No description available.

No description available.

In this thesis chromatographic conditions for the HPLC analysis of ribonucleotides AMP, ADP and ATP were optimized. For their separation chromatographic column, Nucleogel SAX 1000-8, 50 x 4,6 mm, from the German company Marcherey-Nagel was used. The mobile phase of a 0,2 mol/L solution of KH2PO4, its pH was adjusted with 1mol/L with potassium hydroxide to pH = 5,00. As the most suitable flow rate was selected 1,0 mL/min. The analysis was conducted using an isocratic elution. Detection was conducted by a DAD detector at a wavelength of λ = 260 nm. Substances were eluted in the following order AMP (tr = 0,911 min), ADP (tr = 1,667 min) and ATP (tr = 7,262 min). The total analysis time of the mixture of standards, under the conditions mentioned above, lasted for 10 minutes. For the extraction of adenosine-5-ribonucloetides the real matrix frozen and lyophilized leaves of tobacco virginia (Nicotiana tabacum L.) have been used. Two methods of extraction were applied. The first procedure was based on the extraction of AMP, ADP and ATP from the leaves of the tobacco plant by boiling deionized water. In the second method 0,07 mol/L HClO4 for the extraction instead of deionized water was used. Keywords: HPLC, adenosine ribonucleotides, AMP, ADP, ATP, tobacco virginia

Orientador: Ranulfo Monte Alegre / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-05T19:04:18Z (GMT). No. of bitstreams: 1 Ogrodowski_Rodenei_D.pdf: 1087647 bytes, checksum: 05b46b0950dd59d53fe5d62523de3047 (MD5) Previous issue date: 2006 / Doutorado / Doutor em Engenharia de Alimentos

Este trabalho teve como objetivo a utilização de glicerol, principal subproduto da produção de biodiesel, como fonte de carbono para a produção de biomassa e ribonucleotídeos de leveduras. Com este propósito, foi realizado um screening de leveduras para identificar aquelas cepas com maior capacidade de crescimento em glicerol. As leveduras Hansenula anomala CCT 2648 e Kluyveromyces marxianus var. lactis CCT 4086 apresentaram os maiores valores de fator de conversão (YX/S) e produtividade volumétrica em células (QX) sob condições fixas de glicerol (30 g/L), temperatura (30 oC) e pH (5,5). Foi também detectada a produção de etanol e ácidos orgânicos, por algumas das leveduras estudadas em meios contendo glicerol. Com base no potencial de produção de biomassa das cepas selecionadas na etapa anterior, foram realizados estudos para avaliar a influência da concentração de glicerol (10 - 50 g/L), extrato de levedura (1 - 3 g/L), pH(4,5 - 6,5) e temperatura (28 - 40oC) no crescimento destas linhagens, utilizando planejamento experimental 24. De acordo com os resultados, a levedura Hansenula anomala CCT 2648 foi selecionada para a produção de biomassa e ribonucleotídeos em experimentos conduzidos em bioreator por apresentar maior concentração de biomassa (4,59 g/L) quando comparada com a levedura Kluyveromyces marxianus var lactis CCT 4086 (3,37 g/L). Nesta fase, foi avaliada a influência da agitação (300 - 700 rpm), aeração (0,5 - 2 vvm) e concentração de glicerol (10 - 50 g/L) com o objetivo de estabelecer as melhores condições de processo, por meio de planejamento 23. Os maiores valores de YX/S (0,57- 0,60 g/g) foram obtidos nos ensaios conduzidos sob a menor concentração de glicerol (10 g/L), entretanto, o maiores valores de QX (0,44 - 0,62) foram obtidos em cultivos quando se utilizou valores de agitação superiores a 500 rpm. A análise estatística confirmou a significância das variáveis, concentração de glicerol e agitação, e permitiu estabelecer os modelos matemáticos representativos da influência destas variáveis na produção de biomassa e ribonucleotídeos por Hansenula anomala CCT 2648. Os resultados obtidos neste trabalho demonstraram o potencial de utilização do glicerol derivado da fabricação de biodiesel como fonte de carbono de baixo custo para a produção de biomassa e biomoléculas, como por exemplo os ribonucleotídeos. / Glycerol is considered to be the principle sub-product derived from biodiesel production process. In this work, glycerol was used as the only carbon source for biomass accumulation and ribonucleotides production. For this purpose screening of different strains was achieved, where the yeasts defined as Hansenula anomala CCT 2648 and Kluyveromyces marxianus var. lactis CCT 4086 showed high biomass yield (Y X/S) and productivity (QX) as well as better ribonucleotides production, using an initial concentration of glycerol of 30 g/L, temperature of 30oC and pH of 5,5. In addition, ethanol and organic acids were detected during glycerol assimilation by these two studied yeasts. Since these two selected yeast showed a promising potential in biomass and ribonucleotides production, a 24 complete factorial design was employed in order to study the influence of different parameters: glycerol initial concentration (10 - 50 g/L), yeast extract initial concentration (1-3 g/L), pH (4,5 - 6,5) and temperature (28 - 40oC) on the fermentation yield and productivity. According to the obtained results, the yeast Hansenula anomala CCT 2648 achieved higher biomass production (4,59 g/L) when compared to Kluyveromyces marxianus var lactis CCT 4086 (3,37 g/L). Such fact lead to the selection of Hansenula anomala CCT 2648 for a scale-up production of ribonucleotides using a stirred stank bioreactor, where the influence of the following fermentation parameters were studied: agitation (300 - 500 rpm), air input (0,5 - 2 vvm) and glycerol initial concentration (10 - 50 g/L) using a 23 full factorial design. High values of YX/S (0,57 - 0,6 g/g) were observed when the experiments were carried out at lower glycerol concentration (10 g/L), while high values of QX (0,44 - 0,62 g/L.h) were observed at high agitation rates of 500 rpm and above. Mathematical models were also created to confirm this influence of glycerol initial concentration and bioreactor stirring on biomass and ribonucleotides production by Hansenula anomala CCT 2648. Obtained results in this work demonstrated the feasibility of using glycerol derived from biodiesel production as a promising low-value carbon source for biomass and ribonucleotides accumulation.

