Computational analysis of alternative splicing in human and mice

Im ersten Teil wurden Transkript-Spleißstellen untersucht, mit dem Ziel, alternative und Referenzspleißstellen zu unterscheiden. Die Ergebnisse belegen, dass sich beide Klassen von Spleißstellen durch einen Spleißstellen-Score und vermehrtes Auftreten von Spleißfaktor-Bindemotiven in Umgebung der Spleißstellen abgrenzen lassen. Zusätzlich konnte eine positive Korrelation zwischen der Häufigkeit der Nutzung bestimmter Spleißstellen und dem Spleißstellen-Score in beiden Vergleichsklassen nachgewiesen werden. Diese Abhängigkeit impliziert, dass die Genauigkeit der Annotation alternativer Spleißvarianten mit der Anzahl beobachteter Transkripte steigt. Im zweiten Teil wurde das Spleißsignalmotiv GYNNGY untersucht, welches mehr als 40% aller überlappenden Donor-Spleißsignale ausmacht. Mittels in silico Analysen und experimenteller Validierung wurde die Plausibilität dieses subtilen Spleißmusters bestätigt. Der Vergleich mit anderen humanen Spleißvarianten sowie mit Tandem Donoren in Maus-Transkripten zeigte zudem ausgeprägte Unterschiede bezüglich des Spleißstellen-Scores, der Konservierung, sowie dem Vorkommen von Spleißfaktoren-Bindemotiven. Die Verschiebung des Leserasters durch alternatives Spleißen an GYNNGY-Donoren lässt auf eine komplexe Rolle im RNA-Reifungsprozess schließen. Im dritten Teil wurden Reaktionen des spleißosomalen Makrokomples aus publizierten, experimentellen Daten zusammengestellt und mit Hilfe der Petri-Netz-Theorie in einem qualitativen Modell dargestellt. Unter Annahme eines Steady-State Systems wurden minimale, semipositive T-Invarianten berechnet und zur Validierung des Modells herangezogen. Auf Grundlage der vollständigen Abdeckung des Reaktionsnetzwerks mit T-Invarianten konnten weitere Strukturmerkmale, wie Maximal-Gemeinsame Transitions.Mengen und T-Cluster berechnet werden, welche wichtige Stadien des Spleißosomaufbaus widerspiegeln

Clonality in adult T-cell leukaemia/lymphoma

Human T-lymphotropic virus type 1 (HTLV-1) is a retrovirus that persists lifelong within the infected host by driving expansion of infected CD4+T-cells. It is the cause of adult T-cell leukaemia/lymphoma (ATL), an aggressive CD4+ T-cell malignancy, which arises in approximately 5% of individuals typically following decades of asymptomatic infection. The reasons why some individuals develop ATL remain unknown. In this laboratory a novel customised high throughput sequencing and bioinformatic method has been developed in order to map and accurately quantify the proviral integration sites within each host genome in order to identify clonal populations within each host. In this study I aimed first to test the hypothesis that there is a single provirus integrated into each host genome, and secondly to test the hypothesis that the site of retroviral integration determines the risk of leukaemia. In order to quantify the average number of proviral integration sites in each host cell, we isolated infected T-cells from the peripheral blood of infected individuals by limiting dilution cloning. Integration site analysis of these clones revealed that in natural infection each T-cell clone carries a single integrated provirus. This work formed the basis of a publication in the journal Blood (Cook et al 2012). I describe the systematic analysis of the clonality, structure and the integrity of the proviral tax gene in a large cohort of ATL patients (n=197). I correlate these findings with the clinical subtype of ATL and the landscape of the host genome flanking the proviral integration site. Based upon our findings we conclude that the integration site in cis does not directly cause leukaemogenesis and hypothesise that the absolute number of infected clones within an individual, and not oligoclonal proliferation, predisposes to malignant transformation.Open Acces

A database on alternative splice forms on the integrated genetic map service (IGMS)

The IGMS is a comprehensive information system that combines the knowledge from genomic sequence, genetic map and genetic disorders databases. This system is updated weekly and focuses on the analysis of EST data. The IGMS identifies UniGene clusters that are differentially expressed in different types of cancer with respect to different reference tissues. The results can be combined with clinical data to asses the potential relevance of specific genes for patient survival or metastatic spread. The second application maps EST with a specific expression profile. Our third application generates a database of alternative splice forms for nine organisms from EST and mKNA sequence data. The results can be used to find splicing patterns specific for certain tissues or tumour types

