Amphibian chytridiomycosis : a review with focus on fungus-host interactions

Amphibian declines and extinctions are emblematic for the current sixth mass extinction event. Infectious drivers of these declines include the recently emerged fungal pathogens Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans (Chytridiomycota). The skin disease caused by these fungi is named chytridiomycosis and affects the vital function of amphibian skin. Not all amphibians respond equally to infection and host responses might range from resistant, over tolerant to susceptible. The clinical outcome of infection is highly dependent on the amphibian host, the fungal virulence and environmental determinants. B. dendrobatidis infects the skin of a large range of anurans, urodeles and caecilians, whereas to date the host range of B. salamandrivorans seems limited to urodeles. So far, the epidemic of B. dendrobatidis is mainly limited to Australian, neotropical, South European and West American amphibians, while for B. salamandrivorans it is limited to European salamanders. Other striking differences between both fungi include gross pathology and thermal preferences. With this review we aim to provide the reader with a state-of-the art of host-pathogen interactions for both fungi, in which new data pertaining to the interaction of B. dendrobatidis and B. salamandrivorans with the host's skin are integrated. Furthermore, we pinpoint areas in which more detailed studies are necessary or which have not received the attention they merit

Characterization and functional regulation of bioactive peptides in avian macrophages and heterophils

Oligopeptides and low molecular weight polypeptides play central roles as effectors and signal transducers acting as hormones, neurotransmitters, growth factors, toxins, and antimicrobial factors that are important for the survival of the organism. Owing to the ubiquitous involvement of peptides in many key regulatory processes, we have been interested to identify native peptides in different cells and tissues and understand their functions. To conduct our studies, we used avian macrophages and heterophils as models of specialized cells which constitute central components of innate immunity. These studies involved (a) qualitative identification and characterization of the peptides associated with high intensity mass spectral peaks in macrophage and heterophil and (b) the quantitative changes in those peptides under immunomodulating effects of toll-like receptor (TLR) activators. The work presented here describes the identification of thymosin beta 4, an actin binding peptide, in macrophages and its modulation under TLR activation. This dissertation also includes identification of mature avian beta defensin 2 (AvBD2), an antimicrobial peptide in heterophils of 4 different avian species (chicken, turkey, pheasant and quail) and its modulation in chicken heterophils under similar conditions

Konopeptiidide klassifitseermine ja kindlakstegemine varjatud Markovi mudelite ja positsioonispetsiifiliste skoorimaatriksite abil

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Konopeptiidid on soojades meredes elavate koonustigude (Conus sp.) mürgis leiduvad lühikesed valgud. Koonusteod on kiskjad ja toituvad ussikestest, teistest molluskitest või kaladest. Nad tulistavad saaki mürgiga täidetud harpuuniga, mürk muudab saaklooma liikumatuks ja tigu saab ta rahulikult tervelt alla neelata. Mürgi kiirelt uimastav ja halvav toime tuleb paljude erinevate konopeptiidide segust. Konopeptiidid sünteesitakse eellasvalkudena, millel on signaaljärjestus mürgitorusse transportimiseks ja propeptiid, mis aitab mürgipeptiidi õigesti pakkida ja mürgipeptiid. Konopeptiidid jagatakse sarnaste signaaljärjestuste alusel perekondadesse. Teadlased uurivad konopeptiide lootusega leida nende hulgast uusi ravimikandidaate. Konopeptiidid on väga spetsiifilised närvirakkudes leiduvate ioonkanalite modulaatorid ja omavad suurt potentsiaali valuvaigistite või lihastelõdvestajatena. Antud uurimistöö esimeseks eesmärgiks oli välja töötada meetod, mille abil saaks suurtest järjestuste hulkadest välja otsida ja klassifitseerida konopeptiidid. Klassifitseerimine on oluline, sest uuele valgule sarnaste valkude teadasaamine annab palju informatsiooni tema omaduste kohta. Meie valisime sarnasuse võrdlemiseks kahte tüüpi mudelid - profiil-HMM’id ja PSSM’id. Kuna signaalpeptiidid on ühe perekonna piires väga konserveerunud, siis on nende olemasolul järjestuste klassifitseerimine meie mudelitega 100% tundlik ja ka 100% spetsiifiline. pHMM’ide ja PSSM’ide kombineerimisega saavutasime 91% tundlikkuse ka mürgipeptiidide klassifitseerimisel. Töö teiseks eesmärgiks oli otsida konopeptiide koonusteo Conus consors’i genoomist ja mürgitoru transkriptoomist. Konopeptiidide otsimiseks järjestatud transkriptoomist ja genoomist kasutasime teiste meetodite hulgas ka pHMM’e. Me leidsime C. consors’i genoomist 214 konopeptiidi, millest 187 olid uued järjestused. Meil õnnestus teada saada 13-sse erinevasse perekonda kuuluva 15 konopeptiidi geenide ekson-intron struktuur. See on oluline, sest geenistruktuur võib mõjutada konopeptiidide mitmekesisuse teket.Conopeptides are small proteins found in the venom of cone snails (Conus sp.). Cone snails feed on worms, molluscs and fish. They paralyze their prey with venom and swallow it whole. The fast immobilization appears as a result of the mixture of conopeptides in the venom. Conopeptides are synthesized as prepropeptides with a signal sequence for transport into the venom duct, pro-peptide that facilitates proper folding and mature peptide. Conopeptides are grouped into superfamilies according to the signal sequences. Scientists are studying the conopeptides hoping to find new drug candidates. Conopeptides are specific modulators of ion channels in nerve and muscle cells and therefore can be potentially used as painkillers or muscle relaxants. Aim of this study was to develop a method for finding and classifying conopeptides from large amounts of sequences. Classification is important since finding the proteins similar to a newly discovered protein we get a lot of information about it. We used two types of models for classification and identification – profile hidden Markov Models (pHMMs) and position specific scoring matrices (PSSMs). With the signal peptide present the classification is 100% specific and sensitive. By combining the pHMMs and PSSMs we were able to obtain 91% sensitivity also for classification of mature peptides, which is better than with other methods. The second aim of this study was to find conopeptides from the genome and venom duct transcriptome of Conus consors. We used multiple methods, including the pHMMs, to locate the conopeptides. We discovered 214 conopeptides from the genome, 187 of which were novel. We also described the exon-intron structure for 15 conopeptide genes from 13 different superfamilies. Gene structure may influence the propagation of conopeptide diversity

