Despite accounting for approximately half of global primary productivity, photosynthesis in aquatic environments is often limited by the availability of dissolved carbon dioxide (CO$_{2(aq)}$). To overcome the slow diffusion of CO$_{2(aq)}$ as well as kinetic limitations of the primary photosynthetic carboxylase, Rubisco, carbon concentrating mechanisms (CCMs) have evolved in many aquatic photosynthetic organisms to improve photosynthetic efficiency and growth in CO2- limited environments. In the model eukaryotic green alga Chlamydomonas reinhardtii, the CCM is induced under low CO2 in the light and comprises: active inorganic carbon transport systems, carbonic anhydrases and the localisation of Rubisco to a central chloroplast microcompartment called the pyrenoid. In addition to changes in gene expression, acclimation to low CO2 is accompanied by alterations to metabolism, physiology and even cellular ultrastructure. However, mechanisms governing the regulation and interaction of these molecular components to increase CCM activity remain poorly understood. The overall aim of this study was to investigate regulation of the CCM in wild-type and mutant Chlamydomonas strains at both the whole cell and molecular level. Firstly, investigation of CCM induction in synchronised cultures of wild-type cells identified changes in CCM activity that were uncoupled from accumulation of CCM-related mRNA and protein, contrasting with the coordinated response to low CO2 observed in asynchronous control cultures. Pre-dawn induction of the CCM was coincident with preferential localisation of Rubisco and a thylakoid-lumenal carbonic anhydrase (CAH3) to the pyrenoid, highlighting the possible role of endogenous signals and post-translational modifications in modulating CCM activity. Secondly, in order to probe the relationship between pyrenoid formation and CCM induction and activity, CCM expression was investigated in pyrenoid-negative mutants with substituted Rubisco small subunits (RBCS). Low CO2-adapted pyrenoid-less RBCS mutants had impaired growth and low photosynthetic affinity for inorganic carbon (Ci). These pyrenoid-negative strains also showed a specific reduction in the accumulation of several CCM mRNAs, compared to pyrenoid- positive wild-type. Two-dimensional difference in gel electrophoresis (2D-DIGE) was used to compare the soluble proteome of one low CO2-adapted pyrenoid-less RBCS mutant compared to the pyrenoid-positive wild-type. This analysis identified only a few differentially expressed proteins, none of which were directly involved in CCM activity. Two primary metabolic enzymes were more abundant in the wild- type while eight proteins associated with protein synthesis and photosynthesis were more abundant in the pyrenoid-less mutant, suggesting that pyrenoid loss is accompanied by global metabolic, as well as CCM-specific, changes. A shotgun proteomics approach (LC-MS/MS) was used to extend the analysis of the pyrenoid-less RBCS mutant proteome to the whole genome level. Approximately 10% of the total proteins detected using this method were identified as differentially expressed between pyrenoid-negative and pyrenoid-positive strains. Increased abundance of photosynthetic proteins was found in the pyrenoid-less RBCS mutant, confirming the results of 2D-DIGE. In contrast, increased accumulation of CCM and primary metabolic enzymes was detected in the pyrenoid-positive wild-type. Overall, detailed investigation of the phenotype of pyrenoid-negative RBCS mutants indicates that pyrenoid loss leads to impaired induction of the CCM as well as altered metabolism under low CO2 conditions, perhaps as a result of decreased carbon fixation. The results of these studies are explored in the context of the identification of additional CCM components and regulatory mechanisms as well as possible connections between Rubisco aggregation and CCM activity.

No description available.

