Biorreatores, que são explorados desde a antiguidade, são sistemas capazes de realizar a fermentação de compostos orgânicos, continuam sendo amplamente utilizados atualmente devido à diversidade de aplicações. Esses sistemas podem operar em diferentes modos de fermentação, entretanto, os mais utilizados são: fermentação contínua, semicontínua e descontínua. Esse último, juntamente com o processo de digestão anaeróbia (ausência de oxigênio), permitem que uma determinada matéria orgânica seja degradada e transformada em biogás, um dos fatores chave para geração de energia limpa. Percebe-se, portanto, que o estudo de biorreatores em modo de operação descontínuo e em processo de digestão anaeróbia é fundamental para o desenvolvimento de pesquisas relacionadas à geração de energia renovável. Para facilitar o entendimento desse processo, alguns autores propuseram estudos baseados na identificação de parâmetros em modelos não-lineares descritivos, do tipo caixa-branca, que hoje são vastamente utilizados na modelagem de biorreatores. A grande limitação dessa abordagem é que o processo de identificação de sistemas utilizando esses modelos pode ser complexo e demorado, ou, ainda, os parâmetros dos sistemas representados podem não ser identificáveis, inviabilizando o procedimento. Tentando amenizar essas dificuldades, propomos neste trabalho a utilização de modelos polinomiais NAR e NARMA do tipo caixa-preta para a modelagem de biorreatores em modo de fermentação descontínua. Modelos caixa-preta representam sistemas reais por meio de sua saída, sem informação sobre os mecanismos internos desse sistema, simplificando a identificação. Frente a esse contexto, o objetivo deste estudo é investigar a predição e, por consequência, realizar o monitoramento da produção de metano utilizando os modelos caixa-preta propostos em sistemas de biorreatores em modo descontínuo e em processo de digestão anaeróbia. Realizamos estudos que abarcam a investigação de dados simulados e de dados reais. Num primeiro momento são propostos modelos polinomiais dos tipos NAR e NARMA. A partir desses modelos são estimados os parâmetros dos sistemas simulados, com e sem ruído na saída, baseados em condições iniciais propostas na literatura, que denominamos Grupo de Controle. Posteriormente realizamos as validações desses modelos. Em seguida, passamos à etapa de investigação do domínio de validade dos modelos caixa-preta propostos, realizando um estudo em que modificamos as condições iniciais do sistema que representa biorreatores em modo de fermentação descontínua. Por fim, utilizamos dados de um experimento real para realizar o processo de estimação de parâmetros e de validação dos modelos. Os resultados mostraram que os modelos polinomiais NAR e NARMA são bastante adequados para predição de metano em biorreatores em modo de fermentação descontínua em processo de digestão anaeróbia, tanto para os dados simulados quanto para os dados reais. / Bioreactors, which are explored since antiquity, are systems that are capable of performing the fermentation of organic compounds. Nowadays, they are widely applied due to its diversity of applications. These systems can operate in different fermentation modes: continuous, fed-batch and batch. This last fermentation method along with the process of anaerobic digestion allow organic matter to be degraded and converted into biogas, which is a key factor for clean energy generation. It is thus realized that the study of bioreactors in batch mode and anaerobic digestion process is crucial to the development of research related to renewable energy generation. For a better understanding of the process, some authors have proposed studies based on parameters identification in descriptive nonlinear models, white-box models, which are widely used in bioreactors modeling. The main limitation of this approach is that the system identification procedure using these models can be complex and time-consuming, or even the parameters of the systems may not be identifiable. In order to overcome these difficulties, we propose in this work the use of black-box polynomial models for bioreactor modeling in batch mode, with NAR and NARMA model structures. Black-box models represent real systems using its output, without explicitly considering the inner mechanisms of the system, simplifying the identification procedure. Thus, the aim of this work is to investigate the prediction and monitoring methane production using the black-box models proposed using bioreactor systems in batch and anaerobic digestion process. The investigation uses numerical simulation and experimental data. At first, polynomial models of the types NAR and NARMA are proposed. The parameters from these models using simulation data with and without noise at the output, based on initial conditions proposed in the literature, are estimated. Subsequently we perform validations of these models. The next step is the study of the validity domain of the proposed black-box models, which is performed by testing many different initial conditions of the system that represents bioreactors in batch fermentation mode. Finally, we used real experimental data to perform the estimation of the parameters from the process and validation of models. The results, both simulated and experimental, indicate that the polynomial models NAR and NARMA are appropriate for prediction of methane fermentation in batch bioreactors.

