Organisms belonging to the Kingdom Fungi are known to occupy a wide variety of ecological niches and are found globally in virtually all environments. Two members of the smallest of the fungal phylum, the Zygomycota, have also been found to harbor intercellular bacteria initially described as being from or closely related to organisms from the Genus Burkholderia. In this study two microaerophilic members of the species Verticilium from the phyla Ascomycota were characterized. Both appear to carry two bacterial endosymbionts. This is the first evidence of bacterial endosymbionts found within a member of the Ascomycota. Through the use of fluorescent stains, isolation of the intercellular bacteria, DNA analysis and fluorescent in situ hybridization (FISH) it appears that the newly isolated Verticilium sp. fungi contain not one but two bacterial endosymbionts from the family Proteobacteria. One putative symbiont is from the genus Bradyrhizobium, a member of the α-Proteobacteria, and one from the genus Burkholderia, a member of the β-Proteobacteria. This is the first evidence of a fungus containing not one, but two distinct endosymbionts from two separate bacterial families. Additionally the fungi were found to grow from spore across a large pH gradient (pH 1.2 to pH 13.5) and in conditions lacking given nutrient. They were tolerant of concentrations of Fe(II) up to 50mM and grew better with low oxygen levels (1.6%) than without.

The sacoglossan sea slug, Elysia chlorotica, feeds upon and sequesters plastids from the heterokont alga, Vaucheria litorea, and maintains the metabolically active organelles for up to nine months under starvation conditions while utilizing the photosynthate to survive and reproduce. The photosynthetic pigment, chlorophyll a (Chla), is found in all oxygenic photosynthetic organisms and is responsible for capturing photons of light and converting them into chemical energy. Chlorophyll and its associated proteins involved in the light capturing process are subject to photo oxidative damage and must be continually replaced for ongoing photosynthesis to continue; however, genes encoding these proteins are present in the algal nucleus, presenting a conundrum for sustained plastid photosynthetic activity outside the algal cell. One possibility is that Chla is synthesized by the E. chlorotica-kleptoplast association, due to transfer of algal nuclear genes to the sea slug genome. For this study, molecular and biochemical techniques were employed to determine if Chla is synthesized by the animal. Using algal transcriptome sequences for primer design and amplification of target DNA using polymerase chain reaction (PCR), we have identified and sequenced three algal nuclear-encoded gene fragments that correspond to enzymes in the chlorophyll synthesis pathway and one enzyme in the porphyrin synthesis pathway in adult slug and veliger larvae. Sequences from these genes were nearly identical to those present in the alga. Furthermore, these genes are functional; incubation of slugs with radiolabeled 5-aminolevulinic acid (14C-5-ALA), a precursor of chlorophyll biosynthesis, resulted in production of 14C-labeled chlorophyll, as assayed and identified via HPLC resolution of extracts from slugs. In addition, Chla synthesis in the animal occurs for at least six months under starvation conditions. The discovery of chlorophyll synthesis in E. chlorotica is the first animal known to synthesize Chla; moreover, this finding helps elucidate how ongoing photosynthesis can occur in the sea slug after many months in the absence of its algal food.

Thesis (Ph. D.)--University of New Mexico, 2001. / "July, 2001." Includes bibliographical references (leaves 112-121). Color figures, full content, and supplementary materials are available online via www.santafe.edu/p̃th/dss.

