Dissertations / Theses on the topic 'Protozoa'

Apusozoa (Protozoa) is a phylum of heterotrophic gliding zooflagellates of unknown taxonomic affiliation, commonly observed in environmental samples. Almost nothing was previously known about the diversity and ecology of apusozoan species though, as bacterivores, they are probably important functional constituents within microbial assemblages. We explored apusozoan morphological and genetic diversity, ecology, and related methodological questions. By culturing environmental material from a range of habitats, we isolated and maintained monocultures of both previously described apusozoan orders, Apusomonadida (apusomonads) and Planomonadida (planomonads). For planomonads, we present a revised taxonomy based on morphology, ultrastructure, and 18S rDNA genetic differences. We describe nine new species and new genera Nutomonas and Fabomonas, and demonstrate ITS2 rDNA secondary structure analysis for species delineation. During our culturing effort, we also isolated two genotypes of a previously unknown flagellate group, shown here to belong to a novel third apusozoan order, Mantamonadida. We designed molecular probes specific to all three orders and applied them to environmental DNA, detecting novel 18S and ITS1 rDNA lineages in a range of habitats. We mined publically available metagenomic and metatranscriptomic sequence databases using 18S rDNA of described species as seeds, identifying hundreds of sequences with affinities to all three orders. Phylogenies featuring newly retrieved lineages with previously described species suggest that direct sequencing of transcriptomic material is more effective than amplification-dependent methods at detecting rare cells in mixed microbial assemblages. Finally, to test potential future applications of our newly isolated strains, we ran microcosm experiments examining the effect of protozoan (Cercozoa) grazing on the structure of bacterial assemblages, demonstrating that closely related and morphologically similar species can have different impacts on their prey base. Taken together, by combining traditional culturing and modern molecular methods, this thesis drastically improves our understanding of apusozoan diversity and sets the scene for future work using next-generation sequencing and ecologically driven functional experiments.

Submitted by Alessandra Portugal (alessandradf@ioc.fiocruz.br) on 2013-09-17T13:39:42Z No. of bitstreams: 1 Joana Afonso Lima de Oliveira_Dissertação.pdf: 2308780 bytes, checksum: 61baaeaeec93ac8ac32ccd1d9e4c3573 (MD5)<br>Made available in DSpace on 2013-09-17T13:39:42Z (GMT). No. of bitstreams: 1 Joana Afonso Lima de Oliveira_Dissertação.pdf: 2308780 bytes, checksum: 61baaeaeec93ac8ac32ccd1d9e4c3573 (MD5) Previous issue date: 2013-07-28<br>Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil.<br>As vias centrais do metabolismo são responsáveis tanto pela geração e armazenamento de energia, quanto pela formação dos precursores metabólicos que servem como ponto de partida para a biossíntese dos elementos fundamentais, que são polimerizados para formar os constituintes celulares essenciais de todas as células vivas, ou seja, todos os três domínios: Archaea, Bacteria e Eukarya. Vários estudos têm sido feitos nos últimos anos no que se refere a genes associados ao metabolismo de carboidratos (MC) em Protozoários. O objetivo principal deste trabalho foi Inferir os relacionamentos filogenéticos e a diversidade molecular dos genes relacionados ao MC nos genomas de Protozoa. Para isso foram submetidos ao programa OrthoMCL os genomas de 22 espécies de protozoários patogênicos, e utilizando 230 grupos ortólogos listados pelo COG/KOG (NCBI) como pertencentes a categoria “g”, metabolismo de carboidratos, foi feita uma análise de similaridade com o programa BLAST com o intuito de encontrar genes ortólogos aos Protozoa, aos eucariotos e aos procariotos. Destes, foram encontrados 10 grupos ortólogos comuns aos 22 Protozoa, 31 grupos ortólogos entre os eucariotos (KOG) e, 46 grupos ortólogos entre os procariotos (COG). Dos dez grupos ortólogos pertencentes ao MC de protozoários (MC-Núcleo-Protozoa), quatro (enolase, glicose-6-fosfato-isomerase, glicosil transferase e piruvato quinase) foram selecionados para se avaliar o grau de filogenia do MC-Núcleo-Protozoa, através de uma abordagem filogenômica utilizando supermatriz e destes, dois (enolase e glicose-6-fosfato-isomerase) foram escolhidos para se fazer amplificação por PCR com outras espécies de Leishmania e Trypanosoma em bancada para que se pudesse avaliar a diversidade filogenética entre o MC-Núcleo-Protozoa, e MC-COG/KOG. As árvores filogenética feitas com mil replicatas e 80% do valor de corte para cada gene individualmente apoiam a presença de um gene ancestral que se diversificou tanto por especiação quanto por duplicação nos organismos, pois é possível ver que existe uma clara separação entre espécies de Kinetoplastida e Apicomplexa além do que em algumas espécies pode-se observar casos de paralogia. A árvore feita a partir da supermatriz corroborou com o alto grau de conservação dos genes entre as espécies, enfatizando que genes do MC são provavelmente os mais conservados entre os três domínios.<br>The central pathways of metabolism are responsible for both the generation and storage of energy as the formation of metabolic precursors. Such precursors act as a starting point for the biosynthesis of the basic elements which form the essential cellular constituents to all living cells, i.e. in all three domains: Archaea, Bacteria and Eukarya. Several studies concerning the genes associated to carbohydrates metabolism (CM) in Protozoa have been made in the late years. The main objective of this work was to infer the phylogenetic relationships and the molecular diversity of genes related to CM in the Protozoa genomes. For such, it was submitted to OrthoMCL program the genomes of 22 pathogenic protozoan species and, making use of 230 orthologous groups listed by COG/KOG (NCBI) as belonging to the carbohydrate metabolism, it was made a similarity analysis using the BLAST program, intending to find orthologous genes to Protozoa, eukaryotes and prokaryotes. The similarity analysis result presented 10 orthologous groups in common with the 22 Protozoa, 31 orthologous groups among the eukaryotes (KOG) and 46 orthologous groups amidst the procaryotes (COG). From the 10 orthologous groups mentioned above, 4 were selected (enolase, glucose-6-phosphato-isomerase, glycosyl transferase e pyruvate kinase) to evaluate the phylogeny degree of the Core-Protozoa, through a phylogenomic approach using super matrices, and 2 were chosen (enolase e glucose-6-phosphato-isomerase) for amplification by PCR against other Leishmania and Trypanosome species, intending to confirm the phylogeny degree between the Core-Protozoa and COG/KOG. The phylogenetic trees were made with 1000 replicates and a cut-off of 80% for each gene. These trees, individually, support the presence of an ancestor gene which diversified from both speciation and duplication in organisms, because it is possible to observe the existence of a cladistics separation between the Apicomplexa and Kinetoplastida species, besides in some species it is possible to observe paralogy cases. The tree made from de super matrix corroborated with the high conservation degree of the genes among the species, emphasizing that CM genes are, probably, the most preserved among the three domains.

