1,721,289 research outputs found
Genomic epidemiology of malaria vectors in the Anopheles gambiae species complex
Full text linkIn this thesis I ask, how can the study of genome variation within malaria vector populations
contribute to the control of malaria in sub-Saharan Africa.
In the first chapter I provide an introduction to the current situation in malaria control
in sub-Saharan Africa, and the role played by large-scale mosquito control programmes
using insecticide-based interventions. I also introduce high-throughput whole-genome
sequencing and its potential applications to the study and surveillance of malaria vectors.
In the second chapter I introduce the Anopheles gambiae species complex, and provide
historical context by describing how the species complex was discovered, which marked the
introduction of genetic methods into the study and surveillance of African malaria vectors.
I conclude that there are important parallels between past and present efforts towards
malaria elimination, but also new opportunities afforded by genomic epidemiology.
In the third chapter I describe the production of a genome variation data resource
derived from whole-genome sequencing of Anopheles gambiae and Anopheles coluzzii
mosquitoes from 8 African countries, carried out as part of the first phase of the Anopheles
gambiae 1000 Genomes (Ag1000G) Project. This chapter establishes and validates methods
for robust discovery of nucleotide variation from Illumina deep whole-genome sequencing
of individual mosquitoes, and confirms that Anopheles mosquitoes are among the most
genetically diverse organisms in the natural world. Subsequent chapters all perform analyses
using this data resource.
In the fourth chapter I identify genetically distinct populations among the mosquitoes
sampled in Ag1000G phase 1, and quantify genetic diversity within and differentiation
between these populations. I show that there is strong population structure and marked
differences in diversity between populations, suggesting important heterogeneities in
population size and rates of gene flow. These results are an essential foundation on which to
build analyses of recent evolution in subsequent chapters.
In the fifth chapter I search for signals of recent positive selection among the populations
sampled in Ag1000G phase 1, to identify which genes are most important in generating an
adaptive response to the use of insecticides in malaria vector control. I show that there
are strong signals of recent selection both at known insecticide resistance genes and at
previously unknown genes with a plausible link to insecticide resistance.
In the sixth chapter I perform a detailed analysis of the voltage-gated sodium channel
gene, where genetic changes cause target-site resistance to pyrethroid insecticides, the main
ingredient in insecticide-treated bednets. I identify previously unknown mutations within
this gene, and use haplotype data to show that resistance mutations have spread over large
geographical distances and between mosquito species.
In the final chapter, I discuss the potential translation of genome sequencing into
operational malaria vector surveillance and insecticide resistance management systems
Manipulation of the immune response to malaria antigens using bacterial-derived lipoproteins
No full textMalaria is a major cause of illness and death in the world. Much progress has been made in the development of a vaccine against malaria, but there is still no formulation that can induce effective and long-lasting immunity in diverse populations at an affordable cost. Bacterial lipoproteins are potent stimulators of the innate and acquired arms of the immune system. They have been used as vaccines against bacterial diseases, and short lipopeptides have been shown to be potent adjuvants to a diverse range of vaccines against bacterial and viral diseases, and may also be useful in the development of anti-tumour vaccines. Here, the artificial lipidation of selected malaria antigens, MSP119 of Plasmodium chabaudi chabaudi AS and a multi-epitope string comprising mainly P.falciparum epitopes (SGC), is investigated. Direct lipid modification of the antigens was achieved by fusion of the protein coding sequence to a bacterial lipidation signal sequence that directs the post-translational addition of fatty acids to the protein. Antigens were also expressed as fusion proteins to a naturally occurring lipoprotein of Pseudomonas aeruginosa, L-OprI. The inclusion of L-OprI as an admixed adjuvant boosted the antibody response to MSP119, and although this response was not protective against a live blood-stage challenge, neither did it lead to increased parasitaemia and mortality. Immunisation of mice with the multi-epitope string SGC as a fusion to the L-OprI lipoprotein induced an antibody response against the SGC component of the molecule while immunisation with protein alone did not. This antibody response was absolutely dependent on covalent linkage of the L-OprI lipoprotein to SGC; the addition of LOprI as an admixed adjuvant did not induce an antibody response to SGC. SGC-specific IFNγ-secretion by whole spleen cells was induced by immunisation with protein, fusion protein or fusion lipoprotein forms of SGC, but there were no significant differences in the numbers of IFNγ-secreting cells between preparations. There are several technical obstacles that must be overcome before artificial lipoproteins can be produced on a large-scale, but efforts to overcome these obstacles would be worthwhile since lipoproteins appear to represent suitable candidates for further investigation as adjuvants to malaria vaccines
Common genetic variants of the IFN-γ and IFNGR1 regions - disease associations and functional properties
