Trypanosoma cruzi genome plasticity and evolution

Trypanosoma cruzi, a protozoan from the Kinetoplastidae family, is the etiologic agent of Chagas disease, a major public health problem affecting mostly the poorest areas of Latin America. Due to the complex nature of the parasite’s genome it has been impossible to produce a complete reference genome sequence, thus hampering the implementation of post- genomic approaches to unveil the mechanisms of generation of antigenic variation and the identification of new drug targets. My doctoral studies have focused on the application of combined genome sequencing and computational methods to produce a complete reference T. cruzi genome sequence and perform comparative analyses to better understand the mechanisms that allow T. cruzi to evade the mammalian host immune system and to briskly adapt to novel environments. In paper I and II, different genome assembly strategies and second generation sequencing technologies were implemented to perform comparative analyses to identify elements of virulence between T. cruzi and two trypanosomatids that are non-pathogenic to humans: Trypanosoma cruzi marinkellei, a bat-restricted sub-species of the T. cruzi clade and the human avirulent species Trypanosoma rangeli. The studies reveal the expansion of T. cruzi- specific genomic traits specialised in the invasion of mammalian cells. In paper III, using third-generation, PacBio sequencing data it was possible to assemble the complete reference genome sequence of a Trypanosoma cruzi isolate from the DTU-I clade. This breakthrough allowed us - for the first time - to explore in detail the genome architecture of the subtelomeric areas where many parasite virulence factors are encoded. One of the most interesting discoveries was the overrepresentation of interspersed retrotransposons and microsatellites in tandem gene arrays coding for surface molecules, hinting at a retrotransposon-driven mechanism of recombination for generating new sequence variants. Whole genome sequencing of 35 T. cruzi DTU-I isolates, collected from different locations in the American continent, made possible to identify and characterise the mechanisms of adaptability employed by the parasite. Finally, paper IV analyses the mechanisms of genomic hybridisation in T. cruzi and the evolution over time of the hybrid offspring. The analysis revealed that during hybrid formation, the parasite integrates genetic material from each parental strains with the aid of retrotransposons and microsatellites, and the genome of these hybrid isolates moves quickly from a tetraploid to a diploid state. As a result, the hybrid strain has more genetic material, mostly in the subtelomeres, providing the parasite with a pool of new surface molecule genes with the potential to possibly increase its fitness in a new environment. In conclusion, the work presented here has advanced the understanding of parasite biology and provided a genomic resource to be exploited for the identification of drug targets and vaccine candidates

Complex polymorphisms in the plasmodium falciparum Multidrug Resistance Protein 2 Gene and Its Contribution to Antimalarial Response

Complex Polymorphisms in the Plasmodium falciparum Multidrug Resistance Protein 2 Gene and Its Contribution to Antimalarial ResponsePlasmodium falciparum has the capacity to escape the actions of essentially all antimalarial drugs. ATP-binding cassette (ABC) transporter proteins are known to cause multidrug resistance in a large range of organisms, including the Apicomplexa parasites. P. falciparum genome analysis has revealed two genes coding for the multidrug resistance protein (MRP) type of ABC transporters: Pfmrp1, previously associated with decreased parasite drug susceptibility, and the poorly studied Pfmrp2. The role of Pfmrp2 polymorphisms in modulating sensitivity to antimalarial drugs has not been established. We herein report a comprehensive account of the Pfmrp2 genetic variability in 46 isolates from Thailand. A notably high frequency of 2.8 single nucleotide polymorphisms (SNPs)/kb was identified for this gene, including some novel SNPs. Additionally, we found that Pfmrp2 harbors a significant number of microindels, some previously not reported. We also investigated the potential association of the identified Pfmrp2 polymorphisms with altered in vitro susceptibility to several antimalarials used in artemisinin-based combination therapy and with parasite clearance time. Association analysis suggested Pfmrp2 polymorphisms modulate the parasite's in vitro response to quinoline antimalarials, including chloroquine, piperaquine, and mefloquine, and association with in vivo parasite clearance. In conclusion, our study reveals that the Pfmrp2 gene is the most diverse ABC transporter known in P. falciparum with a potential role in antimalarial drug resistance.This work was supported by project grants from the Swedish Development Cooperation Agency, Department for Research Cooperation (SWE 2007-174 and SWE-2009-165). M.I.V. and N.S.O. are recipients of post-doctoral fellowship from Fundacao para a Ciencia e Tecnologia (FCT)/Ministerio da Ciencia e Ensino Superior, Portugal, MCES (SFRH/BPD/76614/2011 and UMINHO/BPD/15/2014, respectively). The Shoklo Malaria Research Unit is part of the Mahidol Oxford University Tropical Medicine Research Unit and is supported by the Wellcome Trust of Great Britain