Iron is recognized as a key element regulating primary production in large regions of the ocean, but nothing is known of its direct effect on higher trophic levels. Growth and metabolism of two species of heterotrophic protozoans fed iron-rich and iron-poor prey were thus examined. Maximum growth rates of Paraphysomonas imperforata and P. butcheri were observed only when Fe quotas of bacterial prey were greater than 70 $ mu$mol Fe:mol C. At lower Fe:C ratios, but at constant prey biomass (C/ml), both species grew significantly slower. Minimum Fe quotas of the flagellates at these slow growth rates ($ sim$10 $ mu$mol Fe:mol C) were similar to those of iron-limited phytoplankton and bacteria. Growth rate reduction was the result of direct elemental limitation by Fe, judging from the protozoans' positive response to Fe additions and from their biochemical characteristics. Filtration and carbon ingestion rates increased under Fe-limitation, but carbon gross growth efficiency (CGGE) decreased when Paraphysomonas imperforata consumed iron-poor bacteria. Ammonium regeneration efficiency was also reduced. The decrease in CGGE was a consequence of reduced activity of the iron-dependent electron transport system, greater DOC excretion, and greater CO$ sb2$ evolution by Fe-limited flagellates. Paraphysomonas imperforata excreted Fe, even when limited by this element, and retained less of the ingested ration and thus had a higher Fe regeneration efficiency than when consuming Fe-rich bacteria. According to recent measurements of biogenic Fe:C in the subarctic Pacific, our results suggest that heterotrophic bacterivorous flagellates may experience iron-limitation in remote oceanic regions. Such limitation could profoundly affect C, N and Fe cycling in the sea.

In order to investigate further the mode of action of these compounds, synthesis of proteomic probes was attempted. Synthetic design and attempts are presented chapter VII. To establish whether there was 24-SMT present in the blood stream form of T. b. brucei, a Northern blot was carried out. This confirmed transcription of the enzyme which was then cloned, over expressed and purified (Chapter VIII). Enzyme assays were carried out against the recombinant enzyme.