On the macroevolution of antipredator defence

This thesis aims to improve our understanding of the macroevolutionary implications of antipredator defences, particularly with regard to how defence impacts biodiversity (including both species and trait diversity). To do this I took a phylogenetic comparative approach and used multiple study systems in an attempt to ensure the generality of my work. I begin by investigating how chemical defence and protective coloration influence ecology by testing for life history and ecological correlates of these defences (Chapter 1). Upon finding evidence for an increased niche space in chemically-defended species, and to some degree in conspicuously-patterned species, I explore whether this leads to increased diversification by increasing speciation rates and/or lowering extinction rates (Chapter 2), as also predicted by escape-and-radiate theory (a major and highly influential framework for the macroevolution of natural enemy interactions). Both conspicuous coloration and chemical defence increased speciation rates, but extinction rates were also raised in chemically-defended lineages, leading to a reduction in net diversification. Macroevolutionary extinction rates may or may not be related to contemporary extinction risk, but if they are then there may be conservation implications by allowing prediction of threat status of species with limited direct information. Consequently, in Chapter 3 I asked whether chemically-defended species are more threatened than those lacking such a defence. In accordance with the macroevolutionary results from Chapter 2, I found that chemical defence is indeed associated with a higher extinction risk even amongst contemporary species. In addition to factors that promote diversity, in this thesis I also investigated convergent evolution as a means of constraining diversity of phenotypic traits, using mimicry as a case study for antipredator defences. Many antipredator defences are convergent to some degree, with examples in the repeated evolution of chemical defences and warning coloration as well as independently derived similarity in protective mimicry. However, methods of quantifying the strength of convergent evolution are lacking, not to mention a conceptual framework to define 'strength' in this context, I began by developing a new method to do this which I called the Wheatsheaf index (Chapter 4). Subsequently, I (in collaboration with a colleague, Amanda Minter) also designed software in the form of an R package (called 'windex') to enable user-friendly implementation of the Wheatsheaf index in a familiar statistical environment to many biologists (Chapter 5). In the final data chapter of this thesis, I apply this method in a case study to explore the patterns of phenotypic convergence that result from the evolution of Batesian and Müllerian mimicry complexes. I find that these two types of protective mimicry are generally characterised by convergence in different broad types of traits, but that the specific traits which converge in a given mimicry complex are less predictable (Chapter 6). Overall, this thesis provides novel insights into the evolutionary patterns and consequences of antipredator defences, develops a framework and methods for the analysis of convergent evolution, and suggests further avenues of research for future studies

