Transcriptional regulation of yir genes in Plasmodium yoelii yoelii infected erythrocytes

In 1998, a large multi gene family was discovered in Plasmodium vivax (del Portillo et al., 1998). This multigene family was termed vir, and later studies showed that vir homologues existed in the three rodent malaria species, Plasmodium chabaudi (cir), Plasmodium berghei (bir) and Plasmodium yoelii (yir). By 5x coverage sequencing of Plasmodium yoelii 17X, 838 yir genes were predicted (Carlton et al., 2002), making this the largest known multi gene family in Plasmodium. YIR proteins are expressed at the surface of infected erythrocytes (Cunningham et al., 2005), and therefore this family is thought to be involved in antigenic variation. The aim of this thesis was to examine how P.yoelii regulates transcription of the yir family. The yir gene structure was verified experimentally, and phylogenetic analysis showed that yir genes could be divided into five supergroups consisting of yir genes with different sizes and subtelomeric localisation. In the blood stages, numerous yir genes were transcribed from all the supergroups in immunocompromised mice. However, a maximum of two yir genes were transcribed in single infected erythrocytes at the Schizont stage, which suggested strong silencing mechanisms. The transcriptional start and polyadenylation sites were identified experimentally, and it was found that both occurred at highly conserved motifs. In addition, the transcription initiation site was located close to an unusual and universally conserved triple-repeat motif, and it was found that all yir transcripts in two populations of parasites initiated downstream of this motif. Transfection experiments were performed in order to examine the role of this motif, but no solid conclusions could be drawn from these. Several alternative splicing events were detected in the yir 5'UTRs, and one of these led to exon 1 skipping of a yir gene. Through bioinformatic analysis of yir 5' intergenic regions, it was found that the UTR introns had a discrete distribution amongst the yir supergroups

Shared Control for Wheelchair Interfaces

Independent mobility is fundamental to the quality of life of people with impairment. Most people with severe mobility impairments, whether congenital, e.g., from cerebral palsy, or acquired, e.g., from spinal cord injury, are prescribed a wheelchair. A small yet significant number of people are unable to use a typical powered wheelchair controlled with a joystick. Instead, some of these people require alternative interfaces such as a head- array or Sip/Puff switch to drive their powered wheelchairs. However, these alternative interfaces do not work for everyone and often cause frustration, fatigue and collisions. This thesis develops a novel technique to help improve the usability of some of these alternative interfaces, in particular, the head-array and Sip/Puff switch. Control is shared between a powered wheelchair user, using an alternative interface and a pow- ered wheelchair fitted with sensors. This shared control then produces a resulting motion that is close to what the user desires to do but a motion that is also safe. A path planning algorithm on the wheelchair is implemented using techniques in mo- bile robotics. Afterwards, the output of the path planning algorithm and the user’s com- mand are both modelled as random variables. These random variables are then blended in a joint probability distribution where the final velocity to the wheelchair is the one that maximises the joint probability distribution. The performance of the probabilistic approach to blending the user’s inputs with the output of a path planner, is benchmarked against the most common form of shared control called linear blending. The benchmarking consists of several experiments with end users both in a simulated world and in the real-world. The thesis concludes that probabilistic shared control provides safer motion compared with the traditional shared control for difficult tasks and hard-to-use interfaces

Investigation of Factors Influencing var Gene Expression in Plasmodium falciparum Parasites from Acute and Chronic Infections