Apicomplexa parasites are single-celled, obligate intracellular cyst-forming protozoa, infecting humans and animals, that pose major threats to world health and global economy. Among the most relevant species for farm animals, Besnoitia besnoiti, Neospora caninum and Toxoplasma gondii are parasites of medical (T. gondii) and veterinary (T. gondii, N. caninum, B. besnoiti) importance in domestic ruminants. Bovine besnoitiosis, caused by Besnoitia besnoiti, is a chronic and debilitating parasitic disease of cattle, characterized by both cutaneous and systemic manifestations, compromising animal welfare and responsible for economic losses on affected farms. In Europe, including Italy, bovine besnoitiosis is an emerging or re-emerging disease, with an increasing geographical distribution and the number of cases of infection. Neospora caninum, a coccidian protozoan, represents an important cause of bovine abortion. It was suggested that the parasite may have adverse effects on fertility and milk production, but only few and contrasting data are available to date. Besides, while only a single genotype of N. caninum exists worldwide, available parasite strains show considerable variation in vitro and in vivo, including different virulence in cattle. Microsatellite markers allow to fingerprint N. caninum isolates or DNAs and undertake population studies. Toxoplasmosis represents an important public health issue, with the consumption of raw or undercooked meat being a major way of human infection. The role of beef in the transmission of the parasite to humans is questioned due to lower quantity of tissue cysts compared with other meat-producing species. However, the habit of consuming raw beef is regionally diffused, and the risk posed by Toxoplasma gondii infection in cattle should not be overlooked. The aim of my doctoral thesis project was to investigate on the epidemiology and molecular characterization of selected protozoa parasites of medical and veterinary importance in domestic ruminants, i.e., B. besnoiti, N. caninum and T. gondii in cattle. A multidisciplinary approach based on clinical features, laboratory tests including serological and molecular techniques, was applied throughout the research project, to achieve a multi-level comprehension of the epidemiology of these parasite infections. Three main research lines were developed: Research Line 1: Exploring bovine besnoitiosis: a multi method approach. A case of bovine besnoitiosis in a dairy farm housing 217 cattle in Italy was reported. A serological screening was performed on the whole herd using the recommended approach of ELISA and confirmatory Western Blot. Seropositive animals were clinically examined to reveal symptoms and lesions of besnoitiosis. Risk factors and the effects of the parasite infection on reproductive and productive performances were evaluated. Histopathology and molecular analyses on tissues from a slaughtered cow affected by the chronic phase of the disease were carried out. An overall seroprevalence of 23.5%, which increased up to 43.5% considering only cows, was recorded. Clinical examination of 33 of the seropositive cows evidenced the presence of tissue cysts in at least one of the typical localizations (sclera, vulva, or skin) in 25 animals. Statistical analysis did not evidence any significative impact of the parasite infection on herd efficiency; however, a decrease of productive parameters was recorded in cows showing cutaneous cysts. Concerning the chronically affected cow, histopathology revealed B. besnoiti tissue cysts in the skin of the neck, rump, hind legs, eyelid and vulva, in the muzzle, in mucosal membranes of the upper respiratory tract, and in the lungs. Parasite DNA was detected also in masseter muscles, tonsils, mediastinal lymph nodes, liver, cardiac muscle, aorta wall, ovaries, uterus, and vulva. Moreover, alterations of laboratory parameters, i.e., hematological and biochemical parameters, enzyme activities and serum cortisol levels in Besnoitia besnoiti naturally infected cows were deeply investigated. Laboratory parameters of 107 cows, 61 seronegative and 46 seropositive to B. besnoiti, including 27 with clinical signs of bovine besnoitiosis, were compared. Generalized Linear Models were used to evaluate the effect of Besnoitia infection on the considered laboratory parameters. Hematological analyses revealed that B. besnoiti infection determined a significant alteration of the leukocyte differential with a higher percentage of neutrophil granulocytes and a lower percentage of lymphocytes in seropositive animals; Erythrocyte and Platelet counts did not show any difference between the considered groups of cows. Biochemistry evidenced that the parasite infection influenced serum protein values in seropositive animals and GLDH in clinically affected cows. No or only slight differences were revealed for all the other biochemical and enzyme activity parameters in B. besnoiti infected animals. Seropositive and clinically affected cows evidenced mild higher concentrations of serum cortisol values if compared to seronegative animals, indicating that bovine besnoitiosis could be related to stress in infected animals and that the parasite could compromise animal welfare and also influence disease onset and progression. Furthermore, a form of generalized demodectic mange in two dairy cows infected with Besnoitia besnoiti was described. Two out of the cows seropositive to B. besnoiti, at clinical examination presented skin nodules, widespread all over the body, and in particular in anterior regions. Skin biopsies from the region of the neck were collected and the nodules were microscopically examined through compression method. B. besnoiti tissue cysts were not revealed but a semi-solid yellowish content was evidenced with the presence of several mites, morphologically identified as Demodex bovis. Histological examination of skin biopsies evidenced slight acanthosis and hyperkeratosis of the epidermis and superficial dermatitis with oedema and macrophagic and eosinophilic infiltration. Cystic formations located in the deep dermis were lined by metaplastic squamous epithelium and severe cellular infiltration. A treatment with eprinomectin was attempted and clinical improvement of both cows was observed, particularly at the fifteenth day after treatment, with nodules reduced in size and mites in there degenerated. Moreover, an additional sub research line from this one was implemented to investigate on these Apicomplexa protozoa, particularly Besnoitia spp. infection, in Italian equids: Research Line 1 bis: Exposure of Italian equids to selected protozoa infections and investigation on clinical besnoitiosis in donkeys. A serosurvey was planned to estimate the prevalence of Sarcocystidae species (Besnoitia spp., Toxoplasma gondii and Neospora spp.) in Italian equids. Serum samples from 268 horses and 18 donkeys raised in Italy were collected and serologically analyzed to detect anti-Besnoitia spp., anti-T. gondii and anti-Neospora spp. antibodies: an approach based on an initial screening by in-house ELISA followed by a confirmatory Western Blot was used. Two horses (0.7%) and four donkeys (22.2%), showed antibodies anti-Besnoitia spp. Ten horses (3.7%) resulted positive to T. gondii and one of these (0.4%) was seropositive also to Neospora spp. This is the first detection of anti-Besnoitia spp. specific antibodies in Italian horses and donkeys. Low prevalence of T. gondii and Neospora spp. in horses raised in Italy was reported. A case of clinical besnoitiosis in two donkeys was reported. Two donkeys, one male and one female, reared in northern Italy, showed suspected skin lesions and poor body condition. The animals were clinically examined, and endoscopy of upper respiratory tract and of the vagina for the female donkey was performed. Blood samples and skin biopsies were collected. Serum samples were analyzed by Western Blot to detect anti-Besnoitia spp. antibodies; a PCR targeting ITS-1 region and sequencing were carried out on DNA extracted from skin biopsies. Moreover, blood samples were examined for hematology, biochemistry, and enzyme activity. Clinical examination revealed numerous scleral pearls in the eyes of both animals; besides, alopecia and hyperkeratosis with skin nodules in the region of the neck, hind leg and on the pinnae were detected. No cysts were evidenced in the nares, in the upper respiratory tract and in the vagina and vulva in the female animal. Both animals resulted seropositive according to Western Blot results. Skin biopsies collected from the donkeys resulted positive for the presence of parasitic DNA. Sequencing demonstrated a homology of 100% with Besnoitia spp. sequences deposited in GenBank. Hematology evidenced light anemia, leukocytosis with eosinophilia, and lymphocytosis, whereas biochemistry and enzyme activity revealed hypoalbuminemia with decreased albumin/globulin ratio and elevated alkaline phosphatase values. This first clinical case of besnoitiosis in donkeys in Italy confirmed the circulation of Besnoitia spp. in Italian and European equids. Research Line 2: Genetic characterization of Neospora caninum isolates in cattle and impact of neosporosis on herd performances. With the aim of evaluating the spread of neosporosis and its effects on herd efficiency, an epidemiological study was designed in two dairy farms recruited as case-study. Both selected farms, located in Lombardy region, performed genetic improvement of Holstein Friesian, and reported cases of abortions ascribable to N. caninum. Blood samples were collected from 540 animals, including cows and heifers above 24 months, and analyzed by indirect immunofluorescent antibody test. Epidemiological data (individual, reproductive and productive data) were noted. Overall, 94 animals (17.4%) resulted positive to N. caninum (15.5% and 18.5% in Farm 1 and Farm 2), with differences between the farms concerning the antibody titres (Chi-square, p-value = 0.04), particularly in cows (Chi-square, p-value = 0.018). Regarding the episodes of abortions, a different pattern was depicted in the two investigated farms. Data on fertility and production were considered. The number of insemination necessary to get an animal pregnant resulted higher in seropositive animals (2.4 and 2.9) than in seronegative animals (2.1 and 2.4 in Farm 1 and 2, respectively). Similarly, particularly in Farm 1, the number of days in lactation of not-pregnant cows resulted higher in seropositive (167.7) than seronegative animals (133.4). Moreover, although the association between N. caninum infection and milk production is still unclear, both the daily production and the mature equivalent milk yield were lower in seropositive (31.02 and 11838.94) than seronegative cows (33.59 and 12274.88) in Farms 1. To genotype N. caninum in aborted bovine foetuses from Lombardy, the proportion of N. caninum PCR-positive aborted foetuses in this area was determined and the available isolates were characterized by multi-locus microsatellite genotyping. Aborted bovine foetuses were collected between 2015 and early 2019 from Italian Holstein Friesian dairy herds suffering from reproductive problems. A total of 198 samples were collected from 165 intensive farms located in Lombardy, northern Italy. N. caninum positive samples were then subjected to multilocus-microsatellite genotyping (MLMG) using ten previously established microsatellite markers. In addition to own data, those from a recent study providing data on five markers were included and analyzed. 55 aborted foetuses were positive for N. caninum by RT-PCR, yielding a prevalence of 27.8%; 43 farms recorded at least one positive fetus (26.1%). Of the 55 samples finally subjected to MLMG, 35 were typed at all or 9 out of 10 loci. Linear regression revealed a statistically significant association between the spatial distance of the sampling sites with the genetic distance of N. caninum MLMGs (P&lt; 0.001). Including data from a previous north Italian study (eBURST analysis) revealed that part of N. caninum MLMGs from northern Italy separate into four groups; most of the samples from Lombardy clustered in one of these groups. Principle component analysis revealed similar clusters and confirmed MLMG groups identified by eBURST. These findings confirm the concept of local N. caninum subpopulations. The geographic distance of sampling was associated with the genetic distance as determined by MLMGs. Research Line 3: Evaluation of Toxoplasma gondii infection in beef cattle raised in Italy. To update information on T. gondii in cattle reared in Italy, a multicentric seroepidemiological survey was designed and implemented in four northern regions (Liguria, Lombardy, Piedmont, and Trentino Alto Adige) and Sardinia. A convenience sampling was performed, collecting 1444 serum samples from 57 beef cattle herds. Thirteen beef breeds were sampled, besides crossbreed; bovines age varied from 3 months to over 12 years. Sera were tested with a commercial ELISA for the detection of anti-T. gondii antibodies. Individual and herd data were analyzed by binary logistic regression analysis. A T. gondii seroprevalence of 10.2% was recorded, with differences among regions and values ranging from 5.3% in Liguria to 18.6% in the Piedmont region (p value = 0.0001). Both young and adult animals and males and females tested positive, without any significant difference (age and gender: p value &gt;0.05). Lower seroprevalence values were recorded in cattle born in Italy (8.7%) if compared with animals imported from abroad (13.4%) (p value = 0.046). To obtain epidemiological and molecular data on T. gondii infection in cattle slaughtered in Italy, 80 animals were sampled from one of the biggest Italian slaughterhouses. Dairy (Holstein Friesian) or dual purpose (Alpine Brown and Pezzata Rossa Italiana) breeds or crossbreeds from 15 farms located in northern Italy were sampled; age of animals varied from six months to three years. Approximately 50 g of diaphragm was collected to obtain meat juice and muscle homogenate samples. Individual data were noted. Meat juice samples were analyzed with a commercial ELISA to detect anti-T. gondii antibodies. DNA extracted from muscle homogenate samples were subjected to B1 real-time PCR. Anti-T. gondii antibodies were found in 10 (12.5%) out of 80 examined animals, whereas parasitic DNA was detected in 26 diaphragm muscle samples (32.5%). Only seven samples scored positive in both test: a fair agreement between ELISA and B1 real-time PCR results was achieved (κ value = 0.254). Nevertheless, higher ELISA S/P% values were recorded in diaphragm samples scoring positive to PCR. Higher number of positive samples were found in younger than older animals considering both ELISA and B1 real-time PCR results. Similarly, considering the provenience, animals that have been acquired from other holdings scored more frequently positive to both ELISA and B1 real-time PCR compared to animals that have never left the holding of origin until slaughter. Statistical analysis showed an effect of ELISA S/P% values on B1 real-time PCR results, increasing the risk of parasitic DNA detection when increasing the S/P% values. The other considered variables (age and provenience) did not show any effect on neither ELISA nor B1 real-time PCR. In conclusion, obtained results from the studies of my PhD project allowed to update information on protozoa of medical and veterinary importance both in domestic ruminants and in equids. It was demonstrated that bovine besnoitiosis continues to spread in Italy: both clinical and laboratory tests are needed for an accurate diagnosis, and thus to implement plans for the control of the disease. Moreover, Besnoitia spp. infection circulates in Italian equids and besnoitiosis may be considered an emerging disease of donkeys in Italy and also in Europe. Serological screening of cows and molecular analysis of aborted foetuses confirmed the role of N. caninum in abortion and reproductive failure in dairy herds in northern Italy; besides, multilocus microsatellite genotyping confirmed the concept of local N. caninum subpopulations. The zoonotic importance of T. gondii should not be underestimated in animal species destined to human consumption, including cattle and horses. Serological data are useful to give an indication of the population exposure to the parasite, whereas molecular methods allow to detect tissue cysts in the edible parts reflecting the risk for human infection.

