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This thesis describes the dynamics of mitochondrial RNA-binding protein 1 and 2 (MRP1/2) complex in different cell lines of Trypanosoma brucei under an optimized immobilized condition. This study reveals the influence of RNA on the complex's dynamics. Furthermore, the function of RNA-binding complex 1 (MRB1) core has been studied via reverse genetic, biochemical and molecular techniques, with its role in RNA editing being proposed.

In last decade, investigations of mitochondria including their various reduced forms such as hydrogenosomes and mitosomes revealed unexpected diversity of this indispensable organelle. Interestingly, the single mitochondrion of parasitic protist Trypanosoma brucei is able to undergo remarkable functional and structural changes reflecting available carbon sources. Moreover, it was proposed that trypanosomes belong among the most ancient eukaryotes and as such, their mitochondria raised high attention of biologists. To contribute to the knowledge of mitochondrial biogenesis and function, we focused on studies of two key mitochondrial processes, the processing of preproteins that are imported to the mitochondria, and mechanism of pyruvate transport to these organelles. Moreover, we also investigated uptake of iron by T. brucei. This metal is essential for function of numerous proteins, particularly for iron-sulfur proteins in mitochondria. Evolutionary history of trypanosomes and their mitochondrion is a question of debates. According to some reports, mitochondrion of trypanosomes represent an ancient form of this organelle, which is supported by identification of putative "archaic" translocase of the outer mitochondrial membrane (ATOM) and finding of only a single type of translocation pore in...

Ultrastructural observations reveal that the spermatozoids of the hornworts Notothylas and Phaeoceros contain two mitochondria and not one as described previously. Mitochondrial ontogeny and nuclear metamorphosis during spermiogenesis in these plants differ from all other archegoniates. The discovery that the posterior region of the coiled nucleus (when viewed from the anterior aspect) lies to the left of the anterior, in striking contrast to the dextral coiling of the nucleus of spermatozoids of other embryophytes, underlines the isolated nature of the hornworts among land plants. As the blepharoplast develops, the numerous ovoid mitochondria initially present in the nascent spermatid fuse to form a single elongated organelle which is positioned subjacent to the MLS and extends down between the nucleus and plastid. At the onset of nuclear metamorphosis, the solitary mitochondrion has separated into a larger anterior mitochondrion (AM) associated with the MLS and a much smaller posterior mitochondrion (PM) adjacent to the plastid. The PM retains its association with the plastid and both organelles migrate around the periphery of the cell as the spline MTs elongate. By contrast, in moss spermatids, where mitochondria undergo similar fusion and division, the AM is approximately the same size as the PM and the latter is never associated with the spline. As in other archegoniates, except mosses, spline elongation precedes nuclear metamorphosis in hornworts. Irregular strands of condensed chromatin compact basipetally to produce an elongated cylindrical nucleus which is narrower in its mid-region. During this process excess nucleoplasm moves rearward. It eventually overarches the inner surface of the plastid and entirely covers the PM.

The mitochondrion contains its own genome. Replication of the mitochondrial DNA (mtDNA) is an essential process that, in most organisms, occurs through the cell cycle with no known mechanism to ensure spatial or temporal constrain. Failures to maintain mtDNA copy number affects cellular functions causing several human disorders. However, it is not clear how the cells control the mtDNA copy number. The mtDNA of trypanosomes, known as kinetoplast DNA (kDNA), is a structurally complex network of topologically interlocked DNA molecules (minicircles and maxicircles). The replication mechanism of the kDNA differs greatly with all other eukaryotic systems. Key features of the kDNA replication mechanism include defined regions for main replication events, coordination of a large number of proteins to drive the replication process, and replication once per cell cycle in near synchrony with nuclear S phase. Two main regions known as the kinetoflagellar zone (KFZ) and the antipodal sites are where main kDNA replication events are known to occur (i.e, initiation, DNA synthesis and Okazaki fragment processing). So far, the localization of the proteins involved in kDNA replication is restricted to two main regions: the KFZ and the antipodal sites. Three mechanisms that directly regulate kDNA replication proteins and serve to control kDNA replication have been proposed: (1) Reduction and oxidation status of the universal minicircle sequence binding protein (UMSBP) controls its binding to the origin sequence, (2) Trans-acting factors regulate the stability of mRNA encoding mitochondrial Topoisomerase II during the cell cycle and, (3) Regulation of TbPIF2 helicase protein levels by a HslVU-like protease to control maxicircle copy number. These mechanisms seem to be protein specific and it appears that a combination rather than a single mechanism regulates kDNA replication. In this study we used Trypanosoma brucei to understand how mitochondrial DNA replication is controlled. We investigated the mechanism of how proteins transiently localize to the sites of DNA synthesis during cell cycle stages. Our data provides a comprehensive analysis of the first two examples of T. brucei kDNA replication proteins that have a cell cycle dependent localization (Ch. 2 and 3). The localization of two of the three essential mitochondrial DNA polymerases (TbPOLIC and TbPOLID) is under tight cell cycle control and not regulated by proteolysis. TbPOLIC and TbPOLID localize to the antipodal sites during kDNA S phase, however, at other cell cycle stages TbPOLIC becomes undetectable by immunofluorescent analysis and TbPOLID disperses through the mitochondrial matrix. In agreement with this data, TbPOLIC and TbPOLID replication complexes were not detected using affinity purification presumably because only a fraction of these proteins are participating in replication at a given time (Ch. 4). We propose that spatial and temporal changes in the dynamic localization of essential kDNA replication proteins provide a novel mechanism to control kDNA replication.

