As sequencing technologies advance genome studies are becoming a basic tool for studying an organism, and with more genomes available comparative genomics is maturing into a powerful tool for biological research. This thesis demonstrates the strength of a comparative genomics approach on a group of understudied eukaryotes, the diplomonads. Diplomonads are a group of single cell eukaryotic flagellates living in oxygen-poor environments. Most diplomonads are intestinal parasites, like the well-studied human parasite Giardia intestinalis. There are seven different G. intestinalis assemblages (genotypes) affecting different hosts, and it’s under debate whether these are one species. A genome-wide study of three G. intestinalis genomes from different assemblages reveals little inter-assemblage sexual recombination, supporting that the different G. intestinalis assemblages are genetically isolated and thus different species. A genomic comparison between the fish parasite S. salmonicida and G. intestinalis reveals genetic differences reflecting differences in their parasitic lifestyles. There is a tighter transcriptional regulation and a larger metabolic reservoir in S. salmonicida, likely adaptations to the fluctuating environments it encounters during its systemic infection compared to G. intestinalis which is a strict intestinal parasite. The S. salmonicida genome analysis also discovers genes involved in energy metabolism. Some of these are experimentally shown to localize to mitochondrion-related organelles in S. salmonicida, indicating that they possess energy-producing organelles that should be classified as hydrogenosomes, as opposed to the mitosomes in G. intestinalis. A transcriptome analysis of the free-living Trepomonas is compared with genomic data from the two parasitic diplomonads. The majority of the genes associated with a free-living lifestyle, like phagocytosis and a larger metabolic capacity, are of prokaryotic origin. This suggests that the ancestor of the free-living diplomonad was likely host-associated and that the free-living lifestyle is a secondary adaptation acquired through horizontal gene transfers.  In conclusion, this thesis uses different comparative genomics approaches to broaden the knowledge on diplomonad diversity and to provide more insight into how the lifestyle differences are reflected on the genetic level. The bioinformatics pipelines and expertise gained in these studies will be useful in other projects in diplomonads and other organismal groups.

Introducción. El estado nutricional de los individuos es considerado un valioso indicador del estado de salud de la población, así como también del accionar de factores socio-económicos y ambientales. Objetivos: Analizar la relación entre el estado nutricional, las enteroparasitosis y las condiciones socioambientales en niños de Aristóbulo del Valle, Misiones. Métodos. Estudio antropométrico transversal y parasitológico en niños de ambos sexos y entre 6 a 11 años de escuelas públicas del Municipio de Aristóbulo del Valle. Para el análisis de desnutrición se utilizó como referencia CDC/NCHS y para exceso de peso IOTF. El análisis parasitológico se realizó mediante la toma de muestras seriadas de materia fecal y escobillado anal. El relevamiento de las condiciones socio-ambientales se realizó mediante encuestas estructuradas. Resultados. El estado nutricional indicó mayor desnutrición crónica (7,5%) que global y aguda y mayor sobrepeso (9,8%) respecto a obesidad. Los niños presentaron alto porcentaje de parasitismo (86%) y las especies más prevalentes fueron Blastocystis hominis, Enterobius vermicularis y Giardia lamblia. La mayoría de las familias de estos niños habitaban viviendas de chapa y madera, con limitado acceso a servicios públicos, altos niveles de desempleo y padres con un nivel educativo básico. / Introduction: The study of the growth and nutritional status of the individuals is considered an important sign of the population health, as well as the action of socio-economic and environmental factors. Objectives: To analyze the relationship among the nutritional status, enteroparasitoses and socio-environmental conditions in children from Aristóbulo del Valle, Misiones. Methods: A cross-sectional anthropometrical and parasitological study was made in children from both sexes between 6 to 11 years old of public school from Municipality of Aristóbulo del Valle. NCHS was employed as reference in the analysis of undernutrition, and IOTF for the excess of weight. The parasitological analysis was realized by faecal samples and anal brushes. Socio-environmental conditions were evaluated through structured interviews. Results: The nutritional status indicated higher chronic undernutrition (7.5%) than underweight and wasting, and higher overweight than obesity (9.8%). Children showed high percentage of parasitism (86%) and Blastocystis hominis, Enterobius vermicularis and Giardia lamblia were the more prevalent species. Most families of these children live in precarious constructions made out of wood and/or masonry, with limited access to public services, high unemployment levels and a basic educational level of parents. Conclusions: The results obtained indicate the impact of the socio-economic, educational and sanitary impairment, in the children health from a sector Aristóbulo del Valle population, that entails to the coexistence of undernutrition, overweight and parasitic infections.

Giardia intestinalis is an anaerobic parasite, that colonizes the small intestine of humans and other vertebrate hosts. This cosmopolitan parasite, which causes diarrhoea, is transmitted by contaminated water or food via a resistant stage, the cyst. The encystation process involves a number of events that lead to a complete reconstruction of the cell into the form of infectious cyst. The aim of this work was to visualize these modifications in vivo by means of enzymatic labelling of proteins. For the purposes of this work, enzymatic tags Y-FAST and HaloTag were chosen, as they enable visualizing live cells under anaerobic conditions. Chimeric protein constructs were created to visualize the dynamics of the encystation vesicles, the structures of endoplasmic reticulum, the adhesive disc and mitosis. Using the developed constructs, we successfully followed the dynamics of the encystation vesicles and the adhesive disc in vivo. Finally, this work has provided novel molecular tools, which will be used to follow the overall redesign of the parasite cell during encystation.