<p>alpha-actinin is a ubiquitous protein found in most eukaryotic organisms. The ability to form dimers allows alpha-actinin to cross-link actin in different structures. In muscle cells alpha-actinin is found at the Z-disk of sarcomeres. In non-muscle cells alpha-actinin is found in zonula adherens or focal adhesion sites where it can bind actin to the plasma membrane.</p><p>alpha-actinin is the shortest member of the spectrin superfamily of proteins which also includes spectrin, dystrophin and utrophin. Several hypotheses suggest that alpha-actinin is the ancestor of this superfamily.</p><p>The structure of alpha-actinin in higher organisms has been well characterized consisting of three main domains: an N-terminal actin-binding domain with two calponin homology domains, a central rod domain with four spectrin repeats and a C-terminal calcium-binding domain. Data mining of genomes from diverse organisms has made possible the discovery of new and atypical alpha-actinin isoforms that have not been characterized yet.</p><p>Invertebrates contain a single alpha-actinin isoform, whereas most of the vertebrates contain four. These four isoforms can be broadly classified in two groups, muscle isoforms and non-muscle isoforms. Muscle isoforms bind actin in a calcium independent manner whereas non-muscle isoforms bind actin in a calcium-dependent manner.</p><p>Some of the protozoa and fungi isoforms are atypical in that they contain fewer spectrin repeats in the rod domain. We have purified and characterized two ancestral alpha-actinins from the parasite Entamoeba histolytica. Our results show that despite the shorter rod domain they conserve the most important functions of modern alpha-actinin such as actin-bundling formation and calcium-binding regulation. Therefore it is suggested that they are genuine alpha-actinins.</p><p>The phylogenetic tree of alpha-actinin shows that the four different alpha-actinin isoforms appeared after the vertebrate-invertebrate split as a result of two rounds of genome duplication. The atypical alpha-actinin isoforms are placed as the most divergent isoforms suggesting that they are ancestral isoforms. We also propose that the most ancestral alpha-actinin contained a single repeat in its rod domain. After a first intragene duplication alpha-actinin with two spectrin repeats were created and a second intragene duplication gave rise to modern alpha-actinins with four spectrin repeats.</p>