The majority of the mitochondrial proteins is synthetized on the cytosolic ribosomes in the form of the protein precursors bearing mitochondrion-targeting signal presequences. Once the protein precursor has reached the mitochondrial matrix the signal presequence is no longer necessary and is cleaved off by heterodimeric mitochondrial processing peptidase (MPP; α/β). Although the crystal structure of MPP is available, the MPP mechanism of function is still matter of discussion. An all atomic, non-restrained molecular dynamics (MD) simulation in explicit water was used to study in detail the structural features of the highly conserved glycine-rich loop (GRL) of the regulatory α-subunit of the yeast MPP. Wild-type and GRL-deleted MPP structures were studied both in the presence and absence of a substrate in the peptidase active site. Targeted MD simulations were employed to study the mechanism of substrate translocation from the GRL to the peptidase active site. We demonstrate that the natural conformational flexibility of the GRL is crucial for the substrate translocation process from outside the enzyme towards the MPP active site. We show that the α-helical conformation of the substrate is important not only during its initial interaction with MPP (i.e. substrate recognition), but also later, at...

Gracilariaceae is a worldwide distributed family that includes numerous economically important species. Currently, five genera are recognized in the Gracilariaceae: Gracilariophila (parasitic), Curdiea, Melanthalia, Gracilaria, and Gracilariopsis. Some species of Gracilaria were taxonomically transferred to Hydropuntia. However, this genus is quite controversial. High-throughput sequencing (HTS) techniques has led to an increase in studies using complete organellar genomes, which have been used to infer phylogenetic relationships in Rhodophyta and the investigation of other aspects of red algal genomes, including gene synteny and horizontal gene transfers (HGT). HTS also facilitated the search for extrachromosomal plasmids and its influence in the organellar genomes by HGT. We applied HTS to assemble and annotate organellar genomes (mitochondria and chloroplast) from seven species of Gracilariaceae using Illumina HiSeq 2500 platform. We also received raw reads of 31 samples of Gracilariaceae from Dr. Goia Lyra that were analysed and included in our work. We used these data, combined with published genomes, to infer phylogenies and compare the genome architecture of these species representing the main lineages in Gracilariaceae. The mitochondrial and chloroplast genomes were highly conserved in gene synteny among the species, and variation mainly occurred in regions where insertions of plasmid-derived sequences (PDSs) were found, which were similar to known red algae extrachromosomal plasmids. In mitochondrial genomes, the PDS insertions were in two regions where the transcription direction changes: between cob and trnL genes, and trnA and trnN genes. PDS insertions in chloroplast genome were in different positions, but generally found between psdD and rrs genes. The bacterial leuC/leuD operon was found in Gracilaria tenuistipitata, G. chilensis, M. intermedia chloroplasts genomes, and also in G. vermiculophylla extrachromosomal plasmid. Phylogenetic trees show two different origins of leuC/leuD: genes found in chloroplasts and plasmids were close to proteobacteria, and genes encoded in the nucleus are close to Viridiplantae and cyanobacteria. Gracilariaceae may be a good model to study the impact of PDS in genome evolution due to the frequent presence of these sequences inserted in organellar genomes. Our phylogenetic analyses demonstrated similar evolutionary histories between the chloroplast and mitochondrial genomes. However, chloroplast phylogeny was better resolved with full support. Our taxonomical sampling supports the presence of three main lineages: Melanthalia/Curdiea, Gracilariopsis and Gracilaria. Melanthalia intermedia was sister to a monophyletic clade including Gracilaria and Gracilariopsis, which were resolved as monophyletic genera. Furthermore, the characteristics of organellar genome architecture, Gracilariopsis and Gracilaria genera are also supported by the loss of the plastid gene petP in Gracilaria and the rearrangement position of the gene trnH in the mitochondrial genome. Beside this, we found no support for the genus Hydropuntia