A biogênese do ribossomo é um processo complexo, altamente ordenado e regulado, no qual o transcrito primário é processado por endo e exonucleases para gerar os RNAs ribossomais maduros. Este processo foi melhor caracterizado em Saccharomyces cerevisiae, porém alguns fatores atuantes em humanos tiveram uma função divergente descrita. Um desses fatores é a proteína NIP7, altamente conservada em eucariotos, que atua na formação da subunidade ribossomal 60S, em levedura, e 40S, em humanos. Assim, esse trabalho propôs a caracterização funcional da proteína CrNIP7, homóloga a NIP7, presente em Chlamydomonas reinhardtii. C. reinhardtii é uma alga verde unicelular ancestral a plantas, utilizada como modelo eucarioto para estudos de fotossíntese e de flagelos. Nesse trabalho, um estudo de complementação funcional foi realizado utilizando duas linhagens de Saccharomyces cerevisiae diferentes e em ambas CrNIP7 complementou a função de Nip7p de leveduras, indicando uma participação na síntese da subunidade 60S do ribossomo. Uma busca por parceiros de interação de CrNIP7 foi também realizada, utilizando CrNIP7 como isca para rastrear uma biblioteca de cDNA de C. reinhardtii em sistema de duplo híbrido em leveduras, o que resultou em dois novos potenciais parceiros de interação. Esses parceiros foram identificados como proteínas preditas conceitualmente no genoma de C. reinhardtii, denominadas Predicted e G-patch. Adicionalmente, a interação entre CrNIP7 e CrSBDS, proteína homóloga a Sdo1 (de levedura) e HsSBDS (de humanos), foi confirmada através de um experimento de duplo híbrido dirigido. A interação entre as proteínas CrNIP7 e CrSBDS foi validada por pull down e um teste preliminar sugeriu que CrNIP7 e Predicted também interagem in vitro. Análises de bioinformática indicam que Predicted, G-patch e CrSBDS tenham regiões intrinsicamente desordenadas, as quais podem se estruturar na interação com seus parceiros. Em conjunto, os resultados desse trabalho contribuem para entendimento do papel de CrNIP7 na biogênese de ribossomos em Chlamydomonas reinhardtii em comparação com outros modelos eucarióticos. / Ribosome biogenesis is a complex, highly regulated and ordered process in which the primary transcript is processed by endo- and exonucleases to generate the mature ribosomal RNAs. This process was best characterized in Saccharomyces cerevisiae, but some factors have been described in humans with different function. One of these divergent factors is NIP7, a highly conserved protein in eukaryotes, which acts in the formation of ribosomal 60S subunit, in yeast, and 40S, in humans. Based on this, this work proposed the functional characterization of CrNIP7 protein, homologous to NIP7, from Chlamydomonas reinhardtii. C. reinhardtii is a green alga, ancestral to plants, that is used as an eukaryote model for photosynthesis and flagella studies. In this study, a functional complementation assay was performed using two different strains of Saccharomyces cerevisiae and, in both approaches, CrNIP7 protein complemented the function of Nip7p from yeast, indicating its participation in the synthesis of the 60S ribosomal subunit. A two-hybrid assay was carried out using CrNIP7 as bait to screen a C. reinhardtii cDNA library in order to find out CrNIP7 interaction partners, wich resulted in two novel potentially partners. The interacting proteins were identified as conceptually predicted proteins in the genome of C. reinhardtii and were called Predicted and G-patch. Additionally, the interaction between CrNIP7 and CrSBDS, a protein homologous to Sdo1 (yeast) and HsSBDS (humans), was confirmed by a direct two-hybrid assay. The interaction between CrNIP7 and CrSBDS proteins was validated by pull down and a preliminary test suggested that CrNIP7 and Predicted also interact in vitro. Bioinformatics analyzes indicate that Predicted, G-patch and CrSBDS have intrinsically disordered regions, which can be ordered in the moment of interaction. Taken together, the results of this work contribute to understand the role played by CrNIP7 in ribosome biogenesis in Chlamydomonas reinhardtii compared to other eukaryotic models.

No description available.