Este trabalho teve como objetivo a utilização de glicerol, principal subproduto da produção de biodiesel, como fonte de carbono para a produção de biomassa e ribonucleotídeos de leveduras. Com este propósito, foi realizado um screening de leveduras para identificar aquelas cepas com maior capacidade de crescimento em glicerol. As leveduras Hansenula anomala CCT 2648 e Kluyveromyces marxianus var. lactis CCT 4086 apresentaram os maiores valores de fator de conversão (YX/S) e produtividade volumétrica em células (QX) sob condições fixas de glicerol (30 g/L), temperatura (30 oC) e pH (5,5). Foi também detectada a produção de etanol e ácidos orgânicos, por algumas das leveduras estudadas em meios contendo glicerol. Com base no potencial de produção de biomassa das cepas selecionadas na etapa anterior, foram realizados estudos para avaliar a influência da concentração de glicerol (10 - 50 g/L), extrato de levedura (1 - 3 g/L), pH(4,5 - 6,5) e temperatura (28 - 40oC) no crescimento destas linhagens, utilizando planejamento experimental 24. De acordo com os resultados, a levedura Hansenula anomala CCT 2648 foi selecionada para a produção de biomassa e ribonucleotídeos em experimentos conduzidos em bioreator por apresentar maior concentração de biomassa (4,59 g/L) quando comparada com a levedura Kluyveromyces marxianus var lactis CCT 4086 (3,37 g/L). Nesta fase, foi avaliada a influência da agitação (300 - 700 rpm), aeração (0,5 - 2 vvm) e concentração de glicerol (10 - 50 g/L) com o objetivo de estabelecer as melhores condições de processo, por meio de planejamento 23. Os maiores valores de YX/S (0,57- 0,60 g/g) foram obtidos nos ensaios conduzidos sob a menor concentração de glicerol (10 g/L), entretanto, o maiores valores de QX (0,44 - 0,62) foram obtidos em cultivos quando se utilizou valores de agitação superiores a 500 rpm. A análise estatística confirmou a significância das variáveis, concentração de glicerol e agitação, e permitiu estabelecer os modelos matemáticos representativos da influência destas variáveis na produção de biomassa e ribonucleotídeos por Hansenula anomala CCT 2648. Os resultados obtidos neste trabalho demonstraram o potencial de utilização do glicerol derivado da fabricação de biodiesel como fonte de carbono de baixo custo para a produção de biomassa e biomoléculas, como por exemplo os ribonucleotídeos. / Glycerol is considered to be the principle sub-product derived from biodiesel production process. In this work, glycerol was used as the only carbon source for biomass accumulation and ribonucleotides production. For this purpose screening of different strains was achieved, where the yeasts defined as Hansenula anomala CCT 2648 and Kluyveromyces marxianus var. lactis CCT 4086 showed high biomass yield (Y X/S) and productivity (QX) as well as better ribonucleotides production, using an initial concentration of glycerol of 30 g/L, temperature of 30oC and pH of 5,5. In addition, ethanol and organic acids were detected during glycerol assimilation by these two studied yeasts. Since these two selected yeast showed a promising potential in biomass and ribonucleotides production, a 24 complete factorial design was employed in order to study the influence of different parameters: glycerol initial concentration (10 - 50 g/L), yeast extract initial concentration (1-3 g/L), pH (4,5 - 6,5) and temperature (28 - 40oC) on the fermentation yield and productivity. According to the obtained results, the yeast Hansenula anomala CCT 2648 achieved higher biomass production (4,59 g/L) when compared to Kluyveromyces marxianus var lactis CCT 4086 (3,37 g/L). Such fact lead to the selection of Hansenula anomala CCT 2648 for a scale-up production of ribonucleotides using a stirred stank bioreactor, where the influence of the following fermentation parameters were studied: agitation (300 - 500 rpm), air input (0,5 - 2 vvm) and glycerol initial concentration (10 - 50 g/L) using a 23 full factorial design. High values of YX/S (0,57 - 0,6 g/g) were observed when the experiments were carried out at lower glycerol concentration (10 g/L), while high values of QX (0,44 - 0,62 g/L.h) were observed at high agitation rates of 500 rpm and above. Mathematical models were also created to confirm this influence of glycerol initial concentration and bioreactor stirring on biomass and ribonucleotides production by Hansenula anomala CCT 2648. Obtained results in this work demonstrated the feasibility of using glycerol derived from biodiesel production as a promising low-value carbon source for biomass and ribonucleotides accumulation.

