Entamoeba histolytica is an important human intestinal parasite that has a major impact on human health and is responsible for approximately 100,000 deaths each year. Entamoeba histolytica is one of several known eukaryotes that harbour strongly reduced mitochondria, called mitosomes, which have lost the vast majority of mitochondrial pathways as well as their organellar genome. While the occurrence and function of mitosomes have been well studied, little is known about their inheritance and division. Mitochondrial division in all studied eukaryotes relies on the participation of dynamin proteins for membrane scission. The central aim of this study was to characterize the dynamin protein family in Entamoeba histolytica and to analyze if they participate in mitosomal division. In relation to this work we studied the occurrence of mitosomes in the distantly related reptilian parasite Entamoeba invadens and revisited the phylogenetic relationships among mitosomal Hsp70, a protein we used for mitosomal localization experiments. Our studies revealed that Entamoeba histolytica contains two classical and two strongly derived members of the dynamin protein family, which we called Drp1, Drp2, Drp3 and Drp4. Drp1 and Drp2 exhibit the classical dynamin protein structure with a GTPase, middle and GTPase effector domain, while Drp3 and Drp4 only appear to contain the dynamin GTPase domain. Using phylogenetic reconstructions we could not identify closely, and thus functionally related, dynamins for Drp1 and Drp2 within the eukaryotic tree of life including the mitochondria‐associated amoebozoan dynamins DymA and DymB. The structurally derived dynamins however, were closely related to amoebozoan and archaeplastidan proteins involved in cytokinesis and chloroplast division. All Entamoeba dynamins are differentially expressed in trophozoites with EhDrp2 appearing to be most abundant and Drp3 expressed the least. We conducted stage conversion experiments using E. invadens to understand the importance of dynamins during cyst formation. During encystation all dynamin expression levels increased. Interestingly, Drp3 expression is strongly upregulated in the mid cyst stages and Drp4 during the late phase of encystation. Thus, Drp3 and Drp4 appear not to be involved in cytokinesis and possibly evolved a novel function in the cyst formation process. We carried out Drp2 enzymatic characterization and localization experiments as well as complementation studies using the related amoebozoan Dictyostelium discoideum in order to understand the role and function of E. histolytica Drp2 in the cell. We found that its kinetic characteristics are comparable to other members of the eukaryotic dynamin protein family by exhibiting low substrate specificity, the ability to oligomerize to higher structures and a substrate dependent cooperative enzyme activity. Drp2 localized to abundant punctate structures in the cytosol but did not colocalize with mitosomes. In addition, Drp2 was not able to complement D. discoideum DymA. Both findings suggest that Drp2 is not directly involved in mitosomal (or mitochondrial) division. We overexpressed Drp2 in E. histolytica and D. discoideum and found a significant effect on cytoskeletal organization. Both strains showed a strong impairment in amoeboid movement, cell‐surface attachment and cell growth. Additionally, the number of nuclei was increased significantly. Our data imply that Drp2 plays an important role for cytoskeletal organization. Additionally in this study, we show that mitosomes are also abundantly present in E. invadens suggesting that mitosomes are characteristic for all Entamoeba spp. Furthermore, we demonstrate that E. invadens cysts contain mitosomes in high abundance comparable to its vegetative life stage. Our studies verify that mitosomal Hsp70 is part of the amoebozoan protein family and of mitochondrial origin as shown by in silico characterization and localization experiments using the homologous Hsp70 antibody.