Ribosomal RNAs (rRNA) undergo several post-transcriptional modifications inside the cell. These modifications can be (1) RNA- independent (enzyme only) and (2) guide RNA-mediated. In the latter mechanism, a group of small, metabolically stable, non-coding RNAs, present as ribonucleoprotein (RNP) particles, modify ribosomal RNAs inside the cell. One of the highly abundant rRNA modifications is pseudouridine (Y) formation. In Archaea and Eukarya, pseudouridine synthases, with the help of small RNAs, form pseudouridines at functionally important regions in rRNA. Cbf5, the pseudouridine synthase, three other core proteins, and a box H/ACA RNA form the ribonucleoprotein complex in sRNP-mediated rRNA pseudouridylation. Certain Ys in rRNAs are evolutionarily conserved from Bacteria to human. Among those, two Ys are present in helix 69 of rRNA and one in helix 90. We successfully deleted Cbf5 in Haloferax volcanii, a haloarchaeon, and showed that the deleted strain was viable. It was the first report where Cbf5 deletion was achieved, because deletion or mutation of cbf5 or of its homologs is lethal in eukaryotes. We also found that the cbf5 deleted strain was unable to produce the three highly conserved Ys in rRNA of H. volcanii (position 1940, 1942 in helix 69, and 2605 in helix 90), whereas the tRNA Ys were intact. To identify the specific structural features of Cbf5 involved in rRNA Ψ formation, we used a cbf5 deleted strain which was complemented with a plasmid borne copy of the gene. Using the crystal structure of Pyrococcous furiosus Cbf5 as template, we created a homology model of H. volcanii Cbf5 (HvCbf5) and identified several residues and motifs/domains of HvCbf5 that might be important to the protein's enzymatic activity. By using an in vivo mutational approach, we confirmed some previously predicted and certain unidentified residues/motifs/domains that serve as positive determinants of rRNA Ys1940, 1942, and 2605 formation inside the cell. A box H/ACA RNA, sR-h45, was bioinformatically predicted before. We confirmed its presence as a double hairpin RNA inside the cell whose level goes down in the absence of Cbf5. We identified that sR-h45 is the guide RNA for sRNP-mediated Ys at the three above mentioned rRNA positions in H. volcanii. Each hairpin of this RNA can independently modify the substrate, both in vivo and in vitro. To characterize the structure of sR-h45, we have used a sR-h45 deleted strain where the function of sR-h45 was complemented with a plasmid-borne copy of the gene. By a combination of in vivo and in vitro mutagenic approaches, we determined specific nucleotides/structures of this RNA, involved in binding to the core proteins and also to the substrate RNA. We also identified that one hairpin of sR-h45 can modify two successive positions (1940 and 1942) in rRNA.