Following their endosymbiotic uptake, plastids undergo profound changes to genome content and to their associated biochemistry. I have investigated how evolutionary transitions in plastid genomes may impact on biochemical pathways associated with plastid gene expression, focusing on the highly unusual plastids found in one group of eukaryotes, the alveolates. The principal photosynthetic alveolate lineage is the dinoflagellate algae. Most dinoflagellate species harbour unusual plastids derived from red algae. The genome of this plastid has been fragmented into small, plasmid-like elements termed “minicircles”. Transcripts of this genome receive a 3’ poly(U) tail and, in some species, undergo extensive sequence editing. Some dinoflagellates have replaced their original plastids with others, in a process termed “serial endosymbiosis”. The major non-photosynthetic alveolates are the apicomplexans, which include the malaria parasite Plasmodium. Apicomplexans are descended from free-living algae and possess a vestigial plastid, which originated through the same endosymbiosis as the ancestral red dinoflagellate plastid. This plastid has lost all genes involved in photosynthesis and does not possess a poly(U) tail addition pathway. I have investigated the consequences of the fragmentation of the red algal dinoflagellate plastid genome on plastid transcription. I have characterised non-coding transcripts in plastids of the dinoflagellate Amphidinium carterae, including the first evidence for antisense transcripts in an algal plastid. Antisense transcripts in dinoflagellate plastids do not receive poly(U) tails, suggesting that poly(U) tail addition may play a role in strand discrimination during transcript processing. I have additionally characterised transcript processing in dinoflagellate plastids that were acquired through serial endosymbiosis. I have shown that poly(U) tail addition and editing occur in the haptophyte-derived serial endosymbionts of the fucoxanthin-containing dinoflagellates Karenia mikimotoi and Karlodinium veneficum. This is the first evidence that plastids acquired through serial endosymbiosis may be supported by pathways retained from previous symbioses. Transcript editing constrains the phenotypic consequences of divergent mutations in fucoxanthin plastid genomes, whereas poly(U) tail addition plays a central role in recognising and processing translationally functional fucoxanthin plastid mRNAs. I have additionally shown that certain genes within fucoxanthin plastids are located on minicircles. This demonstrates convergent evolution in the organisation of the fucoxanthin and red algal dinoflagellate plastid genomes since their endosymbiotic acquisition. Finally, I have investigated transcript processing in the algae Chromera velia and Vitrella brassicaformis. These species are closely related to apicomplexans but are still photosynthetic and apply poly(U) tails to plastid transcripts, as with dinoflagellates. I have shown that poly(U) tails in these species are preferentially associated with translationally functional mRNAs of photosynthesis genes. This is the first plastid transcript processing pathway documented to target a specific functional gene category. Poly(U) tail addition may direct transcript cleavage and allow photosynthesis gene transcripts to accumulate to high levels. The loss of this pathway from ancestors of apicomplexans may have contributed to their transition from photosynthesis to parasitism.

Euglena gracilis is a species of unicellular photosynthetic flagellate that inhibits aquatic ecosystems. E. gracilis belongs to the supergroup Excavata, and are an important component of the global biosphere, have biotechnological potential and is useful biological model due to their evolutionary history and complex biology. Whilst the evolutionary position of E. gracilis is now clear, their relationship with other protists such as Naegleria, Giardia, and Kinetoplastids, remains to be investigated in detail. Investigating and understanding the biology of this complex organism is a promising way to approach many evolutionary puzzles, including secondary endosymbiotic events and the evolution of parasitism, due to their relationship with Kinetoplastids. Here, I report a draft genome for E. gracilis, together with a high quality transcriptome and proteomic analysis. The estimated genome size is ~ 2 Gbp, with a GC content of ~ 50 % and a protein coding potential predicted at 36,526 Open Reading Frames (ORFs). Less than 25% of the genome is single copy sequence, indicating extensive repeat structure. There are evidences for large number of paralogs amongst specific gene families, indicating expansions and possible polyploidy as well as extensive sharing of genes with other non photosynthetic and photosynthetic eukaryotes: red and green algael genes, together with trypanosomes and other members of the excavates. Functional resolution into several of the biological systems indicates multiple similarities with the trypanosomatids in terms of orthology, paralogy, relatedness and complexity. Several biological systems such as nuclear architecture (e.g. chromosome segregation, nuclear pore complex, nuclear lamins), protein trafficking, translation, surface, consist of conserved and divergent components. For instance, several gene families likely associated with the cell surface and signal transduction possess very large numbers of lineage-specific paralogs, suggesting great flexibility in environmental monitoring and, together with divergent mechanisms for metabolic control, novel solutions to adaptation to extreme environments. I also demonstrate that the majority of control of protein expression levels is post-transcriptional and absence of transcriptional regulation, despite the presence of conventional introns. These data are a major advance in the understanding of the nuclear genome of Euglenids and provide a platform for investigation of the contributions of E. gracilis and relatives to the biosphere.