Ticks are obligate haematophagous arthropods that represent a major economic drain upon the world's livestock as well being a significant medical and veterinary risk through the transmission of tick-borne pathogens such as Borrelia burgdorferi, the causative agent of Lyme disease. The tick-host relationship is a function of both ecological and physiological factors. Successful feeding requires the effective acquisition and digestion of a bloodmeal by the tick. Acquisition relies upon the ability of the tick to counteract host immune responses induced by the extended feeding periods of ixodid ticks (up to 2 weeks). The host response to tick infestation and the consequent countermeasures employed by the tick, constitute the tick-host interface. The immune response of hosts to Ixodes ricinus infestations was examined through antigenic profiling. The antigens exposed to the host were shown to vary throughout the feeding period and differed between the different development stages of I. ricinus. It was also shown that different host species infested with I. ricinus recognised different antigens. This was true of both natural and non-natural hosts, and even closely related species. Anti-complement activity was investigated in the salivary glands of Ixodes ticks. This activity was shown to inhibit some host species but not others. The pattern of inhibitory activity varied between the tick species tested in a way that was consistent with known tick host-preferences. The mechanisms of anti-complement activity in I. ricinus salivary glands were explored. The alternative but not the classical pathway of complement was inhibited. Activity was present in unfed ticks and throughout the feeding period. Three targets of the complement system were identified as being modulated by the tick. Digestion of the bloodmeal was explored and a haemolytic activity was associated with the salivary glands of I. ricinus ticks. The activity was demonstrated to be Mg²⁺- dependent. In addition, a subtractive cDNA library enriched for saliva-associated transcripts was successfully produced. Random sampling identified putative differentially expressed genes. The results of this thesis illustrate the complexity of tick-host interactions at the molecular level. It is apparent that the research described poses many more questions than answers.

Root-knot nematodes (RKN) are plant parasites, and a major agricultural pest. RKN are adept at invading and feeding on plant tissue with secretions they release from their gland cells. This thesis compares glandular development in Meloidogyne to the model nematode, Caenorhabditis elegans. Pharyngeal and gland important transcription factors, PHA-4 and HLH-6, respectively, were examined for their role in regulation of parasite-specific genes. Homologues of these genes were identified and sequenced from M. incognita. Phylogenetic analyses revealed a new PPN-specific gene, hlh-6-like. Bioinformatic comparisons of the homologues revealed conservation of the DNA-binding motifs of Ce-HLH-6 and Mi-HLH-6 proteins, as well as conservation of their promoter regions. While HLH-6 antibodies proved inconclusive, expression assays revealed expectant levels of PHA-4 and HLH-6 in Meloidogyne as compared to C. elegans. In identifying genes essential for parasitism we are providing new targets for knockdown during RKN infection. / October 2016

This paper explores the various relationships found in John Steinbeck's "Of Mice and Men," Tennessee Williams's "A Streetcar Named Desire," Franz Kafka's "The Metamorphosis," and F. Scott Fitzgerald's "Tender is the Night." The exploration of each demonstrates the common theme of parasitic relations and the toll this dynamic takes on the persons involved.

Thesis advisor: Marc Muskavitch / The ubiquitin-proteasome system (UPS), composed of classes of proteins central to the process of cellular protein turnover in eukaryotes, is essential to the life cycle of the malaria parasite, Plasmodium falciparum. Although the UPS has been well characterized in other organisms, the extent of its involvement in different stages of P. falciparum growth and development has not been investigated in depth. MG132, a small-molecule proteasome inhibitor known to target the 20S proteasome core (part of the catalytic center for selective protein degradation), has been used successfully in many research studies that require proteasome inhibition. We present data supportive of the conclusion that MG132 is highly effective as a tool for P. falciparum research. In this thesis, I describe the effects of partial and complete proteasome inhibition on parasite growth and development by the use of variable concentrations of MG132. I also assess the effects of MG132 on 20S P. falciparum proteasome enzymatic activities. I have generated parasite lines that exhibit tolerance, or low-level resistance, to MG132, through intermittent compound exposure. Sequencing of the catalytic β-5 subunit of the MG132-tolerant parasites reveals non-synonymous point mutations in three tolerant parasite lines. The use of MG132 as a tool compound for study of the UPS in P. falciparum facilitates research into detailed roles of the proteasome using reversible partial and complete inhibition. MG132-tolerant lines are also valuable tools for studying the genesis of different levels of drug resistance and cross-resistance in parasite evolution. / Thesis (PhD) — Boston College, 2015. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