It is generally accepted that global levels of CO2 will roughly double over the next century. Because of their large population sizes and fast generation times, microalgae may adapt to global change through novel mutations fixed by natural selection, such that future populations may be genetically different from contemporary ones. The prediction that microalgae may respond evolutionarily to rising CO2 was tested using populations of Chlamydomonas reinhardtii grown for 1000 generations at increasing CO2. Laboratory populations grown at high CO2 did not show a direct response to selection at elevated CO2, instead evolving a range of non-adaptive syndromes. In addition, populations selected at elevated CO2 often grew poorly at ambient CO2. The same evolutionary responses were seen in natural populations isolated from CO2 springs. CO2 uptake was measured in a subset of the laboratory selection lines, which were found to have cells that either leaked CO2, had lost the ability to induce high-affinity CO 2 uptake, or both. These phenotypes were tentatively attributed to the accumulation of conditionally neutral mutations in genes involved in the carbon concentrating mechanism (CCM). The high-CO2-selected phenotypes were found to be reversible in terms of fitness when populations were backselected in air, though wild-type regulation of the CCM was not regained. It has been suggested that phytoplankton adaptation to changes in CO2 levels is constrained by selective history. This was tested by culturing genetically distinct populations of Chlamydomonas at decreasing levels of CO2. In this case, divergence between lines was attributable to chance rather than selective history.

A biogênese do ribossomo é um processo complexo, altamente ordenado e regulado, no qual o transcrito primário é processado por endo e exonucleases para gerar os RNAs ribossomais maduros. Este processo foi melhor caracterizado em Saccharomyces cerevisiae, porém alguns fatores atuantes em humanos tiveram uma função divergente descrita. Um desses fatores é a proteína NIP7, altamente conservada em eucariotos, que atua na formação da subunidade ribossomal 60S, em levedura, e 40S, em humanos. Assim, esse trabalho propôs a caracterização funcional da proteína CrNIP7, homóloga a NIP7, presente em Chlamydomonas reinhardtii. C. reinhardtii é uma alga verde unicelular ancestral a plantas, utilizada como modelo eucarioto para estudos de fotossíntese e de flagelos. Nesse trabalho, um estudo de complementação funcional foi realizado utilizando duas linhagens de Saccharomyces cerevisiae diferentes e em ambas CrNIP7 complementou a função de Nip7p de leveduras, indicando uma participação na síntese da subunidade 60S do ribossomo. Uma busca por parceiros de interação de CrNIP7 foi também realizada, utilizando CrNIP7 como isca para rastrear uma biblioteca de cDNA de C. reinhardtii em sistema de duplo híbrido em leveduras, o que resultou em dois novos potenciais parceiros de interação. Esses parceiros foram identificados como proteínas preditas conceitualmente no genoma de C. reinhardtii, denominadas Predicted e G-patch. Adicionalmente, a interação entre CrNIP7 e CrSBDS, proteína homóloga a Sdo1 (de levedura) e HsSBDS (de humanos), foi confirmada através de um experimento de duplo híbrido dirigido. A interação entre as proteínas CrNIP7 e CrSBDS foi validada por pull down e um teste preliminar sugeriu que CrNIP7 e Predicted também interagem in vitro. Análises de bioinformática indicam que Predicted, G-patch e CrSBDS tenham regiões intrinsicamente desordenadas, as quais podem se estruturar na interação com seus parceiros. Em conjunto, os resultados desse trabalho contribuem para entendimento do papel de CrNIP7 na biogênese de ribossomos em Chlamydomonas reinhardtii em comparação com outros modelos eucarióticos. / Ribosome biogenesis is a complex, highly regulated and ordered process in which the primary transcript is processed by endo- and exonucleases to generate the mature ribosomal RNAs. This process was best characterized in Saccharomyces cerevisiae, but some factors have been described in humans with different function. One of these divergent factors is NIP7, a highly conserved protein in eukaryotes, which acts in the formation of ribosomal 60S subunit, in yeast, and 40S, in humans. Based on this, this work proposed the functional characterization of CrNIP7 protein, homologous to NIP7, from Chlamydomonas reinhardtii. C. reinhardtii is a green alga, ancestral to plants, that is used as an eukaryote model for photosynthesis and flagella studies. In this study, a functional complementation assay was performed using two different strains of Saccharomyces cerevisiae and, in both approaches, CrNIP7 protein complemented the function of Nip7p from yeast, indicating its participation in the synthesis of the 60S ribosomal subunit. A two-hybrid assay was carried out using CrNIP7 as bait to screen a C. reinhardtii cDNA library in order to find out CrNIP7 interaction partners, wich resulted in two novel potentially partners. The interacting proteins were identified as conceptually predicted proteins in the genome of C. reinhardtii and were called Predicted and G-patch. Additionally, the interaction between CrNIP7 and CrSBDS, a protein homologous to Sdo1 (yeast) and HsSBDS (humans), was confirmed by a direct two-hybrid assay. The interaction between CrNIP7 and CrSBDS proteins was validated by pull down and a preliminary test suggested that CrNIP7 and Predicted also interact in vitro. Bioinformatics analyzes indicate that Predicted, G-patch and CrSBDS have intrinsically disordered regions, which can be ordered in the moment of interaction. Taken together, the results of this work contribute to understand the role played by CrNIP7 in ribosome biogenesis in Chlamydomonas reinhardtii compared to other eukaryotic models.