The lack of new drugs coming through to the market, to add to those few available, for use against diseases caused by trypanosomatids, calls for ways of safe-guarding the use of the current drugs to prolong their usefulness. This can be achieved by studying mechanisms of resistance to the drugs currently in use. In this thesis, the complementary use of untargeted metabolomic and whole genome sequence analyses was applied to elucidate the mechanisms of amphotericin B (AmB) resistance in L. mexicana promastigotes and isometamidium (ISMM) resistance in bloodstream forms of T. brucei. Resistance to AmB and ISMM was induced by step-wise increase of the drug concentration in the growth medium of L. mexicana promastigotes and bloodstream forms of T. brucei, respectively. Untargeted metabolomics results were used to link a single SNP in the lanosterol 14α-demethylase (CYP51) gene of the sterol (ergosterol) biosynthetic pathway from a multitude of SNPs that were found in generated AmB resistant cells as compared to the parental wild-type cells. The identified SNP was found to be a non-synonymous mutation, causing the change N176I, outside the active sites of CYP51 and was accompanied by accumulation of the enzyme’s product, 4, 4-dimethylcholesta-8, 14, 24-trien-3β-ol. These results suggested a break down in a possible protein-protein interaction of CYP51 with the next enzyme in the pathway, sterol C14-reductase, for efficient flow of the metabolite. Although the main subcellular localisation is different for these enzymes, they had a common localisation in the ER. AmB resistance was accompanied by depleted levels of ergosterol in AmB resistant cells. Expression of the wild-type CYP51 in AmB resistant cells restored ergosterol levels to those of the parental wild-type. Associated with restoration of ergosterol synthesis was reversal of resistance to AmB and susceptibility to pentamidine observed in AmB resistant cells. Thus N176I mutation in CYP51 underlies resistance to AmB in L. mexicana promastigotes. T. brucei resistance to isometamidium was accompanied by loss of the kinetoplast and maintenance of a much reduced mitochondrial membrane potential as compared to the parental wild-type cells. Reduction of the mitochondrial membrane potential was not connected to any apparent alteration of the energy metabolism of related metabolites. However, a perturbation in sphingolipid metabolism was observed to lead to depletion of sphingomyelin and accumulation of its precursor ceramide in the resistant cells. The responsible reaction enzyme, choline phosphorylceramide synthase (SLS4), was found to have 4 non-synonymous mutations which were outside the active site. The ISMM resistant cells exhibited a slow growth phenotype which has also been associated with perturbation of the sphingolipid synthesis pathway. ISMM resistant cells also showed cross resistance to diminazene aceturate and ethidium bromide used for control of African animal trypanosomiasis.

The logistic Lotka-Volterra predator-prey equations with diffusion based on Luckinbill's experiment with Didinium nasutum as predator and Paramecium aurelia as prey, have been solved numerically along with a third equation to include prey taxis in the system. The effect of taxis on the dynamics of the population has been examined under three different non-uniform initial conditions and four different response functions of predators. The four response functions are Holling Type 2 Response, Beddington Type Response or Holling Type 3 Response, a response function involving predator interference and a modified sigmoid response function. The operator splitting method and forward difference Euler scheme have been used to solve the differential equations. The stability of the solutions has been established for each model using Routh - Hurwitz conditions, variational matrix. This has been further verified through numerical simulations. The numerical solutions have been obtained both with and without prey-taxis coefficient. The effect of bifurcation value of prey-taxis coe�cient on the numerical solution has been examined. It has been observed that as the value of the taxis coefficient is increased significantly from the bifurcation value chaotic dynamics develops for each model. The introduction of diffusion in predator velocity in the system restores it back to normal periodic behaviour. A brief study of coexistence of low population densities both with and without prey-taxis has also been done.

Durante o ciclo de vida da Leishmania, amastigotas vivem no interior de fagolisossomas de células fagocíticas de hospedeiros vertebrados, enquanto promastigotas vivem no interior do vetor invertebrado. Proteases intracelulares como as caspases são as principais efetoras no processo apoptótico. Metacaspases (MCAs) são formas evolutivas distantes das caspases de metazoários, presentes em protozoários, plantas e fungos, e vistas como potenciais alvos para combate dos parasitas sem prejuízo do hospedeiro. Ligantes e moduladores das metacaspases são até hoje desconhecidos. O Phage Display é uma técnica baseada na expressão de proteínas sintéticas nos capsidíos de fagos, usada com o propósito de selecionar ligantes de proteínas, células ou tecidos. Produzimos a metacaspase recombinante de Leishmania L. amazonensis e aplicamos Phage Display para buscar peptídeos ligantes dessa enzima. Esses peptídeos permitiram identificar potenciais proteínas ligantes da MCA, como quinases e cinesinas, que fornecem informações sobre a regulação e controle de sua atividade. Futuramente testaremos se peptídeos ativadores da MCA poderão induzir apoptose do parasita e serem usados como drogas para o tratamento da leishmaniose.<br>During its life cycle, Leishmania amastigotes live inside phagolysosomes of phagocytic cells of vertebrate hosts, while promastigotes live inside the invertebrate vector. Intracellular proteases such as caspases are key effectors in the apoptotic process. Metacaspases (MCAs) are distant evolutionary forms of metazoan caspases found in protozoa, plants and fungi, and seen as potential targets to destroy the parasites without damage to the host. Ligands and modulators of metacaspases are so far unknown. Phage Display is a technique based on the expression of synthetic proteins in the phage capsid, and is used for selecting ligands of proteins, cells or tissues. We have produced the recombinant metacaspase of Leishmania (L.) amazonensis and employed Phage Display to find peptide ligands of this enzyme. These peptides led to the identification of potential binding proteins of the MCA, such as kinases and kinesin, which provide information about the regulation and control of MCA´s activity. In the future we will test whether peptide activators of MCA nduce apoptosis of the parasite and can be used as drugs for the treatment of leishmaniasis.

