Analysis of Metacyclic Telomeres in Trypanosoma brucei

Trypanosoma brucei spp. are the agents responsible for African sleeping sickness in man and Nagana in cattle. The organisms have the ability to evade the host's immune system by antigenic variation of their surface coat. The surface coat of the infective forms is composed of a single molecular species, the variant surface glycoprotein (VSG). Each specific VSG is encoded by a separate gene, expression of which occurs in a loose hierarchical order. T. brucei has the coding capacity for approximately 10e3 VSG genes, which are found either in chromosomal clusters or at telomeric loci; it is only from the latter that the gene may be expressed. Telomeric expression sites (ESs) utilized during bloodstream infection are complex, typically between 40 - 60 Kb long, containing several non-VSG expression site associated genes (ESAGs) and preceded by a long barren region devoid of restriction sites. Transcription of telomeric ESs is insensitive to the toxin alpha-amanitin and transcription of a given ES appears to be mutually exclusive of other ESs in vivo. By virtue of their location, chromosomal internal VSG genes need to be transposed to a telomere for expression. The transposition event is duplicative and produces an expression linked copy of the gene. Telomeric VSG genes, however, can be expressed either by duplicative transposition or reciprocal recombination to an active ES, or by in situ activation. The metacyclic stage of the trypanosome life cycle is the first to express VSG genes. The metacyclic form utilizes a highly predictable subset of VSG genes (M-VSGs), comprising only 1-2% of the entire VSG gene repertoire, which appear to be expressed by a distinct and dominant mechanism to that employed during chronic bloodstream infection. Direct molecular analysis of M-VSG gene expression is precluded by the paucity of metacyclic forms in the salivary exudate of the tsetse fly vector and the transient nature of this developmental stage which cannot be cultured in vitro. M-VSG gene expression, however, is still extant in the host bloodstream in the first few days following fly bite. Analysis during this period is compounded by the polyclonal origin of the M-VSG genes expressed by individual organisms and instability of VSG expression. The work described in this thesis focuses on attempts to clone an M-VSG gene telomere and to gain insight into the predictability and stability of the M-VSG repertoire, by analysis of the telomere in a model trypanosome line which circumvents some of the problems associated with previous systems. Cloning and analysis of the BC telomeres for the M-VSG genes for GUTat 7.1 and ILTat 1.22 revealed that each had a remarkably small barren region and shared no sequence homology with ESs used in chronic bloodstream infection, apart from the 70 bp repeat sequence constituting the barren region 5'of the VSG gene. Transcriptional analysis of the ILTat 1.22 metacyclic ES, utilising the model line of trypanosomes expressing the gene in situ, revealed that the ES is extremely short in comparison to bloodstream ESs, extending no more than 3.5 - 4 Kb 5' of the VSG gene. One other region of the ILTat 1.22 BC and GUTat 7.1-2 BC telomeres appeared to be transcriptionally active. This comprised a genomic repetitive element which also was transcribed in procyclic tiypanosomes. The structural individuality of these telomeres was proposed as underlying the stability and physical distinction of the M- VSG repertoire. This hypothesis is supported by an epidemiological analysis of the ILTat 1.22 BC telomere over a 24 year period and spanning diverse epidemic foci of infection. Within Kenyan epidemic foci this telomere is present unaltered in all the stocks investigated over the the period examined and suggests that spread of the disease in East Africa is principally by mechanical transmission; this has important consequences for tackling the disease at source. Cloning and analysis of these telomeres now facilitates characterisation of metacyclic ES control elements and comparisons with other M-ES to be made

The application of artificial intelligence techniques to a sequencing problem in the biological domain

SIGLEAvailable from British Library Document Supply Centre- DSC:DXN002816 / BLDSC - British Library Document Supply CentreGBUnited Kingdo

Establishment of recombinant antibody technologies allowing for the generation of SNAP-tag fusion proteins