published_or_final_version / abstract / Botany / Master / Master of Philosophy

Considerada a mais comum das doenças lisossômicas, a doença de Gaucher é causada por mutações no gene GBA1 que resultam na síntese defeituosa da enzima glicocerebrosidase (GBA), responsável pela hidrólise dos glicocerebrosídios em glicose e ceramida. A deficiência da enzima provoca o acúmulo desses glicolipídios nos macrófagos, principalmente no fígado, no baço e na medula óssea, levando a um fenótipo complexo. O tratamento da doença consiste na administração da enzima GBA humana (HsGBA) exógena e, apesar de sua eficácia, é extremamente oneroso. Assim como outras espécies de microalgas, Chlamydomonas reinhardtii apresenta alto potencial para a produção de grandes quantidades de proteínas recombinantes de forma rápida e a um custo muito inferior ao dos sistemas de expressão tradicionais. O objetivo proposto para o desenvolvimento deste trabalho foi a expressão do gene codificador da HsGBA a partir do genoma nuclear de C. reinhardtii visando à produção da proteína como possível alternativa para a terapia de reposição enzimática. O gene HsGBA artificial foi projetado com códons adaptados para a expressão nuclear em C. reinhardtii e com sítios de restrição. A sequência nucleotídica foi sintetizada pela empresa GenScript USA Inc. e, após seu recebimento em plasmídeo de clonagem, o gene HsGBA foi inserido no plasmídeo pHsp70A/RbcS2-cgLuc por restrição com endonucleases e ligação. Esse vetor foi combinado com o plasmídeo pKS-aph7``-lox para a transferência do cassete de expressão do gene de interesse por recombinação sítio específica mediada pelo sistema Cre/lox, originando o vetor pKS-aph7``-lox::HsGBA, que contém, assim, os cassetes de expressão em tandem para HsGBA e para o marcador de seleção aph7``, codificador da enzima aminoglicosídeo fosfotransferase e capaz de conferir resistência à higromicina B. A linhagem de C. reinhardtii CC-400 cw15 mt+ foi transformada por eletroporação com o plasmídeo resultante linearizado (clivado com EcoRV) e na forma circular. A integração de HsGBA no genoma de cinco linhagens de C. reinhardtii foi comprovada por PCR e sua expressão foi demonstrada em três dessas linhagens de forma qualitativa por RT-PCR. Como HsGBA é controlado por um promotor induzível, hsp70A, diversas condições foram testadas visando à sua máxima expressão. Entretanto, análises por SDS-PAGE e western blot não permitiram a detecção da proteína recombinante. De modo semelhante, a atividade enzimática da HsGBA avaliada em extratos proteicos de linhagens transformadas não foi diferente da observada para linhagens não transformadas de C. reinhardtii. / Considered the most common lysosomal disorder, Gaucher disease is caused by mutations in GBA1 gene that result in defective synthesis of the enzyme glucocerebrosidase (GBA), responsible for the hydrolysis of glucocerebrosides into glucose and ceramide. When the enzyme is defective, these glycolipids accumulate in the macrophages, mainly in the liver, spleen and bone marrow, leading to a complex phenotype. Current treatment consists of enzyme replacement therapy by the administration of exogenous human GBA (HsGBA) and, in spite of its efficacy, it is exceptionally expensive. As other species of microalgae, Chlamydomonas reinhardtii has a high potential for production of large amounts of recombinant proteins rapidly and at a much lower cost than traditional expression systems. In