Im Jahre 2015 gab es weltweit 214 Millionen neue Malariafälle und 438.000 Todesfälle durch Malaria. Der einzellige Parasit Plasmodium falciparum (P. falciparum) verursacht die schwerste Form der Malaria und ist verantwortlich für die Mehrheit der auftretenden Todesfälle. Durch die Expression des Plasmodium falciparum erythrocyte membrane proteins 1 (PfEMP1) auf der Oberfläche von infizierten Erythrozyten können diese an Endothelrezeptoren des Wirts adhärieren, was zu den Symptomen der Malaria führt. PfEMP1 wird von der Familie der hypervariablen var-Gene kodiert. Jeder Parasit besitzt etwa 60 verschiedene var-Gene, von denen jeweils nur eins pro Parasit exprimiert wird. Ein ständiger Wechsel des aktiv transkribierten var-Lokus führt zur Antigenvariation, die es dem Parasit ermöglicht, der Immunantwort des Wirtes zu entgehen. Im ersten Projekt dieser Dissertation konnte gezeigt werden, dass die Moskito- und Humanpassage bei malaria-naiven Individuen die var-Gen Transkription grundlegend verändert. Die in vitro var-Gen Transkription wird maßgeblich durch die Replikationsdauer von P. falciparum im Wirt beeinflusst. Je länger eine Parasitenpopulation der Rezeptorselektion im Wirt ausgesetzt ist, umso mehr verschiebt sich die var-Gen Transkription zugunsten von wenigen, sehr stark transkribierten var-Genen. Des weiteren wurde herausgefunden, dass die var-Gen Transkription in Abwesenheit eines Selektionsdrucks scheinbar vor allem durch ein festgelegtes genetisches Programm definiert wird. Die Daten aus dem zweiten Projekt dieser Dissertation weisen darauf hin, dass PfEMP1 nicht allein für das variable Oberflächensignal von Parasiten in chronischen Infektionen verantwortlich ist, sondern andere variable Oberflächenproteine, wie z.B. die STEVOR und RIFIN Proteinfamilien, am variablen Obeflächensignal beteiligt sind. Zudem scheint die Rezeptorselektion in naiven Wirten und die Antikörperantwort in semi-immunen Wirten die var-Gen Expression zu beeinflussen. Im dritten Projekt dieser Dissertation konnte in zwei von 10 Feldisolaten aus verschiedenen afrikanischen Regionen ein var-Gen gefunden werden, welches ebenfalls konserviert ist. Die Feldisolate wiesen ansonsten eine hohe Diversität in den nichtkodierenden Regionen auf. Dies weist auf eine Selektion gegen die Diversität dieses spezifischen var-Genes hin. In zukünftigen Untersuchungen mit kontrollierten Malariainfektionen (controlled human malaria infections (CHMI)) von malaria-naiven und semi-immunen Individuen könnte der Einfluss der Selektionsdrücke des Wirtes auf die var-Gen-Expression und die Rolle der anderen variable Oberflächenprotein bei der Antigenvariation untersucht werden

Metatranscriptomics captures dynamic shifts in mycorrhizal coordination in boreal forests

Carbon storage and cycling in boreal forests—the largest terrestrial carbon store—ismoderated by complex interactions between trees and soil microorganisms. However,existing methods limit our ability to predict how changes in environmental conditionswill alter these associations and the essential ecosystem services they provide. To addressthis, we developed a metatranscriptomic approach to analyze the impact of nutrientenrichment on Norway sprucefine roots and the community structure, function, andtree–microbe coordination of over 350 root-associated fungal species. In response toaltered nutrient status, host trees redefined their relationship with the fungal commu-nity by reducing sugar efflux carriers and enhancing defense processes. This resulted ina profound restructuring of the fungal community and a collapse in functional coordi-nation between the tree and the dominant Basidiomycete species, and an increase infunctional coordination with versatile Ascomycete species. As such, there was a func-tional  shift  in  community  dominance  from  Basidiomycetes  species,  with  importantroles in enzymatically cycling recalcitrant carbon, to Ascomycete species that have mela-nized cell walls that are highly resistant to degradation. These changes were accompa-nied  by  prominent  shifts  in  transcriptional  coordination  between  over  60  predictedfungal effectors, with more than 5,000 Norway spruce transcripts, providing mechanis-tic insight into the complex molecular dialogue coordinating host trees and their fungalpartners. The host–microbe dynamics captured by this study functionally inform howthese complex and  sensitive biological  relationships may mediate  the carbon  storagepotential of boreal soils under changing nutrient conditions

The Diversity Landscape of <i>Plasmodium falciparum var</i> Genes

Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) is an important group of cytoadhesive multi-domain Plasmodium falciparum surface antigens that are inserted onto the surface of infected erythrocytes and are encoded by the var multi-gene family. PfEMP1 antigens are thought to be important targets of naturally acquired immunity to malaria. This thesis describes nucleotide variation in DBLα sequence tags, a semi-conserved region within the DBLα domain that can be PCR amplified using universal primers. PfEMP1 can be classified using a number of approaches some of which are associated with particular expression patterns in severe, non-severe and asymptomatic malaria. This work compared the classification approaches with an aim of understanding the extent of overlap or discordance between them. To explore non-random distribution of variation in respect to var’s functional specialization, a clustering approach was developed and applied in exploring patterns of nucleotide variation among var subsets from different geographical locations in addition to distribution of predicted B and T-cell epitopes among the var subsets. In summary the sequences showed distinct patterns of nucleotide substitution that suggests that var sequences are under both diversifying and purifying selection

Identification and characterization of T cell receptors reactive to the E7(11-19) epitope of HPV-16