Submitted by Jordan (jordanbiblio@gmail.com) on 2017-04-04T16:50:18Z No. of bitstreams: 1 DISS_2015_Gracieli da Silva Henicka.pdf: 44621169 bytes, checksum: 0564cd576f5da3c9d3914ef63b1ae491 (MD5) / Approved for entry into archive by Jordan (jordanbiblio@gmail.com) on 2017-04-17T16:08:59Z (GMT) No. of bitstreams: 1 DISS_2015_Gracieli da Silva Henicka.pdf: 44621169 bytes, checksum: 0564cd576f5da3c9d3914ef63b1ae491 (MD5) / Made available in DSpace on 2017-04-17T16:08:59Z (GMT). No. of bitstreams: 1 DISS_2015_Gracieli da Silva Henicka.pdf: 44621169 bytes, checksum: 0564cd576f5da3c9d3914ef63b1ae491 (MD5) Previous issue date: 2015-08-24 / Pesquisas apontam que é viável a inserção de conceitos relacionados à Teoria da Complexidade (TC) no ensino médio. O objetivo geral desse estudo foi averiguar a viabilidade e as dificuldades no processo de ensino-aprendizagem dos conceitos e princípios da Teoria da Endossimbiose Sequencial (TES) a luz da TC a partir de um produto educacional fundamentado em princípios da Teoria da Aprendizagem Significativa. O trabalho está fundamentado também na epistemologia de Kuhn, Maturana e Varela. Essa pesquisa investigou os conhecimentos prévios dos estudantes do segundo ano do ensino médio da Educação de Jovens e Adultos (EJA) acerca de conceitos de Ecologia, Evolução, Biologia Geral e Genética. Conceitos estes que apresentaram potencialidade para ancorar os novos conceitos relacionados à TES de Lynn Margulis, que foram abordados em seguida. Ao final da pesquisa produziu-se a título de produto educacional, um guia para o professor com orientações e sugestões de aprofundamento do tema, contendo ainda três apresentações em pptx, três textos-apoio para o professor, três textos-apoio para o aluno e sete testes. O material será disponibilizado no site do programa (http://fisica.ufmt.br/pgecn/) e da pesquisadora (www.alegriaeciencia.com.br). A metodologia foi quali-quanti com intervenção. A pesquisa aconteceu em Alta Floresta, MT e foi organizada em cinco encontros entre 19 de março de 2014 e 16 de abril de 2014. Participaram do estudo 94 alunos da EJA, sendo 37 do 2° ano noturno, 32 do 2° ano matutino e 27 do 2° ano vespertino. O 2° ano matutino apresentou em média, excluindo a entrevista, 66,51% de frequência nas atividades da pesquisa, o 2° ano vespertino 64,55% e o 2° ano noturno apenas 44,40%. A análise de todas as turmas juntas mostrou 57,44% de frequência média, o que sugere que os alunos faltaram muito aos encontros, especialmente os alunos do 2° ano noturno. Somente 77 alunos responderam ao pré-teste A onde se investigou o perfil dos alunos. Desses 63,64% são mulheres e 36,36% são homens. Ao analisar as turmas do dia esse cenário se repete, mas não é o caso da turma da noite, onde a maioria são homens na faixa etária de 18 a 22 anos, enquanto a faixa etária dos alunos do dia é mais diversificada, com a presença de alunos de até 62 anos. No tocante às aspirações pessoais a maioria dos alunos em todas as turmas demonstrou intenção em concluir o ensino médio e dar continuidade em sua formação, fazendo cursos técnicos e/ou faculdade. De maneira geral, os resultados das oficinas apontam algumas dificuldades de leitura e interpretação dos alunos o que dificultou o aprendizado, mas também apontam avanços na evolução da aprendizagem dos mesmos, há bons indícios de captação de significados conceituais que podem ser considerados precursores de aprendizagem significativa para alguns conceitos. Esse estudo demonstrou que é viável de inserção da Teoria da Endossimbiose Sequencial a luz da Teoria da Complexidade no ensino médio da EJA. / Researches show that the inclusion of concepts related to Complexity Theory (CT) is feasible in high school. The overall objective of this study was to investigate the feasibility and difficulties in the teaching-learning process of the concepts and principles of the Theory of Sequential Endosymbiosis (TSE) at the light of CT deriving from an educational product reasoned in the principles of the Theory of Meaningful Learning. The work is based also on Kuhn’s, Maturana’s and Varela’s epistemology. This research investigated the previous knowledge of students of the second year of high school of the Youth and Adult Education (EJA) about the concepts ecology of evolution, General Biology and Genetics. Such Concepts showed potential to anchor the new concepts related to TES by Lynn Margulis, which were covered below. At the end of the survey a teacher's guide with directions and issue of deepening suggestions has been created by way of educational product, still containing three presentations in PPTX, three texts-support for the teacher, three texts-support for the student and seven tests. The material will be available on the program website (http://fisica.ufmt.br/pgecn/) and researcher (www.alegriaeciencia.com.br). The methodology was qualitative and quantitative with participant observation. The research took place in Alta Floresta, MT and was organized in five meetings