as originally proposed / A família Gracilariaceae está globalmente distribuída e inclui várias espécies economicamente importantes. Atualmente, cinco gêneros são reconhecidos em Gracilariaceae: Gracilariophila (parasita), Curdiea, Melanthalia, Gracilaria e Gracilariopsis. Algumas espécies de Gracilaria foram taxonomicamente transferidas para Hydropuntia. Entretanto, esse gênero é bastante controverso. Técnicas de sequenciamento de alta performance (HTS) levaram a um aumento de estudos usando genomas organelares completos, que têm sido usados para inferir relações filogenéticas em Rhodophyta e na investigação de outros aspectos dos genomas de algas vermelhas, incluindo sintenia gênica e transferências horizontal de genes (HGT). O HTS também facilitou a busca por plasmídeos extracromossômicos e sua influência nos genomas organelares por HGT. Nós utilizamos HTS para montar e anotar genomas organelares (mitocôndrias e cloroplastos) de sete espécies de Gracilariaceae usando a plataforma Illumina HiSeq 2500 e recebemos sequências de 31 amostras Gracilariaceae da Dr. Goia Lyra que foram montadas, anotadas e incluídas em nossas análises. Utilizamos esses dados, combinados com genomas publicados, para inferir filogenias e comparar a arquitetura do genoma dessas espécies representando as principais linhagens em Gracilariaceae. Os genomas mitocondrial e plastidial são altamente conservados na sintenia gênica e a variação ocorreu principalmente em regiões onde foram encontradas inserções de sequências derivadas de plasmídeos (PDS), similares aos plasmídeos extracromossômicos conhecidos de algas vermelhas. Nos genomas mitocondriais, as inserções de PDS estavam em duas regiões onde a direção da transcrição muda: entre os genes cob e trnL e os genes trnA e trnN. As inserções de PDS no genoma do cloroplasto estavam em posições diferentes, mas geralmente encontradas entre os genes psdD e rrs. O operon bacteriano leu/leuD foi encontrado nos genomas dos cloroplastos de Gracilaria tenuistipitata, G. chilensis, M. intermedia e também no plasmídeo de G. vermiculophylla. As árvores filogenéticas mostram duas origens diferentes de leuC/leuD: os genes encontrados no cloroplasto e no plasmídeo estavam próximos de proteobactérias, e os genes codificados no núcleo estavam próximos de Viridiplantae e cianobactérias. Gracilariaceae pode ser um bom modelo para estudar o impacto de PDS na evolução de genomas devido à presença frequente de inserções PDS em genomas organelares. Nossas análises filogenéticas demonstraram histórias evolutivas similares entre cloroplasto e mitocondria. No entanto, a filogenia de cloroplasto foi melhor resolvida com valores máximos de Bootstrap em todos os ramos. Nossa amostragem taxonômica corrobora a presença de três linhagens principais: Melanthalia/Curdiea, Gracilariopsis e Gracilaria. Melanthalia intermedia aparece como grupo-irmão do clado monofilético incluindo Gracilaria e Gracilariopsis, que foram resolvidos como gêneros monofiléticos. Além disso, baseado nas características da arquitetura do genoma organelar, os gêneros Gracilariopsis e Gracilaria se distinguem pela perda do gene plastidial petP em Gracilaria e pela posição de rearranjo do gene trnH no genoma mitocondrial. Nós não encontramos evidências para a permanencia o gênero Hydropuntia como originalmente proposto

Photosynthetic electron transport produces ATP and NADPH which support carbon fixation by the Calvin Cycle. To avoid over-reduction of the electron transport chain, plants must balance absorption and consumption of light energy. Mitochondrial alternative oxidase (AOX) is a non-energy-conserving electron sink, making it an ideal candidate to oxidize excess reductant and regulate chloroplastic redox state. Wild-type (WT) and transgenic Nicotiana tabacum lines with enhanced or suppressed AOX protein levels were grown under low light (LL) and moderate light (ML). LL-grown plants were also shifted to ML. AOX transcript and protein levels were enhanced in WT plants under ML. Chlorophyll fluorescence, gas exchange, and contents of chlorophyll, carbohydrate, and malondialdehyde were measured. Lack of AOX protein decreased Photosystem II (PSII) quantum efficiency and CO2 assimilation rates while enhancing PSII excitation pressure compared to WT. These findings suggest a role for AOX in mediating the chloroplast-mitochondrion relationship during acclimation to higher irradiance.