Homo-Oligomerization of the Activating Natural Killer Cell Receptor NKp30 Ectodomain Increases its Binding Affinity for Cellular Ligands

Natural killer (NK) cells are large granular lymphocytes of the innate immune system that spontaneously kill foreign, tumor and virus-infected cells without prior sensitization. In addition, NK cells act as immune regulators by secretion of chemokines and cytokines as well as direct interaction with other immune cells such as dendritic cells. NK cell function is regulated by a balance between inhibitory and activating signals that are transduced into the cell upon target cell interaction. One of the major activating NK cell receptors is the natural cytotoxicity receptor NKp30. Notably, NKp30 plays a unique role since it is the only NK cell receptor involved in triggering NK cell-mediated cytotoxicity as well as shaping the adaptive immune response. Reduced NKp30 expression has clinical implications in patients with acute myeloid leukemia, cervical cancer, and high grade squamous intraepithelial lesions as well as gastrointestinal sarcoma. Furthermore, downregulation of NKp30 expression resulted in an impaired natural cytotoxicity against leukemia cells and was directly correlated with reduced survival. NKp30 is a type I transmembrane protein of approximately 30 kDa comprised of an I-type Ig-like ligand binding domain (LBD), a flexible membrane proximal stalk domain, a single transmembrane helix, and a short cytosolic tail. For intracellular signal transduction, NKp30 associates with the immunoreceptor tyrosine-based activating motif (ITAM)-bearing adaptor molecule CD3zeta via oppositely charged amino acid residues within their transmembrane domains. In 2011, the 3D structure of the NKp30LBD was solved in an unbound and a ligand-bound form. However, so far, only few cellular ligands (BAG-6 and B7-H6) have been discovered, and the molecular details of ligand recognition by NKp30 are poorly understood. Recently, it was shown that the membrane proximal stalk domain of NKp30 is important for efficient ligand binding and signaling with respect to its length and amino acid composition. Additionally, it was demonstrated that proper N-linked glycosylation of the ligand binding domain is essential for ligand binding. But it is still vague, how this germline-encoded receptor is able to recognize multiple nonrelated ligands. Interestingly, a crystallographic dimer of the NKp30 ectodomain was observed arguing for potential intrinsic capability to self-assemble. Moreover, a fraction of NKp30 expressed in E. coli forms oligomers as detected by size exclusion chromatography. The aim of this thesis was to identify structural models and mechanisms, which enable variations of the ligand binding interface of NKp30 to recognize a multiplicity of diverse ligands. In this respect, soluble NKp30 ectodomain variants as well as an anti-NKp30 antibody specifically recognizing an epitope within the LBD of NKp30 were generated for molecular and cellular investigation to address the intrinsic ability of NKp30 to form oligomers, which might impact ligand binding affinity and the efficiency of target cell killing by NK cells. In this thesis, it was demonstrated that baculovirus-infected insect cells were a suitable expression system to produce correctly folded, post-translationally modified and ligand binding-receptive NKp30 proteins, which were functionally equivalent to those derived from human expression hosts. Furthermore, a polyclonal anti-NKp30 antibody, which is specific for an epitope located within the NKp30LBD, was purified from the blood serum of a peptide-immunized rabbit and validated for several molecular and cellular applications. Based on soluble NKp30 proteins comprising either the entire NKp30 ectodomain (LBD and stalk domain) or the ligand binding domain alone, a concentration-dependent formation of NKp30 ectodomain homo-oligomers was found, which was effected by two specific binding sites, one present within the Ig domain of NKp30 and one within its membrane proximal stalk domain. Moreover, both NKp30 ectodomain variant oligomers were functional in ligand binding and contributed to a highaffinity interaction with its cellular ligand B7-H6, which was strongly promoted by the stalk domain. Although both NKp30 ectodomain variant oligomers formed spherical particles of the same diameter in solution, in presence of the stalk domain one oligomer was composed of more monomers. Single particle electron microscopy analyses revealed that the number of calculated 2D classes increased tremendously for the particles of the longer NKp30 ectodomain variant. Therefore, a densest packing of spheres for the monomers within both NKp30 ectodomain variant oligomers is suggested, whereas the flexibility of the stalk domain might allow for a more variable orientation of these monomers within the particle, thereby increasing the heterogeneity of structural arrangements. However, based on decoration experiments with soluble NKp30 ectodomain proteins and primary NK cells facilitating more physiological conditions for NKp30 self-assembly, an ordered arrangement of several NKp30 receptors on the plasma membrane in head-to-head orientation is proposed. Based on these data, oligomerization of the NKp30 ectodomain represents a potent mechanism to modulate the ligand binding affinity of NKp30 for corresponding ligands by increased avidity. Because the degree of oligomerization is dependent on the local receptor concentration, which is upregulated by IL-2 upon NK cell activation, oligomerization of NKp30 might be a molecular transformer of NK cell activation into enhanced cytotoxicity. Future experiments are now focused to investigate NKp30 self-assembly on the plasma membrane of NK cells to enable modulation of the cytotoxicity of NK cell based therapeutics

