Kinetoplastid Genomics and Beyond

This book includes a collection of eight original research articles and three reviews covering a wide range of topics in the field of kinetoplastids. In addition, readers can find a compendium of molecular biology procedures and bioinformatics tools

Tarantulas and social spiders : a tale of sex and silk

Studies of spider silks indicate that they may outperform virtually all synthetic fibres in terms of strength, elasticity and toughness. To date, most silks studied come from only a select few species and likely underrepresent the immense diversity of the clades. Here, protein and mRNA sequence analyses were used to study silk from two types of spider. The first approach used ESI tandem mass spectrometry to sequence peptide fragments of a silk from a tarantula (Mygalomorphae, Theraphosidae), a hitherto neglected family. The results confirm that the common silk types found in araneomorph spiders, Spidroin 1 and Spidroin 2, are also found in mygalomorphs. A putative N-terminal domain that bears a striking similarity to the N-terminus of araneomorph pyriform silk was isolated. If correctly identified, this would be the first ever recorded N-terminal domain for a mygalomorph. The second approach taken was to construct a cDNA library from theraphosid silk glands and adjacent tissue. Sequencing identified a significant number of uniquely truncated rRNAs. These may be the result of specific 'fragile sites' within these transcripts, which would explain the discrete classes of length polymorphisms found. The cDNA library sequences also provided evidence consistent with RNA editing and furthermore identified the presence of both transcribed nuclear pseudogenes and transposable elements. These may reflect past evolutionary horizontal gene transfer events within the spider genome. Similar analysis of next generation sequencing data from the transcriptomes of three Stegodyphus spp. (Araneomorphae) reveal a range of apparent silk types with similarity to major ampullate, minor ampullate and pyriform silks. These were identified by searching for comparative sequence homologies using Microsoft Office Word. No flagelliform silk or recognisable sticky silks were identified, which is consistent with the biology of Stegodyphus species. In addition to studies of silk, previous common conceptions of dimensional morphologies were examined to see if they could adequately sex theraphosid spiders, including the species that was the subject of the silk study already described. An independent samples t-test was conducted to compare morphologies of particular leg hairs and statistical analysis demonstrated that there were significant differences between males and females (t (70) = 9.445, p < .001). This technique may be important in future evolutionary and ecological studies of theraphosids

Tarantulas and social spiders : a tale of sex and silk

Studies of spider silks indicate that they may outperform virtually all synthetic fibres in terms of strength, elasticity and toughness. To date, most silks studied come from only a select few species and likely underrepresent the immense diversity of the clades. Here, protein and mRNA sequence analyses were used to study silk from two types of spider. The first approach used ESI tandem mass spectrometry to sequence peptide fragments of a silk from a tarantula (Mygalomorphae, Theraphosidae), a hitherto neglected family. The results confirm that the common silk types found in araneomorph spiders, Spidroin 1 and Spidroin 2, are also found in mygalomorphs. A putative N-terminal domain that bears a striking similarity to the N-terminus of araneomorph pyriform silk was isolated. If correctly identified, this would be the first ever recorded N-terminal domain for a mygalomorph. The second approach taken was to construct a cDNA library from theraphosid silk glands and adjacent tissue. Sequencing identified a significant number of uniquely truncated rRNAs. These may be the result of specific 'fragile sites' within these transcripts, which would explain the discrete classes of length polymorphisms found. The cDNA library sequences also provided evidence consistent with RNA editing and furthermore identified the presence of both transcribed nuclear pseudogenes and transposable elements. These may reflect past evolutionary horizontal gene transfer events within the spider genome. Similar analysis of next generation sequencing data from the transcriptomes of three Stegodyphus spp. (Araneomorphae) reveal a range of apparent silk types with similarity to major ampullate, minor ampullate and pyriform silks. These were identified by searching for comparative sequence homologies using Microsoft Office Word. No flagelliform silk or recognisable sticky silks were identified, which is consistent with the biology of Stegodyphus species. In addition to studies of silk, previous common conceptions of dimensional morphologies were examined to see if they could adequately sex theraphosid spiders, including the species that was the subject of the silk study already described. An independent samples t-test was conducted to compare morphologies of particular leg hairs and statistical analysis demonstrated that there were significant differences between males and females (t (70) = 9.445, p < .001). This technique may be important in future evolutionary and ecological studies of theraphosids