In all the three domains of life, most RNAs undergo post transcriptional modifications both on the bases as well as the ribose sugars of the individual ribonucleotides. 2'-O-methylation of ribose sugars and isomerization of Uridines to Pseudouridines are two most predominant modifications in rRNAs and tRNAs across all domains of life. Besides 2'-O-methylation of ribose sugars, methylation of pseudouridine (Ø) at position 54 of tRNA, producing m1Ø, is a hallmark of many archaeal species but the specific methylase involved in the formation of this modification had yet to be characterized. A comparative genomics analysis had previously identified COG1901 (DUF358), part of the SPOUT superfamily, as a candidate for this missing methylase family. To test this prediction, the COG1901 encoding gene, HVO_1989, was deleted from the Haloferax volcanii genome. Analyses of modified base contents indicated that while m1Ø was present in tRNA extracted from the wild-type strain, it was absent from tRNA extracted from the mutant strain. Expression of the gene encoding COG1901 from Halobacterium sp. NRC-1, VNG1980C, complemented the m1Ø minus phenotype of the ÄHVO_1989 strain. This in vivo validation was extended with in vitro tests. Using the COG1901 recombinant enzyme from Methanocaldococcus jannaschii (Mj1640), purified enzyme Pus10 from M. jannaschii and full-size tRNA transcripts or TØ-arm (17-mer) fragments as substrates, the sequential pathway of m1Ø54 formation in Archaea was reconstituted. The methylation reaction is AdoMet-dependent. The efficiency of the methylase reaction depended on the identity of the residue at position 55 of the TØ-loop. The presence of Ø55 allowed the efficient conversion of Ø54 to m1Ø54, whereas in the presence of C55 the reaction was rather inefficient and no methylation reaction occurred if a purine was present at this position. These results led to renaming the Archaeal COG1901 members as TrmY proteins. Another aim of this study was to investigate the mechanism of target RNA recruitment to a box C/D sRNP. From data obtained, we have made the following hypothesis- aNop5p, either alone or as a heterodimer with Fibrillarin, binds to single stranded bulges and loops of target RNA. This aNop5p bound target is then hybridized to an assembling guide sRNP complex containing the guide RNA and L7Ae or guide RNA, L7Ae and aNop5p. If the guide:target sequences are complementary to each other, they hybridize and the target nucleotide gets modified. We also think that post modification, the guide and target strands separate, the core proteins rearrange themselves on the guide RNA and then prime the guide RNA for next round of modification. Compared to the general archaeal populations, haloarchaea contain significantly fewer number of box C/D guide RNAs. In archaea, previous studies have underscored the importance of a symmetric assembly of the core proteins on the sRNA. This meant that if the core proteins were unable to bind to either the terminal box C/D or the internal box C'/D' motifs, the sRNP was not efficient to carry out the modification of the target RNA. Essentially the only two haloarchaeal box C/D sRNPs known before had a symmetric architecture. In this study we discovered the first naturally occurring asymmetric box C/D sRNP called sR-41 in Haloferax volcannii. The architecture of Haloferax volcanii sR-41 box C/D sRNP seems to be closer in conformation to eukaryal snoRNPs (eukaryal counterparts of archaeal sRNPs) in which the core proteins assemble asymmetrically on the RNA. Till date, no information regarding the catalytic mechanism of an asymmetrically arranged eukaryal box C/D snoRNPs are available, because of unavailability of any assembly systems or crystal structures. Hence, this archaeal sR-41 guide sRNP provides a unique opportunity to study mechanism of modification in an asymmetrically arranged box C/D sRNP molecule.