The production, expression, and characterisation of insulin and GAD65 recombinant FAB for use in

Ph.D., Faculty of Health Sciences, University of the Witwatersrand, 2006.Objectives. Autoantibodies to the 65kDa isoform of glutamic acid decarboxylase (GAD65Abs) are accepted markers for type 1 diabetes and, together with autoantibodies to insulin (IAA) and a protein tyrosine phosphatase-like islet cell antigen (IA-2), predict the disease. IAA are often the first autoantibodies detected in type 1 diabetics and can be present before the onset of clinical diabetes. These autoantibodies and their epitopes are however not well characterized. We explored the use of monoclonal antibodies and their recombinant Fab (rFab) as reagents for epitope analysis. Methods and Results. Four rFab specific for insulin were cloned from murine monoclonal antibodies (mAbs) 1E2, HB-126, HB-123, HB-127, and one rFab specific for GAD65 was cloned from human mAb IgG antibody DP-D (derived from autoimmune disease patients), to characterise insulin and GAD65 autoantibodies present in the sera of patients with type 1 diabetes. Only rFab 126 and DP-D showed insulin and GAD65 specific binding, respectively in radiobinding assays. In competition experiments with sera positive for autoantibodies to insulin the rFab 126 significantly reduced the binding to 125I-insulin by sera of type 1 (n=35) and type 1.5 diabetes (or LADA) (n=14) patients (p<0.0001). There was no difference in the competition pattern in IAA positive type 1 diabetes patients (n=35) and IAA positive type 1.5 diabetes patients (n=14). The insulin epitope that the rFab binds to was mapped using competitive radiobinding assays with two monoclonal antibodies (mAb 1 and mAb 125) whose epitopes are on the B chain and A chain loop of insulin, respectively. We found the epitope of this recombinant antibody to be located on the A chain loop of the insulin molecule. The 3-dimensional structure of rFab 123, 126 and DP-D were determined using an automated homology modelling programme. Using the computer programme ‘PatchDock’ we attempted to further map the epitope that rFab 126 binds to on insulin. Of the three models generated, only one supported our findings that rFab 126 binds to the A chain loop of insulin. The binding of GAD65Ab in 61 type 1 diabetes patients to GAD65 was analyzed by competitive radioimmunoassays with rFab DP-D to ascertain disease-specific GAD65Ab binding specificities. The median binding was reduced significantly by rFab DP-D (80%) (p<0.0001). The competition pattern in type 1 diabetes patients was different from that in GAD65Ab-positive type 1.5 diabetes patients (n=44), first degree relatives (n=38), and healthy individuals (n=14) (Padoa et al., 2003). Conclusions. We have shown that rFab specific for insulin and GAD65 can be generated using PCR technology and that such agents can be used to determine the insulin/GAD65 epitopes recognized by autoantibodies from type 1 and 1.5 diabetics. These novel findings with GAD65- and insulin-specific rFab support the view that type 1 diabetes is associated with disease- and epitope-specific GAD65- and insulin-autoantibodies and supports the notion that the middle epitope of GAD65 is disease-specific. These GAD65-specific rFab should prove useful in predicting type 1 diabetes. Furthermore, rFabs may be a novel method for blocking autoimmune responses against β cell autoantigens in type 1 diabetics

Microbial Biofilms

In the book Microbial Biofilms: Importance and applications, eminent scientists provide an up-to-date review of the present and future trends on biofilm-related research. This book is divided with four subdivisions as biofilm fundamentals, applications, health aspects, and their control. Moreover, this book also provides a comprehensive account on microbial interactions in biofilms, pyocyanin, and extracellular DNA in facilitating Pseudomonas aeruginosa biofilm formation, atomic force microscopic studies of biofilms, and biofilms in beverage industry. The book comprises a total of 21 chapters from valued contributions from world leading experts in Australia, Bulgaria, Canada, China, Serbia, Germany, Italy, Japan, the United Kingdom, the Kingdom of Saudi Arabia, Republic of Korea, Mexico, Poland, Portugal, and Turkey. This book may be used as a text or reference for everyone interested in biofilms and their applications. It is also highly recommended for environmental microbiologists, soil scientists, medical microbiologists, bioremediation experts, and microbiologists working in biocorrosion, biofouling, biodegradation, water microbiology, quorum sensing, and many other related areas. Scientists in academia, research laboratories, and industry will also find it of interest