High-risk human Papilloma viruses (HR-HPVs) are the main causative agents of cervical, anal, vulvar, vaginal and penile cancer as well as head and neck cancer. The most prevalent HR-HPV types are HPV16 and HPV18. HPV16 is responsible for about 50% of HPV-related cancers and thus it is a preferred type to study. The HPV16 E7 (11-19) peptide is a well-characterized epitope presented by HLA-A*02:01 molecules on HPV16+ tumor cells. Therefore, it represents a tumor-specific antigen and is an ideal target for Adoptive T cell therapy (ATC) due to its viral source and being absent in healthy tissues. Recently, a proof-of-concept study in metastatic cervical cancer patients has shown that administration of HPV16-specific tumor infiltrating lymphocytes (TILs) expanded ex vivo and infused back to the patient induced tumor regression. However, selection and expansion of TILs has some important disadvantages compared to genetically modified T cells. This project was aimed at identifying HPV16 E7-specific TCRs that could be applied as an effective immunotherapy to cancers caused by this virus. We believe that tumor samples from patients at developing stages of HPV+ cancers might not be optimal for finding HPV-specific TCRs, since their TILs react weakly against the E6/E7 oncoproteins. Furthermore, HPV16- specific cytotoxic T cells can be frequently found in peripheral blood of healthy donors. Therefore, in vitro stimulation of CD8+ T cells derived from healthy individuals and subsequent identification of their TCR repertoire could be a better way to obtain efficient TCR candidates for adoptive T cell therapy of HPV16+ cancers. For an efficient in vitro stimulation of T cells, we used a previously constructed and purified recombinant fusion protein composed of an N-terminal fragment of E7 (amino acids 1-30, E71- 30) linked to the N terminus of Flt3L. Immature dendritic cells isolated from PBMCs of healthy donors were pre-incubated with the fusion protein and then co-cultured with autologous T cells. E7-reactive CD8+ T cells (IFN- γ + , CD137+ and HLA-A2-E711-19-Tetramer+ ) were isolated. The exact TCR profile of the E7-reactive T cells was determined by single-cell V(D)J sequencing using the 10X Genomics platform. Our results showed that the E7-Flt3L is a functional protein that can efficiently increase the frequency of CD8+ T cells targeting the HLA-A*02:01-restricted iv HPV16 E7(11-19) epitope among peripheral lymphocytes of healthy donors. Moreover, our workflow combined tetramer binding and activation of T cells to improve the selection of truly reactive T cells. Three TCR candidates were screened in vitro, which showed different patterns of specificity, avidity, and reactivity. Further, we were able to identify several E7(11-19)-reactive motifs in the CDR3β region through in silico characterization of 22 selected TCRs. Importantly, most of these motifs were enriched in patients who cleared HPV16 infection compared to patients with cervical intraepithelial neoplasia grade 3 (CIN3) or higher. Taken together, our study has established an efficient workflow for the identification of TCRs targeting tumor viral antigens which can be an important step toward TCR discovery

A framework for whole lifecycle cost of long-term digital preservation

Digital preservation, also known as digital curation, is the active management of digital information, over time, to ensure its accessibility and usability. Digital preservation is nowadays an active area of research, for many reasons: the rapid evolution of technology, which also results in the rapid obsolescence of old technologies; degradation of physical records; constantly increasing volumes of digital information and, importantly, the fact that it has started to become a legal obligation in many countries. This research project aims to develop an innovative framework estimate costs of long term digital preservation. The framework can lead to generating a cost model that quantifies costs within different business sectors, while capturing the impact of obsolescence and uncertainties on predicted cost. Case studies from financial, healthcare and clinical trials sectors are used to prove the framework concept. Those sectors were chosen because between them they share all file types that are required to be preserved and all are either obliged by European or local laws, e.g. EU Data Retention Directive (2006/24/EC) and/or UK Data Retention Regulations 2014 No. 2042, or interested in preserving their digital assets. The framework comprises of three phases: assessing digital preservation activities, cost analysis and expansion and cost estimation. The framework has integrated two processes that will enable the user to reach a more accurate cost estimate; a process for identifying uncertainties with digital preservation activities and a cost modelling process. In the framework cloud computing was used as an example for storage and compute technologies. Combining different research methodology techniques was used in this research project. Starting with conducting a thorough literature review covering digital preservation and cost modelling. Following the literature review; is a combination qualitative and quantitative approaches, using semi-structured interview technique to collect data from industry experts. Industry experts were chosen from companies, firms and government bodies working with or researching digital preservation. Finalising with validating results by real-life case studies from businesses in selected sectors and experts’ verdict. Comparing the output of the framework to real-life case studies, demonstrated how companies/firms, who target to preserve their digital assets, can utilise it to predict accurately future costs for undertaking such investment. By applying industrially-based cost modelling approaches the framework generates a cost model that predicts single-point and three-points cost estimates, an obsolescence taxonomy, uncertainties identification process and quantifying uncertainties and obsolescence impact on cost prediction. Providing decision makers with all the framework outputs, will provide them with quantifiable information about their future investment, while remaining clear to understand and easy to amend. This makes the framework provide long-term total cost prediction solution for digital preservation to firms; helping, guiding and adding insight into digital preservation added value