Aicardi-Goutières syndrome (AGS) is an autosomal recessive encephalopathy with low incidence. The disease is caused by mutations in the genes encoding for TREX1, SAMHD1, ADAR, IFIH1 and the three genes encoding for the heterotrimeric RNase H2 enzyme. Biallelic mutations in any of the genes cause elevated type I interferon levels in the cerebrospinal fluid (CSF), the hallmark of AGS. In AGS patients, increased type I interferon levels cause massive inflammation in the brain that leads to mental and physical retardation that likely cause death in early childhood. AGS shows significant overlap with the prototypic autoimmune disease systemic lupus erythematosus (SLE). Like AGS patients, SLE patients are also characterized by increased type I interferon levels, anti-nuclear autoantibodies (ANAs) and arthritis. Moreover, heterozygous mutations in TREX1, SAMHD1 and RNase H2 are also found in a small fraction of SLE patients. Due to the genetic, molecular and clinical overlap, AGS is regarded as a monogenic variant of SLE. This overlap allows for the investigation of SLE pathomechanisms using genetically engineered mouse models with AGS-related mutations. In order to generate a mouse model that allows for the identification of pathomechanisms in AGS patients with mutations in the genes encoding for the RNase H2 enzyme, we generated mice with deficiency for the RNase H2 enzyme. Mice with complete deficiency for the RNase H2 enzyme (Rnaseh2c-/- or Rnaseh2bKOF/KOF) died perinatally or were stillborn. Mouse embryonic fibroblasts (MEFs) from E14.5 Rnaseh2bKOF/KOF embryos displayed impaired proliferation that was caused by the accumulation of MEF cells in G2/M of the cell cycle which increased with cultivation time or if MEF cells were isolated from E18.5 Rnaseh2bKOF/KOF embryos. Gene expression analysis of E14.5 fetal liver cells revealed a robust p53-mediated DNA damage response with the cell cycle inhibitor cyclin- dependent kinase inhibitor 1a (Cdkn1a, p21) being the most up-regulated gene. We found increased numbers of phosphorylated histone H2AX (γH2AX) in fetal liver and thymus cells from E18.5 Rnaseh2bKOF/KOF embryos, indicative of DNA double-strand breaks. Finally, we could show increased ribonucleotide loads in genomic DNA from embryos that were completely deficient for the RNase H2 enzyme. Collectively, we have demonstrated that complete RNase H2 deficiency causes persistent genomic ribonucleotide loads that render the DNA instable and prone to DNA strand breaks. DNA damage leads to the activation of p53 that in turn activates the cell cycle inhibitor p21 that inhibits cell cycle progression and likely causes accumulation of RNase H2-deficient cells in G2/M. To bypass early lethality we also generated bone marrow chimera and cell type-specific knockouts of the Rnaseh2b gene. While fetal liver cells of E14.5 Rnaseh2bKOF/KOF embryos could maintain hematopoiesis of irradiated recipient mice for almost one year, bone marrow cells from these primary recipients failed to reconstitute lethally irradiated mice in a secondary transfer. In line with this observation, VavCre-mediated deletion of the Rnaseh2b gene caused a more than hundred fold reduction of peripheral blood B cells, while B cell numbers remained unaltered upon CD19Cre-mediated deletion that occurs much later in B cell development. These data suggested that RNase H2 deficiency leads to the accumulation of genomic ribonucleotides that might cause the accumulation of a so far uncharacterized DNA damage species with increasing cell cycle passages. The data further supported our hypothesis that the impact of RNase H2 deficiency is determined by the number of cell proliferation. Finally, an epidermis-specific knockout of the Rnaseh2b gene displayed the most dramatic phenotype. Knockout mice were characterized by hyperpigmentation, hair loss and spontaneous ulcerations of the skin. Microscopically, these mice displayed moderate thickening of the epidermis and dermal fibrosis as indicated by increased collagen deposition. Macroscopic skin phenotypes were completely dependent on p53 expression, since concomitant deletion of the p53 gene rescued mice from hyperpigmentation, hair loss and ulcerations. This data demonstrated that Rnaseh2b deficiency in the epidermis may also lead to DNA damage and subsequent p53 activation as shown for fetal liver from E14.5 RNase H2-deficient embryos. Preliminary data also indicate an increased incidence of cancer formation in RNase H2/p53 double knockouts, identifying the RNase H2 enzyme as an important tumor suppressor.