Identification and Characterization of Antimicrobial Peptides with Therapeutic Potential

Antimicrobial peptides are key defense molecules adopted by all life forms to prevent infection. They also have other beneficial effects such as boosting immune response, anticancer, and wound healing. The antiviral effects of antimicrobial peptides have laid the foundations for developing new agents to combat seasonal Flu, HIV-1, RSV, Zika, and Ebola. This eBook is constructed to systematically deal with antimicrobial peptides from a variety of natural sources, including fungi, plants, and animals (insects, fish, amphibians, birds, and reptiles). It covers peptide discovery, antimicrobial activity, 3D structure, mechanisms of action and potential applications. Naturally Occurring Antimicrobial Peptides, an eBook published by the journal Pharmaceuticals, provides a helpful introduction to newcomers and refreshes the minds of veterans

Needles in a haystack of protein diversity: Interrogation of complex biological samples through specialized strategies in bottom-up proteomics uncover peptides of interest for diverse applications

Peptide identification is at the core of bottom-up proteomics measurements. However, even with state-of the-art mass spectrometric instrumentation, peptide level information is still lost or missing in these types of experiments. Reasons behind missing peptide identifications in bottom-up proteomics include variable peptide ionization efficiencies, ion suppression effects, as well as the occurrence of chimeric spectra that can lower the efficacy of database search strategies. Peptides derived from naturally abundant proteins in a biological system also have better chances of being identified in comparison to the ones produced from less abundant proteins, at least in regular discovery-based proteomics experiments. This dissertation focused on the recovery of the “missing or hidden proteome” information in complex biological matrices by approaching this challenge under a peptide-centric view and implementing different liquid chromatography tandem mass spectrometry (LC-MS/MS) experimental workflows. In particular, the projects presented here covered: (1) The feasibility of applying a liquid chromatography-multiple reaction monitoring MS methodology for the targeted identification of peptides serving as surrogates of protein biomarkers in environmental matrices with unknown microbial diversities; (2) the evaluation of selecting unique tryptic peptides in-silico that can distinguish groups of proteins, instead of individual proteins, for targeted proteomics workflows; (3) maximizing peptide identification in spectral data collected from different LC-MS/MS setups by applying a multi-peptide-spectrum-match algorithm, and (4) showing that LC-MS/MS combined with de novo assisted-database searches is a feasible strategy for the comprehensive identification of peptides derived from native proteolytic mechanisms in biological systems

Integrated strategies for investigating endocrine mechanisms in Biomphalaria Glabrata as a test organism for androgenic chemical testing

This thesis was submitted for the award of Doctor of Philosophy and was awarded by Brunel University London.Endocrine and metabolic disease or dysfunctions are of growing concern in modern societies across the globe, underlining the need for continued focus on the development of pharmaceuticals. Subsequent scientific research has revealed a trend in the increase of such abnormalities and expansion of chemical industries, highlighting concerns that these disorders may, in part, be caused by exposure to environmental pollutants. This has led to changes in legislation concerning chemicals safety testing involving an increasing number of vertebrate animal tests as a part of environmental risk assessment process, at significant financial and ethical costs. A solution that is appropriate and aligned with the three R’s (reduction, refinement and replacement) in relation to animal research is to exploit the use of small invertebrate organisms as possible replacements for mammals. In line with the above approach/solution, this thesis is based on the null hypothesis that common genes, proteins and processes in gastropod molluscs and humans underlie the response of male reproductive organs to androgenic chemicals. Using a freshwater pulmonate snail, Biomphalaria glabrata, physiological effects of two steroid androgens on the development of mollusc secondary sexual organs were studied. Furthermore, an exhaustive investigation on the mollusc nuclear receptor repertoire and reproductive type neuropeptides was conducted. This also included the study of the evolutionary degree of conservation of these genes in non-model molluscs. The results obtained suggest that the snails did not respond to, and were not affected by exposure to the androgens. These results were supported by the absence of the members of subfamily 3C of nuclear receptors, which includes some of the “vertebrate” steroid hormone targets, suggesting that this mollusc may be an inappropriate model for steroid hormone mediated mammalian endocrine function. The nuclear receptor (NR) repertoire of B. glabrata comprised of 39 nuclear receptors representing all the known subfamilies of the NR superfamily. 21 reproductive type neuropeptide genes were identified encoding precursors that are predicted to release over 124 bioactive cleavage products. The consequence of these findings is significant in the context of the development of alternative model organisms for chemical testing as well as elucidating the taxonomic scope of nuclear receptor mediated endocrine disruption