Photosynthetic electron transport produces ATP and NADPH which support carbon fixation by the Calvin Cycle. To avoid over-reduction of the electron transport chain, plants must balance absorption and consumption of light energy. Mitochondrial alternative oxidase (AOX) is a non-energy-conserving electron sink, making it an ideal candidate to oxidize excess reductant and regulate chloroplastic redox state. Wild-type (WT) and transgenic Nicotiana tabacum lines with enhanced or suppressed AOX protein levels were grown under low light (LL) and moderate light (ML). LL-grown plants were also shifted to ML. AOX transcript and protein levels were enhanced in WT plants under ML. Chlorophyll fluorescence, gas exchange, and contents of chlorophyll, carbohydrate, and malondialdehyde were measured. Lack of AOX protein decreased Photosystem II (PSII) quantum efficiency and CO2 assimilation rates while enhancing PSII excitation pressure compared to WT. These findings suggest a role for AOX in mediating the chloroplast-mitochondrion relationship during acclimation to higher irradiance.

Στην παρούσα εργασία μελετήθηκε η γενετική δομή και οι φυλογενετικές σχέσεις είκοσι τριών πληθυσμών της A.boyeri που προέρχονταν από λίμνες/λιμνοθάλασσες και θαλάσσιες περιοχές του ελλαδικού χώρου, καθώς και ενός πληθυσμού από τη λίμνη Iznik της Τουρκίας. Πιο συγκεκριμένα, προσδιορίστηκε μέρος της αλληλουχίας των μιτοχονδριακών γενετικών τόπων της υπομονάδας I της κυτοχρωματικής οξειδάσης (COI), του βρόγχου εκτόπισης (D-loop) και του κυτοχρώματος b (Cytb). Επιπλέον, προσδιορίστηκε η αλληλουχία ενός πυρηνικού γενετικού τόπου, της ροδοψίνης (Rh). Πραγματοποιήθηκε φυλογενετική ανάλυση των αλληλουχιών και κατασκευάστηκαν φυλογενετικά δένδρα με τις μεθόδους της Σύνδεσης Γειτόνων (NJ), Μέγιστης Φειδωλότητας (ΜΡ) και Μπεϊσιανής Συμπερασματολογίας (ΒΙ). Παρά τη διαφορετική τοπολογία των δένδρων που προέκυψαν, γεγονός που δεν κατέστησε εφικτό τον προσδιορισμό των φυλογενετικών σχέσεων μεταξύ των υπό μελέτη πληθυσμών, η ύπαρξη τριών διαφορετικών τύπων πληθυσμών διαπιστώθηκε τόσο από την ανεξάρτητη όσο και από τη συνδυασμένη ανάλυση των αλληλουχιών. Ο πρώτος τύπος πληθυσμού, ο «λιμναίος», περιελάμβανε τους πληθυσμούς από τις λίμνες/λιμνοθάλασσες καθώς και τους πληθυσμούς από τις θαλάσσιες περιοχές της Καλύμνου, της Κύμης και των Ν. Μουδανιών. Ο δεύτερος τύπος περιελάμβανε όλους τους θαλάσσιους πληθυσμούς (πρώτος «θαλάσσιος» τύπος), με εξαίρεση του πληθυσμούς από την Πρέβεζα, την Εύβοια και την Κω που συνιστούσαν τον τρίτο τύπο πληθυσμού (δεύτερος «θαλάσσιος» τύπος). Επισημαίνεται ιδιαίτερα ότι ο πυρηνικός μοριακός δείκτης που χρησιμοποιήθηκε έφερε αρκετή ποικιλότητα ώστε να επιβεβαιώσει τα παραπάνω αποτελέσματα. Οι «λιμναίοι» πληθυσμοί εμφάνισαν σαφές φυλογεωγραφικό πρότυπο, διακρίνοντας τους πληθυσμούς του Αιγαίου και της Τουρκίας από τους λιμναίους/λιμνοθαλάσσιους του Ιονίου Πελάγους. Επιπλέον, διαπιστώθηκε πως ο πληθυσμός από τον Αμβρακικό διαφοροποιείται αρκετά από τους υπόλοιπους «λιμναίους» πληθυσμούς, ενώ αξιοσημείωτη είναι και η συσχέτιση που βρέθηκε ανάμεσα σε αυτόν τον πληθυσμό και πληθυσμών από την Αδριατική. Όσον αφορά τους θαλάσσιους πληθυσμούς, κανένας από τους δύο τύπους δεν εμφάνισε συγκεκριμένο φυλογεωγραφικό πρότυπο. Επιπλέον, διαπιστώθηκε πως οι πληθυσμοί από την Πρέβεζα, την Εύβοια και την Κω αντιστοιχούν στον εστιγμένο (punctuated) τύπο πληθυσμού, όπως έχει περιγραφεί, ο οποίος δεν έχει έως σήμερα αναφερθεί στην ανατολική Μεσόγειο. Ωστόσο, σύμφωνα με τα αποτελέσματα μας, τα μαύρα στίγματα κάτω από την πλευρική γραμμή δεν μπορούν να αποτελέσουν διαγνωστικό στοιχείο για τη διάκριση των δύο τύπων θαλάσσιων πληθυσμών (punctuated, nonpunctuated), καθώς στον τύπο αυτό εντάσσονται άτομα που είτε φέρουν μαύρα στίγματα είτε όχι, και το αντίστροφο. Από τον