Aicardi-Goutières syndrome (AGS) is an autosomal recessive encephalopathy with low incidence. The disease is caused by mutations in the genes encoding for TREX1, SAMHD1, ADAR, IFIH1 and the three genes encoding for the heterotrimeric RNase H2 enzyme. Biallelic mutations in any of the genes cause elevated type I interferon levels in the cerebrospinal fluid (CSF), the hallmark of AGS. In AGS patients, increased type I interferon levels cause massive inflammation in the brain that leads to mental and physical retardation that likely cause death in early childhood. AGS shows significant overlap with the prototypic autoimmune disease systemic lupus erythematosus (SLE). Like AGS patients, SLE patients are also characterized by increased type I interferon levels, anti-nuclear autoantibodies (ANAs) and arthritis. Moreover, heterozygous mutations in TREX1, SAMHD1 and RNase H2 are also found in a small fraction of SLE patients. Due to the genetic, molecular and clinical overlap, AGS is regarded as a monogenic variant of SLE. This overlap allows for the investigation of SLE pathomechanisms using genetically engineered mouse models with AGS-related mutations. In order to generate a mouse model that allows for the identification of pathomechanisms in AGS patients with mutations in the genes encoding for the RNase H2 enzyme, we generated mice with deficiency for the RNase H2 enzyme. Mice with complete deficiency for the RNase H2 enzyme (Rnaseh2c-/- or Rnaseh2bKOF/KOF) died perinatally or were stillborn. Mouse embryonic fibroblasts (MEFs) from E14.5 Rnaseh2bKOF/KOF embryos displayed impaired proliferation that was caused by the accumulation of MEF cells in G2/M of the cell cycle which increased with cultivation time or if MEF cells were isolated from E18.5 Rnaseh2bKOF/KOF embryos. Gene expression analysis of E14.5 fetal liver cells revealed a robust p53-mediated DNA damage response with the cell cycle inhibitor cyclin- dependent kinase inhibitor 1a (Cdkn1a, p21) being the most up-regulated gene. We found increased numbers of phosphorylated histone H2AX (γH2AX) in fetal liver and thymus cells from E18.5 Rnaseh2bKOF/KOF embryos, indicative of DNA double-strand breaks. Finally, we could show increased ribonucleotide loads in genomic DNA from embryos that were completely deficient for the RNase H2 enzyme. Collectively, we have demonstrated that complete RNase H2 deficiency causes persistent genomic ribonucleotide loads that render the DNA instable and prone to DNA strand breaks. DNA damage leads to the activation of p53 that in turn activates the cell cycle inhibitor p21 that inhibits cell cycle progression and likely causes accumulation of RNase H2-deficient cells in G2/M. To bypass early lethality we also generated bone marrow chimera and cell type-specific knockouts of the Rnaseh2b gene. While fetal liver cells of E14.5 Rnaseh2bKOF/KOF embryos could maintain hematopoiesis of irradiated recipient mice for almost one year, bone marrow cells from these primary recipients failed to reconstitute lethally irradiated mice in a secondary transfer. In line with this observation, VavCre-mediated deletion of the Rnaseh2b gene caused a more than hundred fold reduction of peripheral blood B cells, while B cell numbers remained unaltered upon CD19Cre-mediated deletion that occurs much later in B cell development. These data suggested that RNase H2 deficiency leads to the accumulation of genomic ribonucleotides that might cause the accumulation of a so far uncharacterized DNA damage species with increasing cell cycle passages. The data further supported our hypothesis that the impact of RNase H2 deficiency is determined by the number of cell proliferation. Finally, an epidermis-specific knockout of the Rnaseh2b gene displayed the most dramatic phenotype. Knockout mice were characterized by hyperpigmentation, hair loss and spontaneous ulcerations of the skin. Microscopically, these mice displayed moderate thickening of the epidermis and dermal fibrosis as indicated by increased collagen deposition. Macroscopic skin phenotypes were completely dependent on p53 expression, since concomitant deletion of the p53 gene rescued mice from hyperpigmentation, hair loss and ulcerations. This data demonstrated that Rnaseh2b deficiency in the epidermis may also lead to DNA damage and subsequent p53 activation as shown for fetal liver from E14.5 RNase H2-deficient embryos. Preliminary data also indicate an increased incidence of cancer formation in RNase H2/p53 double knockouts, identifying the RNase H2 enzyme as an important tumor suppressor.