Triple negative breast cancer (TNBC's) is a highly aggressive and invasive subtype of breast cancer, typically characterised by the lack of estrogen receptor (ER), progesterone receptor (PR) and Human epidermal growth factor receptor 2 (HER2) with an inexplicable partiality towards African women. The acute heterogenicity and complexity of TNBC tumours, together with a lack of well-defined molecular targets, complicates prognosis of the diseases resulting in patient reliance on traditional therapies, like chemotherapy, radiotherapy, and surgery, which are associated with elevated incidence of adverse effects and relapse. A major contributor to the heterogenicity of TNBCs is the tumour microenvironment which is composed of tumour infiltrating lymphocytes (TILs), tumour cells, healthy cells, and tumour vasculature. TILs have commonly been used as a prognostic marker and show robust predictive value for TNBC. In-depth analysis of the TIL composition within TNBC tumours may provide greatly beneficial information for the development of newer tumour microenvironment changing therapies and could assist doctors in understanding what therapies a particular patient maybe susceptible to. Thus, the diagnosis and therapy of this disease may greatly benefit from improved molecular profiling and patient stratification. Precision medicine seeks to provide such a solution, by dividing patients into subpopulations based on disease-specific profiles. The identification of new molecular targets would provide the basis for development of novel therapies. To this end, one of the major aims of this thesis was to develop a phage display based screening technique which could be utilised to isolate novel TNBC specific cancer antibodies. Once selected these antibodies could be used to generate TNBC specific therapies. Specific monoclonal antibodies (mAbs) and derivatives thereof, have already been established as a revolutionary tool for drug delivery to cancerous cells. Such antibodies have been conjugated to cytotoxic drugs to form antibody-drug conjugates, which may exhibit multiple advantages over their unconjugated counterparts, but their general use in clinical application has been restricted due to developmental deliberations. Historical conjugation strategies used for the generation of ADCs commonly resulted in heterogeneous mixtures of ADC species, with varying drug-to-antibody ratios resulting in unpredictable pharmacologic characteristics and safety profiles. In more recent time, self-labelling tags such as Snaptag have provided a means of developing homogenised recombinant immunotherapeutics. Snaptag is a modified version of a human DNA repair enzyme, O6 - alkylguanine-DNA-alkyltransferase (AGT) which naturally removes alkyl residues from damaged DNA. The enzyme reacts specifically with benzylguanine (BG) derivatives via irreversible transfer of alkyl groups to cysteine residues forming stable end products. In this thesis, Snaptag technology, together with other antibody discovery and manipulation tools was used to develop a methodology allowing for the generation of disease specific fusion proteins. Specifically, these fusion proteins consist of single-chain antibody fragments genetically fused to snaptag, allowing for the generation of recombinant ADCs that could be used as a drug delivery system carrying any BG-modified drug to a disease specific targets. In addition, snaptag interacts with BG in a 1:1 stoichiometry giving rise to homogenised combination products which when fused to a scFv provides a fail-safe target-specific therapeutic option. In addition to antibody conjugates, one of the most promising of all mAb based therapies currently used, are checkpoint inhibitors. In a balanced immune response, immune activation is counteracted with immunoregulatory pathways such as checkpoint inhibition. These negative regulatory pathways are necessary for maintaining tolerance and preventing hyperactivation, and are governed by cell surface, inhibitory receptors known as ‘'checkpoint inhibitors''. Blocking of checkpoint pathways during chronic infections and cancer has been shown to improve T-cell functions leading to reduced viral load and tumour burden. These findings have been translated into clinical application where checkpoint inhibitors, which are monoclonal antibodies targeting CTLA-4, PD1, PD-L1 or other inhibitory ligands, have been used to block these inhibitory interactions. The main intention of this research was to develop a methodology which could be used to generate snaptag based recombinant fusion proteins with potential diagnostic and therapeutic applications. Several snaptag based fusion proteins were developed using the recommended methodology these included fusion proteins targeting breast cancer specific antigen BCK1, checkpoint inhibitors PDL1, B7.1/CD80 (interacts with CTLA-4),and TIL characterising markers CD3, CD4, CD8, CD19 and CD20. In addition, to demonstrate the versatility and robustness of this methodology we sought to develop a snaptag based fusion protein not targeting breast cancer related antigens. Zika virus, an emerging infectious disease, currently lacking specific therapies was chosen for this purpose. An scFv derived from antibodies targeting the the Zika-DIII envelop protein, which is essential to the viral infection cycle was used in the snap fusion protein. The resulting ZIKA-DII-snap fusion protein demonstrated specific binding to zika virus membrane fractions. This research demonstrates the feasibility of using snaptag technology as a state-of-the-art conjugation strategy capable of bypassing the challenges previously associated with using antibodies as an effective delivery system for therapeutic drugs. By combining the applicability of snaptag technology with other antibody isolation and manipulation tools we were able to generate several functional snaptag based recombinant fusion proteins. Establishment of this methodology represents an important first step in generating medically necessary, pharmaceutically acceptable immunoconjugates that is instrumental in shifting general therapy towards a more personalized precision medicine approach

Capturing the ‘ome’ : the expanding molecular toolbox for RNA and DNA library construction

All sequencing experiments and most functional genomics screens rely on the generation of libraries to comprehensively capture pools of targeted sequences. In the past decade especially, driven by the progress in the field of massively parallel sequencing, numerous studies have comprehensively assessed the impact of particular manipulations on library complexity and quality, and characterized the activities and specificities of several key enzymes used in library construction. Fortunately, careful protocol design and reagent choice can substantially mitigate many of these biases, and enable reliable representation of sequences in libraries. This review aims to guide the reader through the vast expanse of literature on the subject to promote informed library generation, independent of the application

High-Throughput Isolation and Mapping of C. elegans Mutants Susceptible to Pathogen Infection

We present a novel strategy that uses high-throughput methods of isolating and mapping C. elegans mutants susceptible to pathogen infection. We show that C. elegans mutants that exhibit an enhanced pathogen accumulation (epa) phenotype can be rapidly identified and isolated using a sorting system that allows automation of the analysis, sorting, and dispensing of C. elegans by measuring fluorescent bacteria inside the animals. Furthermore, we validate the use of Amplifluor® as a new single nucleotide polymorphism (SNP) mapping technique in C. elegans. We show that a set of 9 SNPs allows the linkage of C. elegans mutants to a 5–8 megabase sub-chromosomal region

Molecular Genetics of S. thermonitrificans ISP5579

The majority of commercially important antibiotic producing Streptomyces are mesophiles, grown at 25-3