the present work we proposed the expression of the HsGBA gene from the nuclear genome of C. reinhardtii aiming the production of the protein as a possible alternative to enzyme replacement therapy. The artificial HsGBA gene, adapted to the nuclear codon usage of C. reinhardtii, was designed with restriction sites and synthesized by GenScript USA Inc. The nucleotide sequence was provided in a cloning vector and the HsGBA gene was inserted into the plasmid pHsp70A/RbcS2-cgLuc by endonuclease digestion and ligation. The resulting vector was fused to the plasmid pKS-aph7``-lox for transferring of the expression cassette of the gene of interest by site-specific recombination mediated by the Cre/lox system, yielding the plasmid pKS-aph7``-lox::HsGBA. As a result, this tandem vector has the expression cassettes for HsGBA and for the selection marker aph7``, which encodes the aminoglycoside phosphotransferase enzyme that confers resistance to hygromycin B. The C. reinhardtii strain CC-400 cw15 mt+ was transformed by electroporation with the resulting plasmid, in the supercoiled and linear (digested with EcoRV) forms. The HsGBA integration into the genome of five strains of C. reinhardtii was confirmed by PCR and their expression was demonstrated qualitatively in three of these strains by RT-PCR. As the HsGBA gene is under the control of the inducible promoter hsp70A, several conditions were tested aiming its higher expression. However, the protein could not be detected by SDS-PAGE and western blot. Likewise, the enzymatic activity of the HsGBA was measured in protein extracts of the transgenic strains but did not differ from the control.

No description available.

Heterothallic, facultatively sexual populations are vulnerable to the loss of a mating type by natural selection during periods of asexual reproduction. Experiments are described which demonstrate a competitive difference between the mating types of Chlamydomonas reinhardtii, a unicellular green alga with two isogamous mating types, mt + and mt $-.$ When grown vegetatively under phototrophic (light) conditions, mt + outcompetes mt $-.$ Assays of the growth parameters of isolated spores suggest that mt + has a higher growth rate than mt $-$ in the light, and that mt $-$ has a higher growth rate than mt + in heterotrophic (dark) growth conditions. / A literature review shows that sampling from natural populations of heterothallic, facultatively sexual species often yields only one mating type or significantly skewed mating-type distributions. This indicates that competition between mating types and the consequent loss of one mating type may be common in these populations. / A discussion of current theories on the evolution of heterothallism as well as the results of a simulation model reveal that heterothallism will spread if any fitness reduction is suffered by in-crossing homothallic individuals. However, fitness differences between the heterothallic alleles allow the invasion of a homothallic allele into a heterothallic population. / The implications of mating type competition on the maintenance and distribution of heterothallic populations in nature are discussed. It is argued that heterothallic, facultatively sexual populations commonly lose the potential for sex because of the loss of one mating-type allele. The prediction is made that homothallism is more common among facultatively sexual organisms than it is among obligately sexual organisms.