Alternative Splicing and Protein Structure Evolution

In den letzten Jahren gab es in verschiedensten Bereichen der Biologie einen dramatischen Anstieg verfügbarer, experimenteller Daten. Diese erlauben zum ersten Mal eine detailierte Analyse der Funktionsweisen von zellulären Komponenten wie Genen und Proteinen, die Analyse ihrer Verknüpfung in zellulären Netzwerken sowie der Geschichte ihrer Evolution. Insbesondere der Bioinformatik kommt hier eine wichtige Rolle in der Datenaufbereitung und ihrer biologischen Interpretation zu. In der vorliegenden Doktorarbeit werden zwei wichtige Bereiche der aktuellen bioinformatischen Forschung untersucht, nämlich die Analyse von Proteinstrukturevolution und Ähnlichkeiten zwischen Proteinstrukturen, sowie die Analyse von alternativem Splicing, einem integralen Prozess in eukaryotischen Zellen, der zur funktionellen Diversität beiträgt. Insbesondere führen wir mit dieser Arbeit die Idee einer kombinierten Analyse der beiden Mechanismen (Strukturevolution und Splicing) ein. Wir zeigen, dass sich durch eine kombinierte Betrachtung neue Einsichten gewinnen lassen, wie Strukturevolution und alternatives Splicing sowie eine Kopplung beider Mechanismen zu funktioneller und struktureller Komplexität in höheren Organismen beitragen. Die in der Arbeit vorgestellten Methoden, Hypothesen und Ergebnisse können dabei einen Beitrag zu unserem Verständnis der Funktionsweise von Strukturevolution und alternativem Splicing bei der Entstehung komplexer Organismen leisten wodurch beide, traditionell getrennte Bereiche der Bioinformatik in Zukunft voneinander profitieren können

Search for biomarkers related to rhinitis and different asthma phenotypes by serum proteomics and immunoassays

sthma is a heterogeneous disease with several clinical phenotypes and molecular endotypes. However, the specific connection between asthma phenotypes and the underlying pathological features is difficult to explain. Thus, the overall aim of this thesis was to search for biomarkers associated with rhinitis and different phenotypes (allergic and non-allergic) and severities (intermittent-mild and moderate-severe) of asthma. To achieve this aim, we studied the role of the immune system through the analysis of certain biomarkers previously related to this disease: CD14 (innate immune system) and CD26/CD126 (adaptive immune system). In addition, in the second part of the present thesis, a prospective, non-target proteomic study aimed to identify new biomarkers associated with different phenotypes of this disease was also performed. This study consisted of the analysis of low abundance serum proteome through iTRAQ-LC-MS/MS and allowed us the identification of several potential biomarkers for allergic (e.g., IGFALS, protein AMBP, or HSPG2) and non-allergic asthma (e.g., CFI), as well as disease severity (e.g., IGFALS)

Role of the complement factor H-related protein 5 in renal disease by protein expression and molecular solution structural studies

Complement Factor H-Related 5 (CFHR5) belongs to the same complement family as the major regulator Factor H. CFHR5 comprises nine short complement regulator (SCR) domains. The duplication of the N-terminal SCR-1/2 domains causes CFHR5 nephropathy, a cause of kidney failure in Cypriots. To clarify the molecular basis of CFHR5 nephropathy, E. coli expression systems were developed for SCR-1 and SCR-1/2 of CFHR5, and recombinant CFHR5 SCR-1/9 was obtained from a commercial mammalian expression system. First, the domain arrangement of CFHR5 SCR-1/9 was studied by analytical ultracentrifugation and X-ray scattering. Sedimentation velocity reported a molecular mass of 134 kDa, indicating that CFHR5 is dimeric. The CFHR5 sedimentation coefficient of 5-6 S decreased with increased NaCl, showing that this became more extended. X-ray scattering also showed that CFHR5 was dimeric. The X-ray mean radius of gyration RG was 5.5 ± 0.2 nm, and its maximum length was 20 nm. This length is low compared to that of 32 nm for monomeric Factor H with 20 SCR domains, indicating that CFHR5 possessed a more compact SCR arrangement than that of Factor H. Atomistic scattering curve modelling of CFHR5 that involved Monte Carlo simulations to generate physically realistic atomistic SCR structures showed that CFHR5 possessed a folded-back compact domain structure. Second, sedimentation velocity showed that SCR-1 was monomeric, while SCR-1/2 was dimeric, thus locating a CFHR5 dimerization site to its N-terminus. In summary, the solution structure of CFHR5 is markedly more compact than previously thought, and its dimerization site was located to SCR-1/2. The perturbation of SCR-1/2 may have a major role in causing CFHR5 nephropathy

An investigation into the biodegradation of peptide cyanotoxins (microcystins and nodularin) by novel gram-positive bacteria.

