Setaria viridis tem sido utilizada como organismo modelo para gramíneas com metabolismo C4, buscando elucidar processos biológicos de plantas. A importância das monocotiledôneas com o metabolismo de C4 deve-se ao melhor balanço fotossintético quando comparado às plantas com metabolismo C3 em condições climáticas com altas temperaturas, como as que prevalecem no Brasil. Setaria viridis representa um sistema modelo de grande importância para a agricultura brasileira, uma vez que tanto a produção brasileira de biocombustíveis como de alimentos está fortemente relacionada aos organismos C4. Os hormônios vegetais são cruciais no estabelecimento de órgãos complexos desde o zigoto até o desenvolvimento da planta adulta. A auxina em particular, desempenha um papel fundamental na formação de um complexo sistema radicular nas plantas. Sabe-se que o transporte polar desse hormônio concentra a auxina em determinados tecidos, desempenhando um papel essencial na expressão diferencial de genes que modulam o desenvolvimento dos vários órgãos da planta. Monocotiledôneas e em particular Poaeceaes exibem um complexo sistema radicular que parece ser chave para o seu vasto potencial adaptativo, mas pouco se sabe sobre os fatores que envolvem as respostas sensoriais e de desenvolvimento que guiam a escolha de quais tipos de raiz as plantas elegem. O objetivo deste estudo foi explorar as vias hormonais e abióticas que influenciam a arquitetura das raízes / Setaria viridis has been used as a model organism for grasses with C4 metabolism, seeking to elucidate plant biological processes. The importance of monocotyledons with C4 metabolism is due to better photosynthetic balance when compared to plants with C3 metabolism in climatic conditions with high temperatures, such as those that prevail in Brazil. Setaria viridis represents a model system of great importance for Brazilian agriculture since both the Brazilian production of biofuels and food is strongly related to C4 organisms. Plant hormones are crucial in the establishment of complex organs from zygote to the adult plant development. Auxin in particular, plays a key role in the formation of a complex radicular system in plants. The polar transport of this hormone is known to concentrate auxin in particular tissues, playing an essential roll in the differential expression of genes that will modulate the development of the various plant organs. Monocots and in particular Poaeceae display a complex radicular system that seems to be key for it vast adaptive potential, but little is known about the factors involving the sensing and developmental responses that guide the choice of which root types to elect. The aim of this study was to explore the hormonal and abiotic cues that influence the root architecture

Setaria viridis tem sido utilizada como organismo modelo para gramíneas com metabolismo C4, buscando elucidar processos biológicos de plantas. A importância das monocotiledôneas com o metabolismo de C4 deve-se ao melhor balanço fotossintético quando comparado às plantas com metabolismo C3 em condições climáticas com altas temperaturas, como as que prevalecem no Brasil. Setaria viridis representa um sistema modelo de grande importância para a agricultura brasileira, uma vez que tanto a produção brasileira de biocombustíveis como de alimentos está fortemente relacionada aos organismos C4. Os hormônios vegetais são cruciais no estabelecimento de órgãos complexos desde o zigoto até o desenvolvimento da planta adulta. A auxina em particular, desempenha um papel fundamental na formação de um complexo sistema radicular nas plantas. Sabe-se que o transporte polar desse hormônio concentra a auxina em determinados tecidos, desempenhando um papel essencial na expressão diferencial de genes que modulam o desenvolvimento dos vários órgãos da planta. Monocotiledôneas e em particular Poaeceaes exibem um complexo sistema radicular que parece ser chave para o seu vasto potencial adaptativo, mas pouco se sabe sobre os fatores que envolvem as respostas sensoriais e de desenvolvimento que guiam a escolha de quais tipos de raiz as plantas elegem. O objetivo deste estudo foi explorar as vias hormonais e abióticas que influenciam a arquitetura das raízes / Setaria viridis has been used as a model organism for grasses with C4 metabolism, seeking to elucidate plant biological processes. The importance of monocotyledons with C4 metabolism is due to better photosynthetic balance when compared to plants with C3 metabolism in climatic conditions with high temperatures, such as those that prevail in Brazil. Setaria viridis represents a model system of great importance for Brazilian agriculture since both the Brazilian production of biofuels and food is strongly related to C4 organisms. Plant hormones are crucial in the establishment of complex organs from zygote to the adult plant development. Auxin in particular, plays a key role in the formation of a complex radicular system in plants. The polar transport of this hormone is known to concentrate auxin in particular tissues, playing an essential roll in the differential expression of genes that will modulate the development of the various plant organs. Monocots and in particular Poaeceae display a complex radicular system that seems to be key for it vast adaptive potential, but little is known about the factors involving the sensing and developmental responses that guide the choice of which root types to elect. The aim of this study was to explore the hormonal and abiotic cues that influence the root architecture