A suite of computational tools to interrogate sequence data with local haplotype analysis within complex ​Plasmodium​ infections and other microbial mixtures

The rapid development of DNA sequencing technologies has opened up new avenues of research, including the investigation of population structure within infectious diseases (both within patient and between populations). In order to take advantage of these advances in technologies and the generation of new types of data, novel bioinformatics tools are needed that won’t succumb to artifacts introduced by the data generation, and thus provide accurate and precise results. To achieve this goal I have create several tools. First, SeekDeep, a pipeline for analyzing targeted amplicon sequencing datasets from various technologies, is able to achieve 1-base resolution even at low frequencies and read depths allowing for accurate comparison between samples and the detection of important SNPs. Next, PathWeaver, a local haplotype assembler designed for complex infections and highly variable genomic regions with poor reference mapping. PathWeaver is able to create highly accurate haplotypes without generating chimeric assemblies. PathWeaver was used on the key protein in pregnancy-associated malaria Plasmodium falciparum VAR2CSA which revealed population sub-structuring within the key binding domain of the protein observed to be present globally along with confirming copy number variation. Finally, the program Carmen is able to utilize PathWeaver to augment the results from targeted amplicon approaches by reporting where and when local haplotypes have been found previously. These rigorously tested tools allow the analysis of local haplotype data from various technologies and approaches to provide accurate, precise and easily accessible results

Ectomycorrhiza Development : Investigation of Selected Ectomycorrhiza Induced Poplar Genes

Mutualistic interaction such as ectomycorrhiza (ECM) is important for forest ecosystem function. Here two kingdoms, plant and fungi, form a symbiosis and exchanges nutrients and carbohydrates. A bottle neck in ectomycorrhizal research is the time demand for transgenic plant generation. Formation of so-called composite plants, where transgenic roots are formed on non-transgenic shoots, is an alternative strategy. An Agrobacterium rhizogenes-mediated root transformation protocol was developed in this work using axenic Populus tremula A tremuloides and P. tremula A alba cuttings. Out of four different A. rhizogenes strains, K599 was found to be the most suitable one. Roots of composite poplars were able to form ectomycorrhiza when inoculated with Amanita muscaria. By using real time quantitative PCR a comparative analysis of transcript levels was done for selected genes in mycorrhized and non-mycorrhized poplar fine roots. A total of 50 ectomycorrhiza-induced genes were chosen based on a genome wide microarray analysis (Nehls, unpublished). As the array oligomers were designed based on Populus trichocarpa genome but the array hybridization was performed using P. tremula x tremuloides cDNA, cross hybridization leading to misinterpretation is feasible. Therefore, the first step was to screen P. tremula/P. tremuloides datasets for the best matching homologs. After primer design and first qRT-PCR tests 14 candidate genes remained. Finally, expression analysis with several independent batches of poplar fine roots and mycorrhizas were obtained for six genes. Two genes, a transcription factor (TF- Potri.008G071100) belonging to the AP2/ERF superfamily and a potential glycosyltransferase (GT- Potri.007G095000) revealing the highest gene expression difference, were selected for further analysis. Fusion with super yellow fluorescent protein revealed a probable subcellular localization of Potri.007G095000 in nucleus and cytoplasm and Potri.008G071100 in nucleus. Following their establishment, composite poplars were used for promoter analysis of the selected genes. For this purpose, around 3 kb promoter fragments were amplified from genomic DNA of P. tremula x tremuloides, successively shortened from the 5' end and the resulting fragments were cloned in front of the coding sequence of a peroxisomal located yellow fluorescent protein. The longest promoter-reporter constructs were used for generation of composite poplars. No reliable ECM induced expression was found for the TF and the GT indicating the need for longer promoter fragments. Furthermore, as the best Arabidopsis TF homolog is known to be auto-regulated, the identification of potential cis-elements in the promoter region was planned. Therefore, protein overexpression in E. coli was initiated. However, all attempts of receiving a natively folded protein in soluble form were unsuccessful