Submitted by MARCOS LEANDRO TEIXEIRA DE OLIVEIRA (marcosteixeira@ufv.br) on 2018-10-04T17:24:07Z No. of bitstreams: 1 texto completo.pdf: 1726508 bytes, checksum: b215614a17770465d875d305244f584b (MD5)<br>Made available in DSpace on 2018-10-04T17:24:07Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1726508 bytes, checksum: b215614a17770465d875d305244f584b (MD5) Previous issue date: 2018-06-29<br>Os microrganismos ruminais têm um papel central na nutrição de ruminantes. Eles têm a capacidade de fermentar componentes do alimento para produzir ácidos graxos voláteis (AGV’s) e crescer (sintetizar proteína microbiana), os quais fornecem a maior parte da energia e aminoácidos exigidos pelos animais. No entanto, quando são fornecidos carboidratos em excesso, a eficiência de crescimento dos microrganismos torna-se baixa porque estes direcionam a energia para outras funções, ao invés de a utilizarem para o crescimento. Diferentes microrganismos respondem a esse excesso de maneiras diferentes. Certas espécies respondem armazenando energia (sintetizando carboidratos de reserva), mas outras espécies respondem dissipando a energia na forma de calor. Para determinar a importância relativa dessas respostas na comunidade microbiana do rúmen, este estudo foi cinduzido com o objetivo de quantificar como os protozoários ciliados e as bactérias responderam à glicose. Teve-se como hipótese que os protozoários ciliados direcionariam mais glicose para a síntese de carboidratos de reserva e desperdiçariam menos energia na forma de calor, em relação as bactérias. Ciliados e bactérias foram isolados do líquido ruminal por filtração e centrifugação, respectivamente. Posteriormente, os ciliados e as bactérias foram suspensos em tampão isento de nitrogênio para limitar o crescimento e dosados com 5 mM de glicose. As amostras foram coletadas ao longo do tempo e, posteriormente, divididas por centrifugação em pellets (células) e sobrenadante. Amostras de pellets foram analisadas quanto à reserva de carboidratos e proteínas, enquanto amostras de sobrenadante foram analisadas para glicose livre, ácido D-L lático, ácido acético, propionato e butirato. Adicionalmente, foi analisado a produção de calor e gases de fermentação (H 2 , CH 4 e CO 2 ). O metabolismo endógeno, a síntese de carboidratos de reserva e o desperdício na forma de calor foram calculados a partir dos dados das análises. A maior parte dos dados foi analisada usando o PROC GLIMMIX do SAS. Teste t de Student foi usado para separar as médias ou determinar se as médias diferiam de 100%. Regressão local (pacote LOCFIT de R; Loader, 1999) foi usada para ajustar os dados das séries no tempo. Em comparação com as bactérias, os ciliados consumiram três vezes mais glicose e sintetizaram carboidratos de reserva quatro vezes mais rápido. Eles incorporaram 53% da glicose em carboidratos de reserva, quase o dobro do valor (27%) obtido para as bactérias. Desperdício de energia na forma de calor não foi detectado para os ciliados, uma vez que toda a produção de calor foi contabilizada pela síntese de reserva de carboidratos e pelo metabolismo endógeno. Em bactérias, a síntese de carboidratos de reserva e o metabolismo endógeno representaram apenas 68% da produção total de calor, assim, elas desperdiçaram grande quantidade de energia por meio da produção de calor (32% da produção total de calor). Esses resultados sugerem que os protozoários ciliados ruminais alteram o curso do metabolismo de carboidratos no rúmen, consumindo glicose mais rapidamente, limitando o uso do excesso de carboidratos pelas bactérias. Essa ação dos ciliados no rúmen provavelmente maximiza a síntese carboidratos de reserva, enquanto minimiza a ocorrência de desperdício de energia na forma de calor.<br>Rumen microbes hold a central role in ruminant nutrition. They ferment feed components to produce volatile fatty acids (VFA) and grow (synthesize microbial protein), which supplies the greater part of energy and amino acids required by the animals. However, when given excess carbohydrate, microbes growth efficiency becomes low because microbes direct energy to non-growth functions, instead of using it for growth. Different microorganisms respond to this excess in different ways. Certain species respond by storing energy (synthesizing reserve carbohydrate), but other species respond by dissipating the energy as heat (spilling energy). To determine the relative importance of these responses in the microbial community of the rumen, this study aims to quantify how mixed ciliate protozoa and bacteria respond to glucose. It was hypothesized that ciliate protozoa would direct more glucose to synthesis of reserve carbohydrate and less to energy spilling than would bacteria. Ciliates and bacteria were isolated from rumen fluid using filtration and centrifugation, respectively. Posteriorly, ciliates and bacteria were resuspended in nitrogen-free buffer to limit growth and dosed with 5 mM glucose. Samples were collected over time and were subsequently divided in pellet (cells) and supernatant by centrifugation. Pellet samples were analyzed for reserve carbohydrate and protein, while supernatant sample were analyzed for free glucose, D- /L-lactic acid, acetic acid, propionate and butyrate. Additionally, were analyzed heat production and fermentation gases (H 2 , CH 4 and CO 2 ). Endogenous metabolism, reserve carbohydrate synthesis and energy spilling were calculated from the data obtained from the analysis data. Most data were analyzed using PROC GLIMMIX of SAS. Student’s t- test was used to separate means or determine if means differed from 100%. Local regression (LOCFIT package of R; Loader, 1999) was used to fit time-series data to smooth curves. Compared to bacteria, ciliates consumed glucose more than 3-fold faster and synthesized reserve carbohydrate 4-fold faster. They incorporated 53% of glucose carbon into reserve carbohydrate, nearly double the value (27%) for bacteria. Energy spilling was not detected for ciliates, as all heat production was accounted by synthesis of reserve carbohydrate and endogenous metabolism. For bacteria, reserve carbohydrate and endogenous metabolism accounted for only 68% of heat production, thus they spilled large amounts of energy (32% of total heat production). These results suggest that rumen ciliates protozoa alter the course of carbohydrate metabolism in the rumen by consuming glucose more rapidly and outcompeting bacteria for excess carbohydrate. This action of the ciliates in the rumen likely maximizes reserve carbohydrate synthesis while minimizing spilling.