Full text linkThere is growing evidence that susceptibility to many inflammatory and infectious diseases may be influenced by our genetic make up. Genetic variants in important immune genes may partially explain variation in susceptibility to common diseases. Interferon-γ (IFNγ) is one of the central mediators of the innate and adaptive immunity and has been implicated in a wide range of infectious and inflammatory disease processes. Severe disruptive mutations in coding regions of the IFN-γ receptor 1 gene (IFNGR1] have been found to be associated with fatal but very rare mycobacterial infections. This study looked at common polymorphisms in potentially regulatory non-coding regions of the IFNγ gene and the IFNGR1 gene and investigated their association with susceptibility to severe malaria, a disease for which there have been indications of a genetic component to susceptibility. Malaria is one of the major causes of childhood deaths in Africa. IFNγ and its receptor have been shown to be critically involved in the host response to the malaria parasites. The promoter regions of IFNGR1 and its neighbouring genes, located on chromosome 6q23, and IFNγ and its neighbours, on chromosome 12ql4, were screened for polymorphisms. Haplotypes and linkage disequilibrium maps were constructed, signatures of natural selection were investigated, haplotype tagging SNPs were dentified, and association with disease was analysed. One of these preliminary results was a putative association between the IFNGRl-470ddel allele and susceptibility to severe malaria in the Mandinka ethnic group. This allele was in strong linkage disequilibrium (LD) with markers which are a considerable distance away which might represent a signature of natural selection. To assess the potential functional significance of the IFNGR1-47Q polymorphism, its effects on DNA-protein interactions and gene expression was investigated further in various cell lines. Evidence of tissue-specific nuclear protein binding to this site which seems to be involved in transcriptional regulation was observed
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Accessing complex genomic variation in Plasmodium falciparum natural infections
Full text linkGenetic polymorphism in Plasmodium falciparum is a considerable obstacle to malaria intervention. Parasites have repeatedly evolved to overcome every front-line antimalarial deployed throughout history, and artemisinin resistant populations are expanding in Southeast Asia. Promising vaccine candidates routinely fail when challenged by the genetic diversity of natural parasite populations, and a recent trial using a blood-stage antigen showed immunity was allele specific. Modern sequencing technologies have revolutionized our understanding of parasite genomics and population genetics by providing access to single nucleotide variation, but characterizing more complex polymorphism remains a key challenge. Solving this problem is important because the selective pressures from drugs and host immunity often create complex polymorphism in the most clinically relevant genes that is missed using standard genotyping methods. In three sections, this thesis is a narrative about 1) encountering complex variation, 2) overcoming it with novel tools, and then 3) innovatively applying those tools to old and new questions. I first show examples of complex variation in a vaccine candidate (EBA-175) and a drug resistance gene (pfcrt) while reporting SNP based analyses of Kenyan and Tanzanian field isolates. While introducing this complex variation I also describe biological insights discovered in these populations. In Kenya I show evidence that chloroquine resistance selects for parasites that are primaquine sensitive, use a GWAS approach to discover new drug resistance loci, and catalogue variation in known resistance genes. In Tanzania I describe the population structure and allele frequencies of parasites from two geographic regions. In the second section of the thesis I develop methods for accessing complex variation and demonstrate their utility by producing de novo assemblies of eba-175, pfcrt, ama1, and msp3.4 from thousands of sequenced samples. Finally, in the third section I apply these tools in depth to eba-175. I comprehensively characterize the SNP and structural variation in eba-175 using an alignment of 1419 de novo assemblies. I use this resource to illustrate the profiles of positive selection across the gene, and corroborate these signals of balancing selection by showing the geographic distribution of the F/C indels and a lesser known 6bp indel positioned between the DBL domains. I then use the alignments to design Sequenom genotyping assays that facilitate a genome wide association study, testing for human associations with the eba-175 indels in the infecting parasite. I close by reporting a potential association on human chromosome 14 with the 6bp indel in eba-175
Genome mapping of malaria resistance genes: the host ligands of PfEMP1
Full text linkErythrocytes infected by mature forms of the Plasmodium falciparum parasite adhere to other components of the vascular space, a behavior considered critical to the pathogenesis of severe malaria. Adhesion is mediated by the P. falciparum erythrocyte membrane protein 1 (PfEMP1), a highly variant antigen expressed by the parasite and subject to switching during the course of an infection. The host ligands of PfEMP1 include CD36, ICAM-1 and the ABO antigens. By employing a series of population- and family-based association studies from multiple African populations, we examined whether variation in the genes underlying these molecules affects susceptibility to severe malaria. Our results suggest that a common frameshift mutation in the ABO glycosyltransferase, responsible for blood group O, is associated with protection from severe malarial phenotypes (P=2x10⁻⁷), particularly severe malarial anaemia. However, we found no significant disease associations with variation in either the ICAM1 or CD36 genes. We focused on two particular functional polymorphisms, the missense ICAM-1Kilifi and the CD36 nonsense mutation T1264G. We genotyped both markers in around 10,000 individuals, but neither demonstrated an association with severe malarial phenotypes. Malaria has been a profound selection pressure shaping human genetic diversity. The last decade has seen the development of several haplotype-based methods to detect signatures of recent positive evolutionary selection. These techniques are potentially