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This study was based on the parasite fauna of three anuran systems. In two of the systems; the spadefoot toads, Scaphiopus bombifrons and S. multiplicatus and the fire and yellow-bellied toads, Bombina bombina and B.variegata, inter-species hybridisation occurs. In the third system an autopolyploid species complex involves the Gray treefrogs, Hyla chrysoscelis and H.versicolor. The status of the Scaphiopus hybrid zone in Arizona was re-assessed by cellulose-acetate gel electrophoresis. Genotypic frequencies were comparable to those previously documented, indicating that a region of 'balanced introgression' may exist. Specimens were collected in areas of host sympatry and the distribution of helminths within hosts of different genetic constitution investigated. From extensive surveys of all three systems, parasite frequency distributions were typically aggregated with no evidence of any change in host genetic predisposition to infection. In addition, the surveys have documented a number of new host and/or parasite locality records for each system. Of particular interest was the discovery of the digenean Clinostomum complanatum in a desert environment, an introduction related to human management of the ecosystem. Particular reference was made to the specificity of the polystomatid monogeneans, Polystoma nearcticum infecting members of the H.chrysoscelis- H.versicolor complex and Neodiplorchis scaphiopodis infecting S.bombifrons and S.multiplicatus. For Polystoma, worms from H.chlysoscelis and H.versicolor could not be separated by morphology, which was supported by preliminary crossinfections. This suggests that P.nearcticum infects both members of this complex. For Neodiplorchis, morphological similarity was confirmed by cross-infections which revealed that N.scaphiopodis can reach sexual maturity and produce fully developed larvae in heterospecific hosts. The neotenic development of P. nearcticum and the initial post-oncomiracidial stages of N.scaphiopodis were documented. Furthermore, certain aspects of transmission dynamics were examined by the experimental infection of H. versicolor tadpoles with P. nearcticum.

Les parasites sont une partie très importante des écosystèmes, néanmoins, dans certains cas tels que l’aquaculture ils causent des épidémies. Dans cette thèse j’ai étudié les interactions hôte-parasite chez les poissons coralliens, dans les milieux naturels et les milieux aquacoles. J’ai étudié l’arrangement des communautés de 13 espèces de monogènes de 34 espèces de poissons papillon dans l’Indo-Pacifique. Les résultats montrent qu’il existe un patron de distribution stable des espèces, ce qui, combiné avec les résultats de l’étude biogéographique, suggère que la spécificité parasitaire observée a pu se développer tout au long des épisodes biogéographiques passés. Notamment, seule une espèce, Chaetodon lunulatus, n’est jamais parasitée. J’ai étudié le mucus des poissons qui est décrit comme la première ligne de défense contre des agressions externes, pour analyser quels facteurs de C. lunulatus pourraient être reliés à l’absence des parasites. Le microbiome et le métabolome du mucus branchial des poissons papillons montrent une grande diversité. C. lunulatus présente une plus grande abondance de Fusobactéries qui est corrélée avec une surexpression de peptides pouvant dériver de la β-hémoglobine. La purification, la synthèse et l’évaluation des activités antiparasitaires des peptides sont en cours au laboratoire. J’ai aussi étudié l’utilisation des plantes médicinales comme alternative à des traitements chimiques dans l’aquaculture. J’ai testé l’activité antibactérienne et immunostimulatrice de plusieurs plantes et algues polynésiennes. L’algue rouge Asparagopsis taxiformis montre de fortes activités antibactériennes sur Vibrio harveyi et Tenacibaculum maritimum et induit une augmentation de l’expression de deux gènes liés à l’immunité chez Platax orbicularis. / Fish parasites are an important part of ecosystems, however, in certain cases such as in aquaculture they can cause severe disease outbreaks. In this thesis I have studied host-parasite interactions in coral reef fishes, both in the natural and culture environments. I have studied the distribution of 13 dactylogyrid species from 34 butterflyfishes in the Indo West-Pacific. Composition of dactylogyrid communities was host specific and together with the biogeography results, where a turnover in the main Haliotrema species was observed, suggest that parasite specificity might result from host-parasite coevolution derived from past biogeographical episodes. Only one butterflyfish species, Chaetodon lunulatus, was never found parasitized by gill monogeneans. I have studied the butterflyfish mucus, which is the first barrier against pathogens, to investigate the C. lunulatus factors that might be related to the monogenean absence. Butterflyfish gill microbiome and metabolome revealed a high diversity. C. lunulatus presented a significantly higher abundance of Fusobacteria which was correlated to a higher expression of potentially derived β-hemoglobin peptides. Synthesis and evaluation of the peptide antiparasitic activities are being performed in the laboratory. I have also studied the use of medicinal plants as an alternative to chemotherapy in fish aquaculture. I investigated the antibacterial and immunostimulant activities of several local Polynesian plants and algae, and I found that Asparagopsis taxiformis displayed a potent antibacterial activity against Vibrio harveyi and Tenacibaculum maritmum and increased expression of two immune-related genes in Platax orbicularis.