between March 19th 2014 and April 16th 2014. The participants were 94 students of EJA, 37 of the 2nd year from night shift, 32 of the 2nd year from morning shift and 27 the 2nd year from the afternoon shift. The morning shift group showed on average, excluding the interview, 66.51% attendance in the activities of research, the evening shift group 64.55% and the night shift group only 44.40%. The analysis of all the groups together showed 57.44% average rate, suggesting that students skipped many of the meetings, especially the students of the night shift. Only 77 students answered the pretest A which investigated the profile of the students. Of these, 63.64% are women and 36.36% are men. In analyzing the courses of the daytime, this scenario is repeated, but it is not the case of the night shift group, where most are men aged 18-22 years old, while the age group of students of the daytime is more diverse, with the presence students up to 62 years old. Regarding personal aspirations most students in each class demonstrated intention to complete high school and continue on their training, making technical courses and/or college. In general, the results from the workshops pointed to some difficulties in reading and interpreting of students hampering the learning, but also point advances in the evolution of learning, there was a good evidence of uptake of conceptual meanings that can be considered significant precursors to some e-learning concepts. This study demonstrated that it is feasible to insert Theory of Sequential Endosymbiosis at the light of Complexity Theory in high school of EJA.

La présence de Wolbachia dans les ovogonies assure la transmission verticale de la bactérie à la descendance de l'hôte. Cependant, nous montrons que chez l'hôte Armadillidium vulgare, l'efficacité de l'infection des descendants tient à un enrichissement en Wolbachia au cours de la maturation des ovaires et des ovocytes dû à une sélection en faveur des ovocytes infectés et/ou à l'entrée secondaire de Wolbachia dans les ovocytes en cours de maturation via l'infection des tissus somatiques. Dans ces tissus, nous avons précisé la localisation de Wolbachia au niveau cellulaire et révélé des morphotypes typiques de chaque tissu. Nous avons également observé Wolbachia chez des hôtes très inattendus; des nématodes non filaires infectant les cloportes, posant la question d'une transmission horizontale, et les A. vulgare mâles, sans qu'ils soient féminisés. Etonnamment, nous avons observé l'infection des gonades mâles dans des lignées d'hôtes chez lesquelles les femelles sont infectées de manière cryptique mais sans que leurs ovocytes ne soient infectés. Le maintien de l'infection entre les générations d'hôtes pourrait alors être dû à une transmission paternelle, inédite pour Wolbachia, ou à une capacité de transmission horizontale très efficace de la bactérie. Par immersion de tissus directement dans des broyats d'organes infectés nous avons en effet démontré que Wolbachia infecte très rapidement des cellules de novo. Les mécanismes d'entrée de Wolbachia dans les cellules sont inconnus mais en monitorant des voies métaboliques clefs de l'hôte nos résultats montrent que l'infection entraine une réponse globale des tissus et implique notamment un détournement de la voie autophagique chez l'hôte. / Wolbachia presence in oogonia ensures bacteria to be vertically transmitted to host offspring. However, in Armadillidium vulgare, we show that the proportion of infected oocytes increases in the course of both ovary and oocyte maturation to reach the transmission rate at the end of ovary maturation. This enrichment can be explained by a preferential selection of oocytes infected with Wolbachia and/or by a secondary acquisition of the bacteria by oocytes. We suspect an acquisition through infected somatic tissues. We localize Wolbachia at the cell level in these tissues and showed particular morphotypes for each tissue. We also observe Wolbachia in unexpected hosts; non filarial nematodes infecting woodlice (suggesting horizontal transmission), and in A. vulgare males (without a feminizing effect of the bacteria). We also observe lineages in which females are cryptically infected. Surprisingly, we observe infected male gonads in these lineages for which female oocytes are uninfected. The infection maintenance across host generations could be due to a paternal transmission of the bacteria (a transmission never described for Wolbachia), or due to an astonishing ability of horizontal transmission. Nevertheless, immersion of uninfected tissues in a solution of crushed infected tissues proves that Wolbachia can quickly infect new tissues. Cellular mechanisms that allow Wolbachia internalization into the cell are still unknown. Thus, we monitor key host metabolic pathways in ovaries and we denote that infection enhances a global response of the entire tissue. Additionally, Wolbachia infection especially implicates a high-jacking of the autophagic pathway.