Genome analysis and comparative genomics of a Giardia intestinalis assemblage E isolate

<p>Abstract</p> <p>Background</p> <p><it>Giardia intestinalis </it>is a protozoan parasite that causes diarrhea in a wide range of mammalian species. To further understand the genetic diversity between the <it>Giardia intestinalis </it>species, we have performed genome sequencing and analysis of a wild-type <it>Giardia intestinalis </it>sample from the assemblage E group, isolated from a pig.</p> <p>Results</p> <p>We identified 5012 protein coding genes, the majority of which are conserved compared to the previously sequenced genomes of the WB and GS strains in terms of microsynteny and sequence identity. Despite this, there is an unexpectedly large number of chromosomal rearrangements and several smaller structural changes that are present in all chromosomes. Novel members of the VSP, NEK Kinase and HCMP gene families were identified, which may reveal possible mechanisms for host specificity and new avenues for antigenic variation. We used comparative genomics of the three diverse <it>Giardia intestinalis </it>isolates P15, GS and WB to define a core proteome for this species complex and to identify lineage-specific genes. Extensive analyses of polymorphisms in the core proteome of <it>Giardia </it>revealed differential rates of divergence among cellular processes.</p> <p>Conclusions</p> <p>Our results indicate that despite a well conserved core of genes there is significant genome variation between <it>Giardia </it>isolates, both in terms of gene content, gene polymorphisms, structural chromosomal variations and surface molecule repertoires. This study improves the annotation of the <it>Giardia </it>genomes and enables the identification of functionally important variation.</p

The Short Non-Coding Transcriptome of the Protozoan Parasite Trypanosoma cruzi

The pathway for RNA interference is widespread in metazoans and participates in numerous cellular tasks, from gene silencing to chromatin remodeling and protection against retrotransposition. The unicellular eukaryote Trypanosoma cruzi is missing the canonical RNAi pathway and is unable to induce RNAi-related processes. To further understand alternative RNA pathways operating in this organism, we have performed deep sequencing and genome-wide analyses of a size-fractioned cDNA library (16–61 nt) from the epimastigote life stage. Deep sequencing generated 582,243 short sequences of which 91% could be aligned with the genome sequence. About 95–98% of the aligned data (depending on the haplotype) corresponded to small RNAs derived from tRNAs, rRNAs, snRNAs and snoRNAs. The largest class consisted of tRNA-derived small RNAs which primarily originated from the 3′ end of tRNAs, followed by small RNAs derived from rRNA. The remaining sequences revealed the presence of 92 novel transcribed loci, of which 79 did not show homology to known RNA classes

Transcription Factor MAFF (MAF Basic Leucine Zipper Transcription Factor F) Regulates an Atherosclerosis Relevant Network Connecting Inflammation and Cholesterol Metabolism