Cyanobacterial secondary metabolites, microcystins (MC) and nodularin (NOD) have become common contaminants in most aquatic ecosystems over recent years presenting a hazard to animal and human health. Unfortunately, these chemically diverse peptide hepatotoxins remain a challenge to most conventional water treatments due to their stable cyclic structures. Over recent years, bioremediation of MC and NOD has become one of the most exciting areas that holds promise for a successful and cost effective solution for water treatment process. The current work presents the biodegradation of MCs and NOD by bacterial isolates from three different bacteria genus Arthrobacter, Brevibacterium and Rhodococcus belonging to Actinobacteria. A total of five isolates representing the three genera have demonstrated an overall metabolism of MC-LR, -LF, -LY, -LW, -RR and NOD in a Biolog MT2 assay. Subsequently, these bacteria were reported to degrade the range of toxins in a separate batch experiment. The bacterial degradation rate of the above cyanobacterial peptides were found to decrease with the multiple subculturing of the bacteria. However, a rapid degradation was discovered when the bacteria were re-exposed to MC or other prokaryotic peptides demonstrating an inducible bacterial biodegradation. Utilising latest molecular biology techniques, the gene responsible for production of MC degrading enzymes was successfully elucidated and its activity was evaluated. Analysis of the degradation products of MC-LR revealed a glutathione conjugate detoxification mechanism involved during the degradation of MC-LR by Rhodococcus sp. (C1). A novel MC degradation pathway was proposed. Further studies were suggested to fully characterise the degradation pathway and to evaluate the MC detoxification mechanism in bacteria

In vitro expanded human CD4+CD25+ regulatory T cells suppress effector T cell proliferation.

Regulatory T cells (Tregs) have been shown to be critical in the balance between autoimmunity and tolerance and have been implicated in several human autoimmune diseases. However, the small number of Tregs in peripheral blood limits their therapeutic potential. Therefore, we developed a protocol that would allow for the expansion of Tregs while retaining their suppressive activity. We isolated CD4+CD25 hi cells from human peripheral blood and expanded them in vitro in the presence of anti-CD3 and anti-CD28 magnetic Xcyte Dynabeads and high concentrations of exogenous Interleukin (IL)-2. Tregs were effectively expanded up to 200-fold while maintaining surface expression of CD25 and other markers of Tregs: CD62L, HLA-DR, CCR6, and FOXP3. The expanded Tregs suppressed proliferation and cytokine secretion of responder PBMCs in co-cultures stimulated with anti-CD3 or alloantigen. Treg expansion is a critical first step before consideration of Tregs as a therapeutic intervention in patients with autoimmune or graft-versus-host disease

Isolation and characterisation of novel DNA aptamers against Mycobacterium tuberculosis biomarkers: new tools for tuberculosis diagnostics

Tuberculosis is a curable disease with an average treatment success rate of 86 %. Despite this, there were an estimated 1.5 million deaths due to tuberculosis in 2013, most of which occurred in low and middle income countries. In order to overcome tuberculosis in developing countries innovation in diagnostics is key to administering treatment. While detection of whole mycobacteria has been favoured in the past to diagnose tuberculosis, culturing mycobacteria is costly and microscopy is often not sensitive enough due to low bacterial loads. Detection of Mycobacterium tuberculosis biomarkers in urine, a safe and easy specimen to test, could offer a cost effective and simple solution to identify patients with tuberculosis. Enzyme linked immunosorbent assays (ELISAs) were performed on concentrated tuberculosis patient urine to detect two M. tuberculosis biomarkers: lipoarabinomannan (LAM) and early secreted antigen-6 kDa (ESAT-6). Concentrating urine improved the detection of LAM in human immunodeficiency virus (HIV) negative patients and patients with a CD4 count > 200 cells/µl. ESAT-6 was not detected by ELISA due to a high background signal caused by the available antibodies cross reacting with a human protein present in urine which was identified by western blot and mass spectrometry. Targeted mass spectrometry did not detect ESAT-6 or its dimer partner, culture filtrate protein-10 kDa (CFP-10) in tuberculosis positive patient urine. Since concentrating urine samples is impractical in a clinical setting a more sensitive diagnostic is needed to detect LAM in urine and ESAT-6 or CFP-10 in other samples. Aptamers can be packed more densely on biosensor surfaces increasing the dynamic range of detection while matching the affinity that an antibody has for a biomarker. Chemically modified DNA aptamers were isolated for LAM and the ESAT-6.CFP-10 dimer. The aptamers were characterised by enzyme linked oligonucleotide assays (ELONAs) and biolayer interferometry. One aptamer bound with high affinity to ESAT-6 while one aptamer bound with low affinity to LAM. The use of aptamers as capture agents for detecting biomarkers in biological specimens thus appears to be a viable option for diagnosing tuberculosis, although availability and concentration of individual biomarkers seems likely to remain key to the choice of specimen in which to make diagnostic measurements