RNAs undergo different posttranscriptional chemical modifications, which affect their structural stability and functional diversity. RNA methylation is a very common type of post-transcriptional modification and is present in all domains of life: Archaea, Eukaryotes and Bacteria. Some of these methylations are catalyzed either by a RNA-protein complex or by stand-alone enzymes. The RNA-protein complex (Ribonucleoprotein complex) is comprised of a small RNA known as the guide RNA (Box C/D RNA) and core proteins (L7Ae, Nop5, and Fibrillarin). Box C/D RNAs contain conserved regions, called box C and box D near their 5’ and 3’ termini, respectively, and their imperfect copies called box C’ and box D’, internally. A short stretch of sequence between these Boxes are known as the guide/spacer regions, as the guide region helps in recruiting and positioning a specific target RNA for modification. Both in Archaea and Eukarya, box C and box D, as well as box C’ and box D’ together can form a structure called a Kink-turn (K-turn) that is characterized by a canonical Watson-Crick base-paired stem on one side, and a non-canonical stem on the other, separated by a 3-nucleotide loop. In Archaea box C’ and D’ can also form a K-loop, where the canonical stem of K-turn is replaced by a loop. Archaeal L7Ae binds first to the K-turn or K-loop and allows the recruitment of other proteins to form the complex. The presence of a unique box C/D RNA of Haloferax volcanii, called sR-tMet has been reported previously to guide the 2’-O-methylation of C34 in elongator pre-tRNAMet. Here we tried to characterize the structure-function relationship of this guide RNA under in vivo conditions. This RNA lacks a conventional K-turn or K-loop at its C’/D’ motif. We have created an H. volcanii strain that has a genomic deletion of sR-tMet. The sR-tMet gene is not essential for H. volcanii but this sR-tMet deleted strain lacks the 2’-O-methylation of C34 of its elongator tRNAMet. Unlike the close sR-tMet homologs (sR8 from Methanocaldococcus jannaschii and sR49 from Pyrococcus abyssi), the Box C’/D’ motif of sR-tMet is neither a K-turn nor a K-loop. The introduction of proper K-loop in the Box C’/D’ motif (sR-tMet with K-loop) abolished its Cm34 modification function in ΔsR-tMet strain. Direct interaction between L7Ae and the K-loop is not an absolute requirement for its function. However, disruption of the G/A and A/G pairing in Box C/D motif and Box D’ suggests the importance of these non-Watson crick base pairings in respect to sR-tMet’s function. Several other mutational studies have revealed that peculiar sR-tMet guide RNA from H. volcanii, behaves more like a Eukaryotic Box C/D RNA (where the K-loop is not required and presence of longer spacer length) than regular Archaeal one. Pseudouridine synthase 10 (Pus10) is the most recently identified Ψ synthase, found only in higher eukaryotes and Archaea. Archaeal Pus10 produces either tRNA Ψ55 or both tRNA Ψ54 and Ψ55 modifications. In Human, its Ψ synthase activity is not yet confirmed and interestingly it has been implicated in apoptosis. Herein for the first time we revealed that this putative RNA Ψ synthase protein, Human Pus10 (HuP10), translocates from the nucleus to the cytoplasm in TRAIL induced apoptosis. This nucleo-cytoplasmic movement of HuP10 occurs through the CRM1 mediated nuclear export pathway and Caspase 3 influences this movement. HuP10 also mediates crosstalk between the extrinsic and intrinsic pathways during TRAIL-induced apoptosis. Other than its involvement in apoptosis, we have also uncovered that HuP10 is involved in regulation of cell proliferation. Depletion (knockdown) of this protein in different cancer cell lines, promotes cell migration and anchorage-independent cell growth in the absence of any apoptotic stimulation.