BACKGROUND: Coronary artery disease (CAD) is a multifactorial condition with both genetic and exogenous causes. The contribution of tissue-specific functional networks to the development of atherosclerosis remains largely unclear. The aim of this study was to identify and characterize central regulators and networks leading to atherosclerosis. METHODS: Based on several hundred genes known to affect atherosclerosis risk in mouse (as demonstrated in knockout models) and human (as shown by genome-wide association studies), liver gene regulatory networks were modeled. The hierarchical order and regulatory directions of genes within the network were based on Bayesian prediction models, as well as experimental studies including chromatin immunoprecipitation DNA-sequencing, chromatin immunoprecipitation mass spectrometry, overexpression, small interfering RNA knockdown in mouse and human liver cells, and knockout mouse experiments. Bioinformatics and correlation analyses were used to clarify associations between central genes and CAD phenotypes in both human and mouse. RESULTS: The transcription factor MAFF (MAF basic leucine zipper transcription factor F) interacted as a key driver of a liver network with 3 human genes at CAD genome-wide association studies loci and 11 atherosclerotic murine genes. Most importantly, expression levels of the low-density lipoprotein receptor (LDLR) gene correlated with MAFF in 600 CAD patients undergoing bypass surgery (STARNET [Stockholm-Tartu Atherosclerosis Reverse Network Engineering Task]) and a hybrid mouse diversity panel involving 105 different inbred mouse strains. Molecular mechanisms of MAFF were tested in noninflammatory conditions and showed positive correlation between MAFF and LDLR in vitro and in vivo. Interestingly, after lipopolysaccharide stimulation (inflammatory conditions), an inverse correlation between MAFF and LDLR in vitro and in vivo was observed. Chromatin immunoprecipitation mass spectrometry revealed that the human CAD genome-wide association studies candidate BACH1 (BTB domain and CNC homolog 1) assists MAFF in the presence of lipopolysaccharide stimulation with respective heterodimers binding at the MAF recognition element of the LDLR promoter to transcriptionally downregulate LDLR expression. CONCLUSIONS: The transcription factor MAFF was identified as a novel central regulator of an atherosclerosis/CAD-relevant liver network. MAFF triggered context-specific expression of LDLR and other genes known to affect CAD risk. Our results suggest that MAFF is a missing link between inflammation, lipid and lipoprotein metabolism, and a possible treatment target

Shotgun Sequencing Analysis of Trypanosoma cruzi I Sylvio X10/1 and Comparison with T. cruzi VI CL Brener

Trypanosoma cruzi is the causative agent of Chagas disease, which affects more than 9 million people in Latin America. We have generated a draft genome sequence of the TcI strain Sylvio X10/1 and compared it to the TcVI reference strain CL Brener to identify lineage-specific features. We found virtually no differences in the core gene content of CL Brener and Sylvio X10/1 by presence/absence analysis, but 6 open reading frames from CL Brener were missing in Sylvio X10/1. Several multicopy gene families, including DGF, mucin, MASP and GP63 were found to contain substantially fewer genes in Sylvio X10/1, based on sequence read estimations. 1,861 small insertion-deletion events and 77,349 nucleotide differences, 23% of which were non-synonymous and associated with radical amino acid changes, further distinguish these two genomes. There were 336 genes indicated as under positive selection, 145 unique to T. cruzi in comparison to T. brucei and Leishmania. This study provides a framework for further comparative analyses of two major T. cruzi lineages and also highlights the need for sequencing more strains to understand fully the genomic composition of this parasite

Draft Genome Sequencing of Giardia intestinalis Assemblage B Isolate GS: Is Human Giardiasis Caused by Two Different Species?

Giardia intestinalis is a major cause of diarrheal disease worldwide and two major Giardia genotypes, assemblages A and B, infect humans. The genome of assemblage A parasite WB was recently sequenced, and the structurally compact 11.7 Mbp genome contains simplified basic cellular machineries and metabolism. We here performed 454 sequencing to 16× coverage of the assemblage B isolate GS, the only Giardia isolate successfully used to experimentally infect animals and humans. The two genomes show 77% nucleotide and 78% amino-acid identity in protein coding regions. Comparative analysis identified 28 unique GS and 3 unique WB protein coding genes, and the variable surface protein (VSP) repertoires of the two isolates are completely different. The promoters of several enzymes involved in the synthesis of the cyst-wall lack binding sites for encystation-specific transcription factors in GS. Several synteny-breaks were detected and verified. The tetraploid GS genome shows higher levels of overall allelic sequence polymorphism (0.5 versus <0.01% in WB). The genomic differences between WB and GS may explain some of the observed biological and clinical differences between the two isolates, and it suggests that assemblage A and B Giardia can be two different species

How a specific game affects pupil's reading ability. : An intervention study.