SNARE proteins play essential role in most membrane fusions taking place in eukaryotic cell. They are responsible for all fusions that occur across endocytic and secretory pathways. Apart from these processes stand mitochondria and plastids. Fusion of these organelles is directed by specific protein machineries. In this work we review up-to-date information on SNARE mediated membrane fusion and fusion of outer and inner mitochondrial membranes with an emphasis on situation in flagellated protozoan parasite Giradia intestinalis. It was suggested that one of typical SNARE protein in Giardia (GiSec20) is localised to its highly reduced mitochondria called mitosomes. This protein is also essential for surviving of Giardia trophozoites. In this work we show that mitosomal localization of Gisec20 is caused by episomal expression however the protein is localised to endoplasmic reticulum under physiological conditions. Using GFP tag we were able to characterize its targeting signal which showed to be localised in transmembrane domain of GiSec20. This signal targets the protein to mitosomes of G. intestinalis and S. cerevisiae, respectively. Mitosomal localization was prevented by adding 3'UTR to gene sequence and its episomal expression. This suggests existence of targeting mechanism based on information...

Giardia intestinalis is a protozoan causing diarrhea worldwide. Beside its medical importance, it is evolutionary distant protist with two nuclei within a cell adapted for parasitic life in the environment poor of oxygen. Its genome is small and compact in term of gene content and size. It is therefore an attractive model organism for studies of minimal requirements for cellular processes. Present work brings new partial information on different levels of chromosome integrity maintenance of this parasite. Our study presents characteristics of chromosome termini and their protection. We localized telomeres during all stages of the trophozoite cell cycle and determined the length of Giardia telomeres ranging from 0.5 to 2.5 kb, we proved an existence of an active telomerase enzyme synthesizing telomeric repeats in in this parasite, despite the fact that giardial telomerase is structurally divergent. Present data support the view that the chromosomal termini in Giardia are maintained in a conservative manner that is common to other eukaryotes. We described effects of commonly used drug for treatment of anaerobic infections, metronidazole, on DNA and cell cycle progression in susceptible and resistant cell lines. Incubation of cells with this drug causes phosphorylation of histone H2A in cell nuclei...

The diplomonads are a diverse group of eukaryotic microbes found in oxygen limited environments such as the intestine of animals were they may cause severe disease. Among them, the prominent human parasite Giardia intestinalis non-invasively colonizes the small intestine of humans and animals where it induces the gastrointestinal disease giardiasis. Two of the eight genetic groups of G. intestinalis, assemblage A and B, are known to infect humans and have zoonotic potential. At the start of project, genome scale data from assemblage B-H was either sparse or entirely missing. In this thesis, genome sequencing was performed on the assemblage B isolate GS (Paper I) and the P15 isolate (Paper III) of the hoofed-animals specific assemblage E to investigate the underlying components of phenotypic diversity in Giardia. Comparisons to assemblage A isolate WB revealed large genomic differences; entirely different repertoires of surface antigens, genome rearrangements and isolate specific coding sequences of potential bacterial origin. We established that genomic differences are also manifested at the transcriptome level (Paper VIII). In a follow up analysis (Paper IV) we concluded that the Giardia assemblages are largely reproductively isolated. The large genomic differences observed between Giardia isolates can explain the phenotypic diversity of giardiasis. The adaptation of diplomonads was further studied in Spironucleus barkhanus (Paper II), a fish commensal of grayling, that is closely related to the fish pathogen Spironucleus salmonicida, causative agent of systemic spironucleosis in salmonid fish. We identified substantial genomic differences in the form of divergent genome size, primary sequence divergence and evidence of allelic sequence heterozygosity, a feature not seen in S. salmonicida. We devised a transfection system for S. salmonicida (Paper VI) and applied it to the study of the mitochondrial remnant organelle (Paper VII). Our analyses showed that S. salmonicida harbor a hydrogenosome, an organelle with more metabolic capabilities than the mitosome of Giardia. Phylogenetic reconstructions of key hydrogenosomal enzymes showed an ancient origin, indicating a common origin to the hydrogenosome in parabasilids and diplomonads. In conclusion, the thesis has provided important insights into the adaptation of diplomonads in the present and the distant past, revealing hidden diversity.

Underdiagnosis of intestinal parasites in patients with diarrhea A compilation from the Swedish public health authority indicates that infections caused by Cryptosporidium spp. increased in Sweden from 47 cases in 2004 to 594 cases in 2016 and Giardia intestinalis causes around 1300 infections per year. The primary aim of this study was to investigate the prevalence of parasites in patients with diarrhea. Furthermore, the study investigated whether samples taken with E-swab could be analyzed with real-time polymerase chain reaction (PCR) for detection of Cryptosporidium spp., Dientamoeba fragilis, Entamoeba histolytica/dispar and G. intestinalis rather than Sodium acetate-acetic acid-formaline fixative (SAF-fixative). Prevalence of parasites in fecal samples was collected from 200 samples from patients with bacterial issue ordered. For evaluation of E-swab, 22 frozen, unfixed samples that were positive for intestinal parasites was used. Twelve positive E-swab samples was used as comparative positive controls. This was analyzed using real-time PCR. Bacteria was counted for 9.5% of the infections whilst parasites counted for 14% of the infections. The conclusion was that E-swab could replace SAF-fixative in the diagnosis of intestinal parasites and that there is that an underdiagnosis of intestinal parasites. Keywords: Cryptosporidium spp, Dientamoeba fragilis, Entamoeba histolytica, Entamoeba dispar, Giardia intestinalis, real-time PCR, E-swab, prevalence.