of ThrRS identified BGCs encoding potentially novel natural product ThrRS inhibitors

Theoretical studies of apolipoprotein interactions

This thesis describes molecular modelling simulations of the cyclic apoC-II(60-70) peptide (cyc(60-70)). Cyclic apoC-II(60-70) is a known inhibitor of amyloid aggregation by apoC-II(60-70). This project involves Molecular Dynamics simulations of several related peptides of cyc(60-70). The first study aims to structurally characterise cyc(60-70) and explore the effects of temperature on cyc(60-70). The cyclic peptides are then compared to the NMR obtained structures to identify consistent features. Using the NMR structures, an ensemble trajectory was obtained through MD simulations where further analysis revealed three important features: 1) cyc(60-70) is extremely flexible; 2) cyc(60-70) preferentially adopts an elongated orientation; and 3) cyc(60-70) tends to form a distinct hydrophilic/hydrophobic face. To understand the inhibition activity of cyc(60-70), a scrambled cyc(60-70) peptide was generated. ThT fluorescence revealed that scrambled cyc(60-70) exhibits no inhibitory activity as opposed to the unscrambled peptide. The scrambled peptide was simulated through MD and was found to be inconsistent with the three main features of cyc(60-70). The scrambled peptide was extremely rigid, adopting a spherical orientation with slightly less distinction between hydrophilic/hydrophobic regions. This suggests that these features abolish the inhibitory activity of cyc(60-70). The final study involved simulating cyc(60-70) in the presence of apoC-II(60-70). The simulations revealed that apoC-II(60-70) was more dynamic in the presence of cyc(60-70) however, cyc(60-70) was no longer flexible. Based on this result, a capping mechanism of inhibition was proposed. The proposed mechanism suggests that cyc(60-70) binds strongly to the hydrophobic region of apoC-II(60-70), generating a hydrophilic shell that discourages further peptides self-association

Prediction of novel biochemical class disease related proteins and microRNAs by machine learning approach

Ph.DDOCTOR OF PHILOSOPH