Antigenic variation in the African trypanosome: molecular mechanisms and phenotypic complexity

Antigenic variation is an immune evasion strategy that has evolved in viral, bacterial and protistan pathogens. In the African trypanosome this involves stochastic switches in the composition of a variant surface glycoprotein (VSG) coat, using a massive archive of silent VSG genes to change the identity of the single VSG expressed at a time. VSG switching is driven primarily by recombination reactions that move silent VSGs into specialized expression sites, though transcription-based switching can also occur. Here we discuss what is being revealed about the machinery that underlies these switching mechanisms, including what parallels can be drawn with other pathogens. In addition, we discuss how such switching reactions act in a hierarchy and contribute to pathogen survival in the face of immune attack, including the establishment and maintenance of chronic infections, leading to host-host transmission

The dynamics and nature of the hierarchy of VSG expression during Trypanosoma brucei infection

Trypanosoma brucei is a tsetse fly-transmitted kinetoplastid protozoan that parasitises a wide range of mammals in sub-Saharan Africa. The chronic infections typical of trypanosomes have a profile characterised by intermittent parasitaemic peaks, that become smaller and further apart. A key component that facilitates the survival of the parasite within the bloodstream of the host, and the generation of chronic infections, is antigenic variation. This process involves the inherent switching of the surface-expressed variant surface glycoprotein (VSG), and the new variable antigen types (VATs) in order to preempt the host immune response. There are approximately 1,000 genes in the VSG repertoire, and the observation that certain VATs appear at similar times within infections, has led to the conclusion that the expression of VSGs is semi-predictable and semiordered. This hierarchical system allows optimal use of the VSG repertoire, and is highly likely to be a significant factor in generating chronicity. It has been established that VSGs encoded by subtelomeric genes tend to appear early in infection, whilst those encoded by chromosomal internal genes tend to appear later. The first aim of this thesis was to examine the timing of expression of a subset of VSGs within replicate infections in mice and cattle, and to link the timing of expression to the genetic locus of the silent, donor, VSG gene. The VSGs examined represented all VSG locus types, incorporating 1 bloodstream expression site (BBS) gene (the transcription site for VSG genes in the bloodstream stage), 2 metacyclic expression site (MBS) genes, 2 minichromosomal genes, and 2 chromosomal internal genes. The infections were initiated with pleomorphic trypanosomes that switch at a high rate, and are a close-to-field, 'wild type' strain. The infections in mice confirmed that there was a statistically significant difference in the timing of onset of VAT-specific immune responses across replicate batches of infections with two separate analyses; one by comparing the average time of onset of the immune response (General Linear Model [GLM]; F6,106=7.49, p < 0.0001), and the second by ranking the onset of the immune response by sequence of appearance (GLM; Fe,112=8.03, p<0.0001). The appearance of VSGs within a restricted period of time was confirmed by directly analysing the parasite population in cattle, using FSG-specific reverse transcriptase polymerase chain reaction (RT-PCR). These findings allow further dissection of the hierarchical expression of VSGs, and provide statistically significant confirmation of the existence of semi-ordered expression in high-switching pleomorphic trypanosomes for the first time. A mathematical model was formulated (with the assistance of Dr. K. Lythgoe, University of Edinburgh) in order to simulate the dynamics of trypanosome infections. The model incorporated and built upon successful aspects of previous studies, and included measured biological parameters that are known to affect in vivo parasite kinetics. Manipulations were undertaken to investigate the effect, in silico, of differential VSG switching rates. The effect of varying the intrinsic rate of growth of the immune response was also analysed. The simulations indicated areas for potential further experimental studies. In particular, results suggested that the rate of growth of the immune response may be extremely important in shaping the profile, and duration, of an infection. The outcome of modelling whereby VSG switching was manipulated, suggested that there are distinct subsets of VSGs, which have differing probabilities in switching to each other. The significance of genomic position of the silent, donor, VSG, and also sequence homology between the donor VSG and the expressed VSG were investigated. The proportional influence of genomic locus and homology-driven switching was suggested to be the most important aspect to be elucidated, with respect to further resolving the hierarchical switching of VSGs

Product quality improvement internship with Auckland Biosciences

Auckland BioSciences (ABS) produces adult bovine serum for use in cell culture. Adult bovine serum is the liquid fraction of clotted blood collected from adult cattle. ABS produce this by collecting whole blood directly from the animals, allowing it to clot naturally, and extracting the serum via a centrifugation process. One of the quality parameters for serum is haemoglobin. Haemoglobin is a protein that exists inside red blood cells, and is released into the surrounding blood fluid when blood cells are ruptured, a phenomenon known as haemolysis. Haemolysis increases the amount of haemoglobin measured in the end product. High levels of haemoglobin are considered to be undesirable in serum. Current Situation ABS currently produces serum with a haemoglobin specification of< 45 mg/dL. This is relatively high compared to the standard industry expectations for adult serum, which range from 25-45 mg/ dl . ABS occasionally have issues with producing serum batches that exceed the specification of 45 mg/dl, which has occurred in 10 batches over the past 12 months, resulting in product that must be discarded or sold at less than cost price. ABS is seeking to lower the haemoglobin levels measured in the end product, which will prevent production batches from exceeding 45 mg/ml, and also potentially meet the higher market haemoglobin specification of 30 mg/dl. This would enable ABS to sell serum at a higher average price. This project was carried out using a DMAIC quality improvement framework. A literature review was carried out on this framework, which confirmed its suitability, due to its data driven, iterative approach that enables a person with limited subject knowledge to effectively carry out a quality improvement project . Scope of Project The scope of this project includes : Identifying significant causes of haemolysis in ABS' production process. Identifying ways to eliminate or reduce haemolysis in a commercially feasible manner

Biokinetics Of microbial consortia using biogenic sulfur as a novel electron donor for sustainable denitrification

In this study, the biokinetics of autotrophic denitrification with biogenic S0 (ADBIOS) for the treatment of nitrogen pollution in wastewaters were investigated. The used biogenic S0, a by-product of gas desulfurization, was an elemental microcrystalline orthorhombic sulfur with a median size of 4.69 µm and a specific surface area of 3.38 m2/g, which made S0 particularly reactive and bioavailable. During denitritation, the biomass enriched on nitrite (NO2–) was capable of degrading up to 240 mg/l NO2–-N with a denitritation activity of 339.5 mg NO2–-N/g VSS·d. The use of biogenic S0 induced a low NO2–-N accumulation, hindering the NO2–-N negative impact on the denitrifying consortia and resulting in a specific denitrification activity of 223.0 mg NO3–-N/g VSS·d. Besides Thiobacillus being the most abundant genus, Moheibacter and Thermomonas were predominantly selected for denitrification and denitritation, respectively

A regulated high negative voltage generator for single-photon avalanche photodiodes

In this work, a regulated high negative voltage generator for biasing single-photon avalanche photodiodes (SPAD) was developed. The circuit provides up to -70 V from a positive voltage source. This circuit allows users to control the negative output voltage using a positive voltage rail, thus eliminating the requirement of a negative voltage for the negative voltage control. This approach simplifies the setting of the output and facilitates integration in miniaturized photon counting systems. The testing on a fabricated PCB of this circuit show that the output voltage can be accurately controlled up to -70 V with ripples of less than 80 mV. A SPAD based experimental setup was also built and the experimental results show that the circuit is able to maintain a stable bias voltage for a planar SPAD at both low and high counting rates