La symbiose est l'un des principaux moteurs de l'évolution. Le génotype du symbiote est capable d'altérer le phénotype de l'hôte, et vice-versa : c'est le « phénotype étendu ». Dans ce contexte, les endosymbioses à Wolbachia sont remarquables. Cette bactérie intracellulaire est un parasite de la reproduction capable d'induire la féminisation des mâles génétiques ou l'incompatibilité cytoplasmique chez ses hôtes crustacés isopodes terrestres. Actuellement, aucun mécanisme moléculaire régissant ces effets n'est connu. Dans le but d'identifier des gènes impliqués dans la féminisation, nous avons utilisé une approche intégrative qui combine à la fois des analyses génomiques, d'expression de gènes et phénotypiques. Nous avons tout d'abord analysé l'évolution moléculaire de la voie de la recombinaison homologue dans les génomes de Wolbachia, source importante de plasticité génomique pouvant être liée à la diversité des phénotypes. Ensuite, afin d'effectuer des études comparatives qui augmenteraient considérablement la compréhension des mécanismes de la féminisation, nous avons établi un système où la souche féminisante wVulC féminise deux hôtes isopodes (hôte naturel : Armadillidum vulgare : hôte hétérologue : Cylisticus convexus) présentant un timing différent de la différenciation sexuelle. En effet, l'effet féminisant étant supposé avoir lieu avant ou pendant la différenciation sexuelle, il est important de distinguer l'effet de Wolbachia dû à la différenciation sexuelle de celui dû au développement. Enfin, une approche par gènes candidats (du séquençage de génome bactérien à l'analyse comparative d'expression de gènes bactériens durant le développement de l'hôte) a permis de déterminer une liste réduite de 29 gènes (parmi les 1885 gènes de wVulC) dont la probabilité qu'ils soient impliqués dans la féminisation est élevée. Le rôle potentiel de ces gènes candidats comme effecteurs supposés de la féminisation induite par wVulC est ensuite discuté. Ce travail contribue grandement à l'identification de facteurs potentiels d'endosymbiotes qui ont un impact évolutif sur la détermination du sexe de leurs hôtes. / Symbiotic interactions are a major driver of evolution. The symbiont genotype is able to alter the host phenotype, and the other way round: it is called "the extended phenotype". In this respect, Wolbachia endosymbiosis is remarkable. This intracellular bacterium is a well-known reproductive parasite able to induce feminization of genetic males or cytoplasmic incompatibility in its terrestrial isopod crustacean hosts. Currently, no molecular genetic basis of these reproductive manipulations has been described. In order to identify genes involved in feminization, we used an integrative approach that combines genomic, gene expression and phenotypic studies. We first analysed the molecular evolution of the homologous recombination pathway in Wolbachia genomes, an important source of genomic plasticity that can be linked with phenotypic diversity. Then, in order to perform comparative studies that will substantially improve the understanding of the molecular mechanisms of feminization, we established a system in which the feminizing strain wVulC feminizes two different isopod hosts (natural host: Armadillidium vulgare ; heterologous host Cylisticus convexus) that have a different sexual differentiation timing. Indeed, as feminization is thought to happen before or during sexual differentiation, it is important to distinguish the effect of Wolbachia due to sexual differentiation from that due to development. Finally, a gene candidate approach (from bacterial genome sequencing to comparative bacterial gene expression during host developement) allowed us to determine a reduced list of 29 genes (among the 1885 genes of wVulC) that have a high probability to be involved in feminization. The potential roles of these candidate genes as putative effectors of feminization induced by wVulC is then discussed. This work substantially contributes to the identification of putative endosymbiont factors that have an evolutionary impact on sex determination of their hosts.