πολλαπλασιασμό με τη μέθοδο της αλυσιδωτής αντίδρασης πολυμεράσης (PCR) του μιτοχονδριακού τμήματος Cytb και τον προσδιορισμό της αλληλουχίας του, διαπιστώθηκε η ύπαρξη μίας ένθεσης ανάμεσα στο tRNA του γλουταμινικού οξέος και το Cytb. Η ένθεση αυτή είχε μέγεθος περίπου 200bp στα «λιμναία» άτομα και λίγο μικρότερο (~150bp) στα άτομα που εμφάνισαν το πρότυπο punctuated (εστιγμένα) (Πρέβεζα, Εύβοια, Κως), ενώ απουσίαζε από τους υπόλοιπους θαλάσσιους πληθυσμούς. Βάσει των παρόντων αποτελεσμάτων και σε συνδυασμό με αντίστοιχα αποτελέσματα προηγούμενων μελετών επιβεβαιώνεται η άποψη σύμφωνα με την οποία η παρουσία ή απουσία της συγκεκριμένης ένθεσης μπορεί να χρησιμοποιηθεί ως διαγνωστικός χαρακτήρας για τη διάκριση των τριών διαφορετικών τύπων πληθυσμών της A.boyeri. / The genetic differentiation and the phylogenetic relationships of twenty three Atherina boyeri populations originating from lakes/lagoons and marine sites of Greece, as well as from a population from lake Iznik in Turkey were investigated. More specifically, the partial sequence of the mitochondrial genes cytochrome oxidase I (COI), cytochrome b (cytb) and control region were determined. The partial sequence of the nuclear proteincoding gene rhodopsin (Rh) was determined, as well. Phylogenetic analysis of the sequences was conducted using the methods: Neighbor-Joining (NJ), Maximum Parsimony (MP) and Bayesian Inference (BI). Even though trees presented different topologies, and thus the phylogenetic relationships among populations were not able to be resolved, the presence of three main population forms was observed both from the separate and combined analyses of datasets. The first population type (‘lagoon’ type) contains all the lagoon/lake populations, as well as three populations (Kymi, Kalymnos and Nea Moudania) originating from marine environment. The second type includes nearly all marine populations (first ‘marine’ type), excluding specimens collected from three geographical distant areas (Preveza, Evvoia and Kos), which form the third A. boyeri population type (second ‘marine’ type). It is worth mentioning that the nuclear marker used was proved to carry sufficient diversity in order to confirm the above results. The ‘lagoon’ type populations presented a clear phylogeographic pattern, with the populations from Aegean Sea and Turkey forming a distinct group from the populations from the Ionian Sea. Moreover, the population from Amvrakikos appeared to be distinct from the rest ‘lagoon’ type populations and a remarkable correlation of this population and populations from Adriatic Sea was found. On the other hand, none of the two ‘marine’ type populations presented a specific phylogeographic pattern. Additionally, it was found that the populations from Preveza, Evvoia and Kos correspond to the ‘punctuated’ marine type, as has been described, which was not previously reported in eastern Mediterranean Sea. However, the fact that in the present study a population (Evvoia) without black spots on the flanks is clustered with the punctuated form and vice versa (specimens from Limnos presented black spots but was clustered with the non-punctuated ‘marine’ type populations), suggests that differences in color pattern do not seem to be a sufficient marker for discriminating the two marine types. From the PCR amplification of cytb and determination of its sequence the presence of approximately 200bp intergenic spacer between tRNA glutamic acid (tRNAGlu) and cyt b genes was observed among the ‘lagoon’ type specimens and a smaller one among the specimens from Preveza, Evvoia and Kos. The intergenic spacer was absent from the rest marine populations. Thus, our results support the suggestion that this character is conserved and capable to be used for distinguishing the different forms.