Orientador: Helio Langoni / Coorientador: Silvio Luis de Oliveira / Banca: Maria Cecilia Rui Luvizotto / Banca: Juliana Giantomassi Machado / Banca: Simone Baldini Lucheis / Banca: Lisiane Almeida Martins / Resumo: O objetivo do estudo foi avaliar a resposta humoral, a carga parasitária e os aspectos histopatológicos presentes no fígado em camundongos BALB/c experimentalmente infectados por L. (L.) chagasi e imunossuprimidos. 96 camundongos foram subdivididos em quatro grupos: controle (I) - sem tratamento; imunossuprimidos (II) - tratamento com dexametasona (DXM) e pentoxifilina (PTX); infectado (III) - infecção por L (L.) chagasi e infectados e imunossuprimidos (IV) - infecção por L (L.) chagasi e tratados com DXM e PTX. O dia de infecção foi considerado como o dia zero e a imunossupressão ocorreu 60 dias pós-infecção. As amostras foram obtidas de cada grupo, em momentos distintos 15, 30, 60, 75 e 90 dias pós-infecção, em que se coletou sangue total, para detecção de IgG1 e IgG2a pelo ensaio imunoenzimático (ELISA), fragmentos de baço e fígado, para detecção da carga parasitária pela técnica de microtitulação em cultura e fragmentos de fígado para o exame histopatológico. Houve diferença significativa na produção das imunoglobulinas e IgG1 foi à subclasse de imunoglobulina mais produzida pelos grupos (P < 0,0001). Quando comparadas a subclasses de imunoglobulinas dentro do mesmo grupo, IgG1 também apresentou médias maiores de produção nos grupos III e IV (P = 0,014 e P = 0,009). E quando se considera o momento, não houve diferenças significativas entre IgG1 e IgG2a. Aos 90 dias pós-infecção foram encontradas diferenças significativas entre as médias de produção de IgG1 e IgG2a, entre os grupos III e IV e o grupo III produziu mais as duas subclasses (P = 0,02). Nos momentos 75 e 90 dias pós-infecção houve produção maior de IgG1 e IgG2a. O Grupo IV apresentou maior carga parasitária (P < 0,005). A carga parasitária no fígado foi mais elevada 15 e 30 dias pós-infecção... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: This study aimed to evaluate the humoral response, the parasite load and liver histopathological features in BALB/c experimentally infected by L. (L.) chagasi and immunosuppressed Ninety-six mice divided into four groups: control (I) - no treatment; immunosuppressed (II) - Treatment with dexamethasone (DXM) and pentoxifylline (PTX), infected (III) - infection by L. (L.) chagasi and infected and immunosuppressed (IV) - infection by L (L.) chagasi and treated with PTX and DXM. The days of infection was considered as day zero and immunosuppression occurred 60 days post-infection. Samples were obtained from each group at different times 15, 30, 60, 75 and 90 days post-infection, which collected whole blood for detection of IgG1 and IgG2a by test immunoassay (ELISA), fragments of spleen and liver, to detect the parasite load in the technique microtiter culture and fragments of liver for histopathology. Significant differences in production of immunoglobulins, IgG1 was the subclass immunoglobulin produced by most groups (P<0.0001). When comparing the immunoglobulin subclasses within the same group, IgG1 also showed higher average production in groups III and IV (P=0.014 and P=0.009). When one considers the time, there were no significant differences between IgG1 and IgG2a. At 90 days post-infection were significant differences between the means of production of IgG1 and IgG2a between group III and IV and group III produced over the two subclasses (P=0.02). In periods of 75 and 90 days post-infection there was increased production of IgG1 and IgG2a. Group IV showed a higher parasite load (P <0.005). The parasite load in the liver was higher 15 and 30 days post-infection and 60 post-infection in spleen (P = 0.0001, P = 0.004, P=0.01), regardless of group. Considering the group IV, 15 days post-infection... (Complete abstract click electronic access below) / Doutor