invaluable tools in our hunt for genetic variants that protect from life threatening malaria. We used simulations and empirical data from the International HapMap Project to demonstrate the validity of searching for long regions of haplotype homozygosity, as an approach to finding alleles undergoing selective sweeps. We analysed genetic data from a range of populations, particularly those utilized by HapMap, to investigate whether our candidate genes were associated with signals of recent positive selection. We characterized the distribution of a selection event associated with the CD36 1264G allele, focused in Central-West Africa, and demonstrated a novel signal of low population differentiation at the ABO gene, suggestive of longstanding balancing selection. Our work confirms that variation in the host ligands of PfEMP1 modulates severe malaria susceptibility, and highlights the value of using signals of selection, along with functional experiments and genetic association studies, to dissect the biology of severe malaria
Plasmodium falciparum population genetics in northern Ghana
Full text linkThe main thrust of this thesis was to characterize P.falciparum genetic diversity in northern Ghana. To do this, I used simple techniques to purify P. falciparum DNA from clinical samples across a rural setting for whole-genome sequencing. The goal was to provide a framework for exploring host-parasite genetic interactions. Utilizing Illumina deep sequencing data for 277 isolates I analyzed P. falciparum genetic diversity and described within-host diversity across this area. I observed random mating (ie no population structure) in the local parasite population, and a high genetic diversity indicative of high out-crossing. Moreover, when I aggregated my data with similar published data from Burkina Faso and Mali (sites ≈500km apart), no population structure was evident. In contrast, sites sampled in Cambodia and Thailand (≈ 800km apart) were found to have greater population structure and high potential for inbreeding. This may be driven by differences in transmission intensity between the sites sampled in West Africa and southeast Asia. To demonstrate the utility of deep sequencing data, I focused on the genomic regions of pfdhfr, pfdhps and pfcrt, known to be under antimalarial drug selection. I surveyed the full diversity of point mutations already characterized in these genes and discovered previously unknown variants. However, in order to provide a means to follow up on new variants or interesting candidate regions in large clinical samples with limited parasite DNA, I assessed the Sequenom iPLEX platform for high-throughput genotyping of P. falciparum polymorphisms. This necessitated developing a method appropriate for assigning genotypes in haploid genome mixtures common in natural infections. Finally, I used this method to type host and parasite markers in a case-control sample set from this region for exploring host-parasite genetic interactions. I found that children who have the sickle-cell trait and carry parasites that have pfdhfr resistant alleles lose their protection against severe malaria as compared to children who have normal haemoglobin and are infected with parasites with these resistant alleles
Global and local patterns of population structure and their role in the evolution and demography of Plasmodium falciparum
Full text linkIn this thesis, I study the role of genetic population structure in the evolution and demography of Plasmodium falciparum by focusing on the recent onset of artemisinin resistance in Southeast Asia, an alarming event for global public health. I describe the population structure of Plasmodium falciparum in the Thai-Cambodian border region, characterizing sympatric but differentiated subpopulations associated with artemisinin resistance. I show evidence that they are the product of recent founder events and seem the primary force spreading resistance. Next, I study a superset of the kelch13 mutations associated with artemisinin resistance, assessing their relationship with population structure and recent founder effects. Each resistant subpopulation possesses a distinct kelch13 allele that, in conjunction with a particular genetic background, seem to have driven recent founder effects. I examine the demography of these resistance alleles using patterns of haplotype sharing and show that the primary mode of spread consists of independent mutational events, with limited gene flow within countries in East Southeast Asia. Subsequently, I assess the origin of kelch13 mutations observed in African isolates, concluding that they are indigenous and have originated independently. These observations undermine localized resistance containment as a strategy for malaria control and suggest that population structure and founder effects may predate and facilitate the emergence of resistance. Therefore, monitoring these phenomena could warn about the development of resistance before phenotypic evidence materializes. Next, given the importance of demographic inference to inform malaria control programs and the advent of large genomic datasets, I develop a fast and scalable method to build the ancestral haplotype graph. I show that this data structure, composed of a collection of local haplotype trees, is informative about the recent genealogical history of the sequences and can be used to summarize and study shared haplotype patterns along the genome. I describe a set of algorithms with quasilinear time complexity as a first step in the development of scalable demographic inferential methods that can be applied to several thousands of sequences. I also evaluate how mixed infections affect the analysis of deep sequencing data and review the FWS statistic, a relative measure of inbreeding and complexity of infection. In doing so, I show that the original FWS estimator discards the diversity encoded by rare variants and provide an alternative estimator without such bias that is simpler, more intuitive and has a better resolution.</p
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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