The role of miR-23a~24~27a cluster in the pathogenesis of treatment resistant rheumatoid arthritis

Background: Rheumatoid arthritis (RA) is a symmetric polyarthritis arising from autoimmune dysregulation leading to severe disability and increased risk of co- morbidities and death. A chronic disproportionate inflammatory process lies at the heart of disease pathogenesis. Breach of self-tolerance, subsequent immune effector cell activation in the context of abundant expression of effector cytokines all contribute to uncontrolled inflammation. Molecular safeguards that normally operate to promote immune regulation appear defective in RA. Intensive basic and translational research over the last 30 years have contributed the emergence of an array of new therapeutics for the treatment of RA, which has transformed patient outcomes. The identity of the cytokine targeting treatments that have been most successful elucidates a functional hierarchy that implicates elements of both innate and adaptive immunity. In particular, dysregulation of TNFα and IL-6 biology are at the core of effector pathways and as such unravelling their detailed regulation is of critical importance. Moreover their primary synthesis places myeloid cells, and, in particular, blood-derived monocytes at the heart of pathogenic circuitry. Best current clinical practice is to treat early disease and deploy aggressive treatments directed towards restoration of immune balance in virtually all patients. However only a proportion of such patients will actually have poor prognosis disease and in reality merit such aggressive interventions - the identification of such clinical endotypes is a major challenge for the next decade. The field of epigenetics and consequent regulatory control of inflammatory cells offers rich potential in this regard. Examples of such regulatory elements are small RNA species – microRNAs (miRs), which serve as negative regulators of cellular transcription and thereby repress protein translation. Importantly they do so across functionally integrated pathways, operating beyond individual moieties. A growing body of evidence implicates a significant role of miRs in the regulation of inflammatory processes in the context of RA. Objectives: To identify miR species that are differentially regulated in patients with poor response to therapeutic intervention, compared to patients with well- controlled disease and healthy controls. Thereafter, to characterise candidate 2 miRs arising from these investigations to thereby determine their functional significance. Together these studies will shed light on a substantially ignored area of RA biology, namely the underlying mechanisms that subserve drug resistance in RA. Key Results: Microarray profiling of CD14+ monocytes derived from patients with drug resistance upon receipt of DMARDs or biologic treatments, compared with good responders or matched healthy controls identified the miR-23a~24-2~27a cluster to be significantly repressed in monocytes from resistant RA. Further analysis identified that two members of the cluster, miR-23a and miR-27a are implicated in a feedback loop regulating the IL-6 pathway. Thus IL-6 stimulation of primary monocytes suppresses the expression of this miR cluster, permitting expression of their direct molecular target, namely IL-6R, thus sensitising cells to further IL-6 signalling. I also observed that cells lacking miR-23a and miR-27a express higher levels of the pro-inflammatory cytokines TNFα and IL-6 when stimulated with LPS, further confirming that lack of these miRs has direct implications for chronic inflammatory processes. The remaining member of this miR cluster, miR-24, was shown to directly target methylene tetrahydrofolate reductase but not dihydrofolate reductase enzymes, implicating it in the target pathway of methotrexate (MTX), the most commonly used anchor DMARD. Although this is unlikely to confer disease resistance, this interaction suggests that miR-24 levels could be predictive of tolerability of methotrexate use. The potential biomarker capabilities of miR-24 in relation to MTX use, or miR-23a and miR-27 with regards to responsiveness to anti-IL-6 or JAK signalling inhibition therapeutics will be evaluated in my future work. Conclusion: This series of studies has elucidated highly novel pathways that mediate amplification of inflammatory responses in blood-derived monocytes through feedback pathways operating via regulatory miRs. Furthermore, analysis of a distinct cohort of RA patients allowed identification of miR species that have the potential to be utilized as clinical biomarkers for treatment efficacy or tolerability evaluation. Although a separate validation study is required, the detailed investigation of the role of these miRs performed here provides a clear mechanistic insight into their function and will certainly support future discovery