Les Wolbachia sont des α-Protéobactéries endocellulaires transmises maternellement et qui manipulent la reproduction des Arthropodes pour augmenter leur transmission. Chez le moustique Culex pipiens, Wolbachia induit l'incompatibilité cytoplasmique (IC) qui se traduit par une forte mortalité embryonnaire lors de croisements entre individus infectés par des souches incompatibles de Wolbachia. Ce moustique se caractérise par une forte diversité génétique de ses Wolbachia (nommées wPip) et par des patrons d'IC complexes. Nous avons examiné les mécanismes qui façonnent la dynamique de cette association symbiotique aux niveaux génomique, phénotypique et populationnel. Nous avons montré que les souches wPip ont une origine génétique commune récente et qu'elles s'organisent en groupes génétiques présentant une structuration géographique. Nous avons mis en évidence des évènements de recombinaison entre souches wPip qui pourraient jouer un rôle majeur dans la diversité génétique des Wolbachia et dans l'évolution rapide des patrons d'IC. En croisant des lignées de moustiques d'origines géographiques diverses et infectées par des souches de différents groupes génétiques, nous avons montré que les IC (i) évoluent très rapidement chez Cx. pipiens; (ii) sont contrôlées par plusieurs déterminants génétiques, et (iii) qu'il y a une relation entre les patrons d'IC et les groupes génétiques des Wolbachia. Dans les populations naturelles, il apparaît que les IC sont contre sélectionnées au sein d'une population mais qu'une zone de contact entre populations infectées par des souches incompatibles peut se maintenir de façon stable. / Wolbachia are maternally inherited endocellular α-Proteobacteria that manipulate the reproduction of Arthropods to promote their own transmission. In the mosquito Culex pipiens, Wolbachia induce cytoplasmic incompatibility (CI) which results in high embryonic mortality in crosses between mosquitoes infected with incompatible Wolbachia strains. This mosquito is characterized by high genetic diversity of its Wolbachia (referred as wPip strains) and by complex CI patterns. We examined mechanisms that shape the dynamics of this symbiotic association at genomic, phenotypic and field population levels to understand how it evolves. We showed that wPip strains have a unique and recent evolutionary origin and that their diversity clusters into distinct genetic groups with a geographic structure. We revealed the existence of extensive recombinations among wPip strains, which could influence their adaptive dynamics by creating new wPip strains and thus allow the rapid emergence of new CI patterns. The analysis of crossing relationships between mosquito lines from different geographic origins and infected with wPip strains belonging to different genetic groups showed that CIs (i) evolve rapidly in Cx. pipiens; (ii) are controlled by several genetic factors, and (iii) there is a significant relationship between CI patterns and genetic divergence of wPip strains. In field populations, it appears that CIs are selected against within a population but a contact zone between populations infected by incompatible Wolbachia strains can be stably maintained.

Actinorhizal plants are in symbiosis with the nitrogen-fixating soil bacterium Frankia, which forms nodules in the plant root. However, several studies also report other endophytic bacteria appearing in the nodules, but their function and interaction with the host plant or Frankia is not yet understood. This thesis used a bioinformatic approach to investigate the metagenomes of eighteen actinorhizal nodule samples to find out which bacteria are present, how the microbiomes differed from each other, and if the genomes of non-Frankia inhabitants could give indications of any functions. The results showed that the bacterial composition, richness, and diversity differed among the samples, especially between the samples sequenced from the field versus those primarily cultivated in a greenhouse. All samples had a substantial number of sequencing reads belonging to potential endophytes, such as strains of Enterobacteria, Pseudomonas, Streptomyces, Micromonospora, Mycobacteria and Pseudonocardia. There seemed to be a common microbial community shared among the plants on a family level, since no significant difference was found in the core microbiomes between the field and greenhouse groups. Some sequences found in the metagenomes were annotated as potential functions of the fellow travellers, such as antibiotic synthesis, proteins involved in regulating abiotic stresses, but also probable plant damaging compounds rather associated with pathogens than symbionts.