No description available.

Raman optical activity (ROA) is a powerful probe of the structure and behaviour of biomolecules in aqueous solution for a number of important problems in molecular biology. Although ROA is a very sensitive technique for studying biological samples, it is a very weak effect and the conditions of high concentration and long data collection time required limit its application for a wide range of biological samples. These limitations could possibly be overcome using the principle of surface enhanced Raman scattering (SERS). The combination of ROA with SERS in the form of surface enhanced ROA (SEROA) could be a solution for widening the application of ROA. In the last few years, the generation of reliable SEROA spectra of biomolecules has been problematic due to non-homogenous colloidal systems forming and low signal-to-noise ratios which complicated detection of the true SEROA signal from the analyte. L- and D-enantiomers give full or partially mirror image chiroptical spectra, this property of enantiomers can be employed to prove the chiroptical activity of the SEROA technique. In this thesis we employed a hydrophilic polycarbopol polymer as stabilising media which has led to the first report of mirror image SEROA bands for enantiomeric structures. This new technique of incorporating the hydrogel polymer as a means to stabilise the colloidal system has proven to be reliable in obtaining high quality SEROA spectra of D- and L-enantiomers of ribose and tryptophan. In an extension of the hydrogel-stabilised SEROA work, we also demonstrate that single nanoparticle plasmonic substrate such as silver silica nanotags can enhance the weak ROA effect. These dye tagged silica coated silver nanoparticles have enabled a chiral response to be transmitted from a chiral analyte to the plasmon resonance of an achiral metallic nanostructure. The measurement of mirror image SERROA bands for the two enantiomers of each of ribose and tryptophan was confirmed for this system. The generation of SEROA for both systems was achieved and confirmed SEROA as a new sensitive tool for analysis of biomolecular structure. In a related project, Raman and ROA spectra were measured for adenosine and seven of its derivative ribonucleotides. Both of these spectroscopic techniques are shown to be sensitive to the site and degree of phosphorylation, with a considerable number of marker bands being identified for these ribonucleotides. Moreover, the SERS studies of these ribonucleotides were also performed. The obtained SERS spectra were shown similar features that confirm these analytes interact with the surface in a similar manner, hence limiting the structural sensitivity of this method towards phosphate position. Short dipeptides such as diketopiperazine (DKP) have been investigated during the last decades as both natural and synthetic DKPs have a wide variety of biological activities. Raman and ROA spectra of linear and cyclic dialanine and diserine were measured to charecterize their solution structures. Density functional theory (DFT) calculations were carried out by a collaborator to assist in making vibrational band assignments. Considerable differences were observed between the ROA bands for the cyclic and linear forms of both dialanine and diserine that reflect large differences in the vibrational modes of the polypeptide backbone upon cyclicization. In this study, the ROA spectra of cyclic dialanine and diserine have been reported for the first time which demonstrated that ROA spectroscopy when utilised in combination with computational modelling clearly provides a potential tool for characterization of cyclic peptides.