The effect of competing food sources on termite consumption of hexaflumuron bait and subsequent mortality was examined. Also, the effect of hexaflumuron on the termite gut fauna was evaluated to determine if hexaflumuron could kill termites via a secondary mode of action. Hexaflumuron consumption in no-choice and choice tests was evaluated at 2d and 5d. Total diet consumption was not different between the treatment groups. Hexaflumuron consumption was reduced by a factor of 3 in the presence of a competing control diet and reduced by a factor of 57 in the presence of an inulin diet. In the choice test, termites preferred inulin over hexaflumuron. Termite mortality after hexaflumuron consumption was quantified at 5d, 15d, 20d, 25d, and 35d in three treatment groups. Mean mortality for termites fed only hexaflumuron diet was significantly higher than termites fed only control diet. Mean mortality for termites given a choice was no different than mortality for termites fed only hexaflumuron or control diet. LT50s for termites fed control diet, hexaflumuron diet, or both diets in the choice test, were 24.4d, 18.7d, and 20.6d, respectively. The choice test LT50 did not differ from the LT50 of either no-choice test. Termites fed only hexaflumuron diet had an earlier LT50 than termites fed only control diet. The effect of hexaflumuron on gut fauna survival was evaluated at 5d and 15d. No significant differences were found in total numbers of protozoa in termites fed either hexaflumuron or control diet. Pyrsonympha was the only protozoa significantly reduced by hexaflumuron consumption.<br>Master of Science

Thesis (Ph.D.)--University of Waterloo, 2004.<br>"A thesis presented to the University of Waterloo in fulfillment of the thesis requirement for the degree of Master of Science in Biology." Includes bibliographical references.

Orientador: Karin Werther<br>Banca: Nádia Regina Pereira Almosny<br>Banca: Marcos Rogério André<br>Banca: Lúcia Helena O'Dwyer de Oliveira<br>Banca: Estevam Guilherme Lux Hoppe<br>Resumo: Testudines atuam como hospedeiros intermediários para hemogregarinas (Haemogregarina spp. e Hemolivia spp.) e hemosporídeos (Haemoproteus spp. e Haemocystidium spp.). Esses hemoprotozoários pertencentes ao Filo Apicomplexa são parasitas heteroxenos que afetam principalmente hemácias. Sua presença, intracitoplasmática, em extensões sanguíneas de répteis, pode ser eventualmente observada. A identificação morfológica não é suficiente para a classificação de espécie, sendo recomendada a utilização de métodos moleculares. Com o objetivo de pesquisar a presença de hemogregarinas e hemosporídeos em Phrynops geoffroanus (cágados-de-barbicha) e Podocnemis expansa (tartarugas-da-Amazônia) ambos de vida livre, foram utilizados métodos morfológicos e moleculares. Nas extensões sanguíneas coradas e em cortes histológicos (de fígado, pulmão, baço e coração de P. geoffroanus) foi investigada a presença de hemoparasitas. A partir de amostras de sangue e de tecidos foi realizada a pesquisa de material genômico de hemoparasitas por meio da Reação em Cadeia da Polimerase (cPCR), com base nos genes 18S rRNA (hemogregarinas) e Citocromo b (hemosporídeos). Em extensões sanguíneas de 100% (7/7) de P. expansa foram observadas estruturas sugestivas para hemogregarinas. Observou-se amplificação de fragmentos baseados no gene 18S rRNA para 100% (7/7) de P. expansa. As análises filogenéticas de Máxima Verossimilhança (MV) e Bayesiana foram obtidas de sequências de amostras sanguíneas de P. expansa. Tais... (Resumo completo, clicar acesso eletrônico abaixo)<br>Abstract: Testudines act as intermediate hosts for hemogregarines (Haemogregarina spp. and Hemolivia spp.) and hemosporidia (Haemoproteus spp. and Haemocystidium spp.). These haemoprotozoans belongs to the phylum Apicomplexa are heteroxenous parasites that affect erythrocytes. Their presence in reptiles' blood extensions can eventually be observed. Morphological identification is not enough for the classification, so molecular methods are recommended. In order to investigate the presence of hemogregarines and hemosporidia in free-living Phrynops geoffroanus (geoffroy's side-necked turtle) and Podocnemis expansa (giant Amazon turtle), morphological and molecular methods were used. The presence of hemoparasites was investigated at stained blood smears and histological slides (liver, lung, spleen and heart from P. geoffroanus). Genomic material of hemoparasites was detected by Polymerase Chain Reaction (cPCR), based on the 18S rRNA genes (hemogregarines) and Cytochrome b genes (hemosporidia). Hemogregarines-like structures were observed in stained blood smears from 100% (7/7) P. expansa. Amplification of fragments based on 18S rRNA gene was observed at 100% (7/7) P. expansa samples. Maximum Likelihood and Bayesian phylogenetic analyzes were obtained from sequences from P. expansa blood samples. These sequences were grouped into a monophyletic clade and clustered close Haemogregarina genus sequences, obtained from fresh water turtles from North America, Asia, Europe and Africa. This situation put forward molecular investigation of more sequences of hemogregarines from freshwater turtles from geographically close South American countries, with the view to collaborate in a robust affirmation of the emergence of a new clade of the genus Hemogregarina.<br>Doutor