Målet med denna studie var att undersöka och utvärdera effekten av systematiserad träning av avkodnings- och ordläsningsförmåga med ett lässpel hos elever i årskurs två med låg läsförmåga. Sex elever som presterade inom stanine 1-3 på ordavkodning i mitten av årskurs två deltog i studien. Studien hade en single subject design med två beselinegrupper med tre elever i varje grupp. På testet före interventionen presterade ingen av eleverna över stanine 3 på ordläsning. Testen efter interventionen visade att alla eleverna hade förbättrat sin avkodning och läsförmåga. Detta indikerar att en intervention med systematisk träning av avkodning och ordläsningsförmåga i den här åldern har god effekt

Spelet bakom sportsidorna - En studie om sportjournalisters påverkan på nyhetsurvalet

The purpose of this study was to investigate why Swedish sports journalists choose to report more about male sport than female sport. Studies have shown that the news selection in sports journalism is dominated by male sport and we found that this subject hadn’t been covered from a Swedish perspective. The aim of this study was therefore to map and specify the reasons behind the male dominated news selection in Swedish sports journalism. To achieve this, we created a survey that was sent out to eight Swedish sport publications. After receiving a total of 56 responses we started analyzing them through four determinants: the control fields, the environment at the newsdesk, gender and gatekeeping. The theories we used to analyze were: the theory about the control fields, Melin-Higgins theories about survival at a newsdesk and the gatekeeping theory. The results showed that the control field of interest are a possible determinant in Swedish sports journalists news selection. A majority of the respondents answered that they weigh in their public’s interest in the news selection and that they find their public’s interest as male dominated. Here we also found that journalist at a commercial newsdesk rates the control field of interest as more important compared to the journalists at a non-commercial newsdesk. Regarding gender as a determinant we came to the conclusion that the male sports journalist’s covers male sport in a larger scale than female sports journalist. The female respondents also answered that they find it more prestigious to cover male sport. These were the only determinants we found relevant based on the results. Both the environment at the newsdesk and gatekeeping appeared to be irrelevant as determinants in our study. Considering the results, we found that the control field of interest and gender appears to be two reasons behind the male dominated news selection in Swedish sports journalism

Jag fick lära mig hur man inte ska göra : Sjökaptensstudenters upplevelser av handledning under fartygsförlagd utbildning

Syftet med denna undersökning var att förstå och beskriva sjökaptensstudenters upplevelser av handledning vid utförandet av uppgifter som krävs för att uppnå lärandemålen under delkursen Fartygsförlagd utbildning 1 (1FF01K). Halvstrukturerade personliga intervjuer genomfördes med åtta förstaårsstudenter mellan den 25:e mars och den 5:e april 2013. Dessa spelades in och transkriberades för att sedan tolkas och struktureras i olika kategorier beroende av upplevelser. Strategiskt urval användes för att välja ut informanterna vilka intervjuades med hjälp av ”critical incident technique”. Resultatet av undersökningen visade att en handledningsmetod som innebär tydliga instruktioner eller demonstrationer följt av korrigering och bedömning upplevdes positivt vid utförandet av vissa uppgifter. I andra fall har ökat eget inflytande upplevts positivt. Uppgiftens karaktär och krav påverkar i hög grad upplevelsen av handledningsmetoden. I studien har också framkommit att avsaknad av tillräcklig handledning riskerar att göra det svårare för studenten att utföra sina uppgifter i arbetsjournalen och därmed uppnå lärandemålen för fartygsförlagd utbildning.The purpose of this study was to understand and describe master mariner students’ experiences of supervision during the performance of tasks necessary to achieve the intended learning outcomes for the first module of on board practical training. Semi structured personal interviews were made with eight freshman students between 25th of March and 5th of April 2013. These interviews were recorded and transcribed before being interpreted and structured into different categories. Strategic selection was used to select interviewees and these were interviewed using ”critical incident technique”. The result of this study shows that a supervision method that includes distinct instructions or demonstrations followed by correction and assessment is experienced in a positive way during the performance of certain tasks. In other cases, increased own influence has been experienced positively. The characteristics and requirements of a task affect the experience of the supervision method to a high degree. The study also shows that a lack of sufficient supervision can make it more difficult for the student to perform the tasks in the on board training manual and achieve the intended learning outcomes for on board practical training