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Studies of ribosomal reading frame maintenance are often based on frameshift mutation suppression experiments. In this thesis, suppression of a frameshift mutation in Salmonella enterica serovar Typhimurium by a tRNA and a ribosomal protein are described. The +1 frameshift mutation hisC3072 (that contains an extra G in a run of Gs) is corrected by mutations in the argU gene coding for the minor tRNAArgmnm5UCU. The altered tRNAArgmnm5UCU has a decreased stability and reduced aminoacylation due to changed secondary and/or tertiary structure. Protein sequencing revealed that during the translation of the GAA-AGA frameshifting site the altered tRNAArgmnm5UCU reads the AGA codon inefficiently. This induces a ribosomal pause, allowing the tRNAGlumnm5s2UUC residing in the ribosomal P-site to slip forward one nucleotide. The same frameshift mutation (hisC3072) was also suppressed by defects in the large ribosomal subunit protein L9. Single base substitutions, truncations, and absence of this protein induced ribosome slippage. Mutated ribosome could shift to the overlapping codon in the +1 frame, or bypass to a codon further downstream in the +1 frame. The signal for stimulation of slippage and function of L9 needs to be investigated. During the search for suppressors of the hisD3749 frameshift mutation, a spontaneous mutant was isolated in the iscU gene that contained greatly decreased levels of the thiolated tRNA modifications ms2io6A and s2C. The iscU gene belongs to the iscR-iscSUA-hscBA-fdx operon coding for proteins involved in the assembly of [Fe-S] clusters. As has been shown earlier, IscS influences the synthesis of all thiolated nucleosides in tRNA by mobilizing sulfur from cysteine. In this thesis, it is demonstrated that IscU, HscA, and Fdx proteins are required for the synthesis of the tRNA modifications ms2io6A and s2C but are dispensable for the synthesis of s4U and (c)mnm5s2U. Based on these results it is proposed that two distinct pathways exist in the formation of thiolated nucleosides in tRNA: one is an [Fe-S] cluster-dependent pathway for the synthesis of ms2io6A and s2C and the other is an [Fe-S] cluster-independent pathway for the synthesis of s4U and (c)mnm5s2U. MiaB is a [Fe-S] protein required for the introduction of sulfur in ms2io6A. TtcA is proposed to be involved in the synthesis of s2C. This protein contains a CXXC conserved motif essential for cytidine thiolation that, together with an additional CXXC motif in the C-terminus may serve as an [Fe-S] cluster ligation site.

Die Fusionsreaktion des Coronavirus MHV wird vom S-Protein vermittelt. In der vorliegenden Arbeit wurde der Eintrittsweg von MHV-A59 in Mauszellen untersucht. Die Infektivität kann durch lysosomotrope Substanzen und Inhibitoren der Clathrin-abhängigen Endozytose gehemmt werden. Der Eintritt von MHV-A59 in Mauszellen erfolgt über die Clathrin-abhängige Endozytose und setzt die anschließende Fusion der viralen und zellulären Membran bei niedrigem pH-Wert voraus. Fluoreszenzmikroskopische Studien zur Interaktion fluoreszenzmarkierter MHV-A59 Partikel mit Mauszellen bestätigen, dass MHV-A59 über Endozytose aufgenommen wird. Nach Bindung der Viren an die Zellen und anschließende Erniedrigung des pH-Wertes kommt es zur Färbung der Plasmamembran. Die Erniedrigung des pH-Wertes in Abwesenheit des Rezeptors führt zu einer irreversiblen Konformationsumwandlung im viralen Fusionsprotein, die die Inaktivierung der Fusionsaktivität und den Verlust der Infektivität zur Folge hat. Die Ergebnisse deuten auf die Beteiligung des endozytotischen Weges für den viralen Eintritt von MHV-A59 hin. Ein niedriger pH-Wert eines zellulären endosomalen Kompartiments induziert vermutlich die Konformationsänderung im S-Protein und löst die Fusionsreaktion aus. Ein vorläufiges 3D-Modell des S-Proteins von MHV-A59 konnte erstellt werden. Darüber hinaus wurde die Struktur und Fusionsfähigkeit des S-Proteins von SARS-CoV analysiert. Ein Zell-Zell-Fusionsassay konnte nachweisen, dass die Fusion zwischen S-Protein und ACE2-exprimierenden Zellen sowohl abhängig vom pH-Wert als auch von der proteolytischen Spaltung in S1- und S2-Untereinheit ist und durch Erniedrigung des pH-Wertes zusätzlich verstärkt werden kann. Im abschließenden Teil der Arbeit wurde auf Basis theoretischer Untersuchungen eine Vorhersage des mutmaßlichen Fusionspeptids des S-Proteins von MHV-A59, welches alle wesentlichen Merkmale eines internen Fusionspeptids aufweist, vorgenommen. Es liegt nahe der "Heptad-Repeat" (HR) Domäne HR1. / Fusion of the Coronavirus MHV-A59 is mediated by the viral S-protein. The entry pathway of MHV-A59 into murine cells was studied in this work. Infection was strongly inhibited by lysosomotropic compounds and substances interfering with clathrin-dependent endocytosis, suggesting that MHV-A59 is taken up via endocytosis and delivered to acidic compartments. Fluorescence microscopy of labeled MHV-A59 confirmed that the virus is taken up via endocytosis. When the virus was bound to cells and the pH was lowered to 5.0, we observed a strong labeling of the plasma membrane. Electron microscopy revealed low pH triggered conformational alterations of the S-ectodomain. These alterations are likely to be irreversible because low pH-treatment of viruses caused an irreversible loss of fusion activity. The results imply that endocytosis plays a major role in MHV-A59 infection and that the acidic pH of the endosomal compartment triggers a conformational change of the S-protein mediating fusion. Furthermore the conformation of the trimeric spike protein of the murine hepatitis virus A59 was characterized by cryoelectron microscopy. A preliminary 3D-reconsruction of the native structure could be accomplished. Besides we studied the structure and fusion capability of the spike protein expressed by SARS-CoV. The cell-based fusion assay revealed that fusion of spike protein and ACE2-receptor expressing cells was strongly dependent on low pH and on proteolytic cleavage of the S-protein into S1 and S2 subunit. Additionally fusion could be significantly increased by lowering of the pH. The theoretical part of the thesis allowed the identification of the putative fusion peptide, which showed main characteristics of internal fusion peptides. It allows the heptad regions of the spike protein to assemble in the six-helix bundle structure (6HB). This structure is of great importance to initiate the approximation of viral and cellular membrane and thus to induce fusion.