Submitted by Anderson Silva (avargas@icict.fiocruz.br) on 2012-09-19T13:39:08Z No. of bitstreams: 1 renato_s_junior_ioc_bp_0048_2011.pdf: 2131023 bytes, checksum: c67826382b4b8b6c00b329a3f8c712d2 (MD5)<br>Made available in DSpace on 2012-09-19T13:39:08Z (GMT). No. of bitstreams: 1 renato_s_junior_ioc_bp_0048_2011.pdf: 2131023 bytes, checksum: c67826382b4b8b6c00b329a3f8c712d2 (MD5) Previous issue date: 2011<br>Universidade Federal Fluminense. Centro de Estudos Gerais. Instituto de Biologia. Niteroi, RJ, Brasil<br>A família Trypanosomatidae inclui parasitas de uma grande variedade de vertebrados, invertebrados (principalmente insetos), plantas e algumas espécies de protozoários, sendo, depois do nematóides, os de maior distribuição de hospedeiros na natureza. No presente trabalho, um novo isolado foi obtido por coprocultivo de trato intestinal de Leptoglossus stigma Herb, 1784 (Hemiptera, Coreidae), capturado no município de Itaguaí/RJ. O isolado original e três clones (obtidos por citometria de fluxo) foram depositados na "Coleção de Flagelados do Laboratório de Transmissores de Leishmanioses." Um clone foi caracterizado por diversas abordagens em comparação com espécies de referência de diferentes gêneros. Foi analisado o crescimento em meio de cultivo em intervalos de 24 h, entre 48-144 h, a diferenciação celular e a morfometria dos principais estágios evolutivos encontrados. A análise de isoenzimas foi realizada utilizando-se os seguintes sistemas: GPI, PGM, 6PGDH, HK, ACON, MPI, FUM, IDH, MDH e ACON. RAPD-PCR foi realizado utilizando-se 6 iniciadores, conjuntamente com outros tripanosomatídeos. Os dados destas análises foram processados numericamente e submetidos à análise computacional utilizando-se o coeficiente de associação Jaccard e o algoritmo de agrupamento UPGMA. Realizou-se seqüenciamento do amplicon do gene de SSU rRNA e o fragmento analisado foi alinhado com vinte e uma seqüencias depositadas no GenBank, gerando uma árvore filogenética resultante do pareamento. O conjunto de resultados deste trabalho sugere que a amostra obtida constitui nova espécie, pertencendo a um novo gênero, nomeada Vickermania itaguaiensis.<br>The Trypanosomatidae family includes parasites of a wide variety of vertebrates, invertebrates (mainly insects), plants and some species of protozoa, and after the nematodes, the highest distribution of hosts in nature. In this study, an isolate was obtained by coproculture from Leptoglossus stigma Herb, 1784 (Hemiptera, Coreidae) intestinal tract, captured in the city of Itaguaí/RJ. The original isolate and clones (obtained by flow cytometry) were deposited in the "Coleção de Flagelados do Laboratório de Transmissores de Leishmaniose". One clone was characterized by several approaches in comparison with the reference species of different genus. Growth was analyzed in culture medium at 24 h intervals between 48-144 h, cell differentiation and morphometric parameters of the main evolutionary stages matches. The isoenzyme analysis was performed using the following systems: GPI, PGM, 6PGDH, HK, ACON, MPI, FUM, IDH, MDH and ACON. RAPD-PCR was performed using 6 primers, together with other trypanosomatids. The analysis of these data were numerically processed and submitted to computer analysis using the Jaccard association coefficient and UPGMA clustering algorithm. We carried out sequencing of the amplicon from SSU rRNA gene and the fragment analyzed was aligned with twenty-one sequences deposited in GenBank, generating a phylogenetic tree resulting from the pairing. The result set of this work suggests that the sample obtained represents a new species belonging to a new genus, named Vickermania itaguaiensis.

The purpose of this research was to examine the colonization process and relationship of physico-chemical parameters to diatom and protozoan communities colonizing polyurethane foam (PF) artificial substrates in lentic habitats. This was the first study to utilize multivariate techniques for comparison of protozoan and diatom communities The following hypotheses were examined in this study: 1. diatom and protozoan species accrual is similar because the organisms are approximately the same size and share similar ecological conditions, 2. protozoan assemblages are influenced by the physicochemical parameters of their environment, and 3. diatoms and photosynthetic protozoans are more closely related to the physico-chemical parameters of their environment than are the protozoans of all trophic groups. PF substrates were placed in the littoral zone of lentic habitats. Substrates were sampled through a time series and examined for their diatom and protozoan species' presence-absences. The first hypothesis was tested by using the MacArthur-Wilson equilibrium model and by fitting the data to the model by non·linear least squares regression. Protozoan species accrual fit the model in most cases, while diatom species accrual did not. The second part of the research dealt with five lentic habitats in northern lower Michigan which were sampled as described above and concurrent with organismal sampling several physico-chemical parameters were sampled. These environmental parameters included pH, alkalinity, conductivity, temperature, and concentrations of dissolved oxygen, chloride, silica, ammonia, and total and ortho-phosphate. Protozoan communities were examined using reciprocal averaging ordination. It was found that the bog and marsh had distinct communities, while the three lakes did not. Several physicochemical parameters and factors correlated significantly with axes generated by samples in species space. The final section tested the degree of relationship among diatoms, autotrophic protozoans, and protozoans to the physicochemical parameters and factors. pH had the highest correlations with the first axes for each group. Diatom communities had the greatest degree of relationship to the physico-chemical parameters, evidence for this is provided by the greatest number of correlations between ordination axes and the physico-chemical parameters and factors.<br>Ph. D.<br>incomplete_metadata

Robinson's xenic parasite culture method was used to investigate the prevalence of Dientamoeba fragilis and Blastocystis hominis in faecal samples submitted to the National Public Health Service for Wales (NPHS) laboratory at Aberystwyth. Culture, in combination with a commercial trichrome stain, gave a D. fragilis positivity-rate of 2.6% (25/976), and a B. hominis positivity-rate of 8.0% (78/976). Isolates of B. hominis and D. fragilis were typed using riboprinting of the amplified SSU rRNA gene. Sixty six B. hominis and thirty three D. fragilis isolates produced a product after amplification. The B. hominis typing results confirmed the extensive genetic variation reported by other workers, and six new ribodemes were found among the human isolates. Two animal Blastocystis isolates were typed; a monkey isolate was ribodeme 1, and an ovine isolate was a new ribotype. All D. fragilis isolates that were riboprinted were found to be genotype 1.