Biorreatores, que são explorados desde a antiguidade, são sistemas capazes de realizar a fermentação de compostos orgânicos, continuam sendo amplamente utilizados atualmente devido à diversidade de aplicações. Esses sistemas podem operar em diferentes modos de fermentação, entretanto, os mais utilizados são: fermentação contínua, semicontínua e descontínua. Esse último, juntamente com o processo de digestão anaeróbia (ausência de oxigênio), permitem que uma determinada matéria orgânica seja degradada e transformada em biogás, um dos fatores chave para geração de energia limpa. Percebe-se, portanto, que o estudo de biorreatores em modo de operação descontínuo e em processo de digestão anaeróbia é fundamental para o desenvolvimento de pesquisas relacionadas à geração de energia renovável. Para facilitar o entendimento desse processo, alguns autores propuseram estudos baseados na identificação de parâmetros em modelos não-lineares descritivos, do tipo caixa-branca, que hoje são vastamente utilizados na modelagem de biorreatores. A grande limitação dessa abordagem é que o processo de identificação de sistemas utilizando esses modelos pode ser complexo e demorado, ou, ainda, os parâmetros dos sistemas representados podem não ser identificáveis, inviabilizando o procedimento. Tentando amenizar essas dificuldades, propomos neste trabalho a utilização de modelos polinomiais NAR e NARMA do tipo caixa-preta para a modelagem de biorreatores em modo de fermentação descontínua. Modelos caixa-preta representam sistemas reais por meio de sua saída, sem informação sobre os mecanismos internos desse sistema, simplificando a identificação. Frente a esse contexto, o objetivo deste estudo é investigar a predição e, por consequência, realizar o monitoramento da produção de metano utilizando os modelos caixa-preta propostos em sistemas de biorreatores em modo descontínuo e em processo de digestão anaeróbia. Realizamos estudos que abarcam a investigação de dados simulados e de dados reais. Num primeiro momento são propostos modelos polinomiais dos tipos NAR e NARMA. A partir desses modelos são estimados os parâmetros dos sistemas simulados, com e sem ruído na saída, baseados em condições iniciais propostas na literatura, que denominamos Grupo de Controle. Posteriormente realizamos as validações desses modelos. Em seguida, passamos à etapa de investigação do domínio de validade dos modelos caixa-preta propostos, realizando um estudo em que modificamos as condições iniciais do sistema que representa biorreatores em modo de fermentação descontínua. Por fim, utilizamos dados de um experimento real para realizar o processo de estimação de parâmetros e de validação dos modelos. Os resultados mostraram que os modelos polinomiais NAR e NARMA são bastante adequados para predição de metano em biorreatores em modo de fermentação descontínua em processo de digestão anaeróbia, tanto para os dados simulados quanto para os dados reais. / Bioreactors, which are explored since antiquity, are systems that are capable of performing the fermentation of organic compounds. Nowadays, they are widely applied due to its diversity of applications. These systems can operate in different fermentation modes: continuous, fed-batch and batch. This last fermentation method along with the process of anaerobic digestion allow organic matter to be degraded and converted into biogas, which is a key factor for clean energy generation. It is thus realized that the study of bioreactors in batch mode and anaerobic digestion process is crucial to the development of research related to renewable energy generation. For a better understanding of the process, some authors have proposed studies based on parameters identification in descriptive nonlinear models, white-box models, which are widely used in bioreactors modeling. The main limitation of this approach is that the system identification procedure using these models can be complex and time-consuming, or even the parameters of the systems may not be identifiable. In order to overcome these difficulties, we propose in this work the use of black-box polynomial models for bioreactor modeling in batch mode, with NAR and NARMA model structures. Black-box models represent real systems using its output, without explicitly considering the inner mechanisms of the system, simplifying the identification procedure. Thus, the aim of this work is to investigate the prediction and monitoring methane production using the black-box models proposed using bioreactor systems in batch and anaerobic digestion process. The investigation uses numerical simulation and experimental data. At first, polynomial models of the types NAR and NARMA are proposed. The parameters from these models using simulation data with and without noise at the output, based on initial conditions proposed in the literature, are estimated. Subsequently we perform validations of these models. The next step is the study of the validity domain of the proposed black-box models, which is performed by testing many different initial conditions of the system that represents bioreactors in batch fermentation mode. Finally, we used real experimental data to perform the estimation of the parameters from the process and validation of models. The results, both simulated and experimental, indicate that the polynomial models NAR and NARMA are appropriate for prediction of methane fermentation in batch bioreactors.

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This diploma thesis addresses the practical and theoretical aspects of piercing jewellery and related accoutrements. The practical part describes the design and construction of a series of ceramic jewellery boxes for piercing jewellery. These three boxes are functional and unique jewellery in their own right. The theoretical part of this thesis examines body piercing art, piercing techniques and piercing jewellery. A visual section displays the piercing jewellery and documents the making of the jewellery boxes and their final design.

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