GLK1 and GLK2 transcription factors have been suggested to be involved in the regulation of chloroplast development, organic nitrogen signaling, disease resistance and circadian rhythmicity (Waters et al. 2009; Gutiérrez et al. 2008; Savitch et al. 2007; Sprott et al. 2010). This implies that multiple factors may play roles in regulation of GLK genes. In the present study, transcriptional regulation of GLK1 and GLK2 in Arabidopsis by various endogenous and environmental stimuli was investigated with the objective of elucidating the primary signalling pathway affecting expression of these two genes. Collectively, results of GLK1 and GLK2 expression in response to the experimental treatments of Arabidopsis point to the regulation of the two genes by changes in photosynthetic metabolism and reactive oxygen species (ROS) levels, and by organic nitrogen signalling. Changes in ROS levels and organic nitrogen signalling may also affect the two genes indirectly by interfering with or altering photosynthetic metabolism.

Electrodeposition method is used to deposit diamond-like carbon (DLC) films on copper substrates via anodic oxidation at low temperature. These films are characterized using Raman spectroscopy, Fourier transform infrared spectroscopy and scanning electron microscopy. Homogeneity of these films is studied using Raman spectroscopy and scanning electron microscopy. Scotch tape peel tests indicate adherent film on copper substrate. Carbon phase transformation is studied using thermal annealing experiments in conjunction with Raman spectroscopy and scanning electron microscopy. A cathodic electrochemical method is also studied to deposit diamond-like carbon films on copper substrates. However, films deposited by the cathodic route have poor adhesion and quality compared to anodically deposited films. It is also possible to grow diamond phase on copper substrates using acetylene in liquid ammonia via electrodeposition route. An electrochemical method is proposed for boron doping into DLC films.

Submitted by Nadir Basilio (nadirsb@uninove.br) on 2016-05-25T16:04:47Z No. of bitstreams: 1 Nicole Cristine Rigonato de Oliveira.pdf: 1240943 bytes, checksum: 14fa67b6692555f3bc76fb1f34186220 (MD5) / Made available in DSpace on 2016-05-25T16:04:47Z (GMT). No. of bitstreams: 1 Nicole Cristine Rigonato de Oliveira.pdf: 1240943 bytes, checksum: 14fa67b6692555f3bc76fb1f34186220 (MD5) Previous issue date: 2014-12-19 / The acute respiratory distress syndrome (ARDS) is a disease characterized by respiratory failure due to an inflammatory response, which has high morbidity and mortality. Several cytokines seem to orchestrate the acute and chronic processes, mainly mediated by toll like receptors (TLRs). The literature demonstrates that aerobic exercise (AE) is capable of decreasing the secretion of pro-inflammatory cytokines in the lungs mediated increased release of interleukin 10 (IL-10), and AE modulate expression of TLRs and antioxidant enzymes. The objective of this study was to evaluate whether the anti-inflammatory effects of AE in an experimental model of intra and extrapulmonary ARDS are mediated by IL-10. For this, the animals were subjected to physical training on a treadmill, moderate for 4 weeks. 24 hours after the last physical test, animals received LPS by intratracheally (it) (10ug / animal) or intraperitoneally (ip) (100ug / animal). After 24 hours, the animals were assessed for the number of cells and pro- and anti-inflammatory cytokines in bronchoalveolar lavage fluid (BAL) and serum were the number of neutrophils in the lung parenchyma and expression of TLR4, TLR7, SOD, ânion and QL in lung homogenates. AE reduced accumulation of neutrophils in the lung parenchyma, so it LPS and LPS ip (p <0.01) and BAL (p <0.01). AE attenuate the levels of proinflammatory cytokines in BAL and serum (p <0.05), as measured by ELISA. AE had levels of anti-inflammatory cytokine IL-10 increased in the serum and BAL (p <0.05). AE also reduced the expression of TLR4 in lung homogenates (p <0.05) and increased the expression of TLR7 in group LPS + AE evaluated by western blotting (p <0.05). The results for the antioxidant enzyme SOD showed a significant increase in the groups submitted to AE. It follows that the impact of reduced LPS-induced ARDS, regardless of etiology, and these effects appear to be mediated by modulation of the AE secretion of anti-inflammatory cytokines, especially IL-10, modulating TLR4 and TLR7 and increased expression of SOD (p <0.05). / A síndrome do desconforto respiratório agudo (SDRA) é uma doença caracterizada pela insuficiência respiratória decorrente a uma resposta inflamatória, que apresenta alta morbi-mortalidade. Diversas citocinas parecem orquestrar os processos agudo e crônico, mediados principalmente por receptores toll like (TLRs). A literatura demonstra que o exercício aeróbio (EA) é capaz de diminuir a secreção de citocinas pró-inflamatórias nos pulmões, mediado pelo aumento da liberação de interleucina 10 (IL-10), além de o EA modular a expressão de TLRs e enzimas antioxidantes. Assim, o objetivo desse estudo foi avaliar se os efeitos anti-inflamatórios do EA em modelo experimental da SDRA intra e extrapulmonar são mediados por IL-10. Para isso, os animais foram submetidos ao treinamento físico em esteira, de intensidade moderada, durante 4 semanas. Após 24 horas ao último teste físico, os animais receberam LPS por via intra-traqueal (it) (10ug/animal) ou por via intra-peritoneal (ip) (100ug/animal). Após 24 horas, os animais foram avaliados para o número de células e de citocinas pró e anti-inflamatórias no fluído do lavado broncoalveolar (LBA) e no soro, número de neutrófilos no parênquima pulmonar e expressão do TLR4, TLR7, SOD, ânion e QL nos homogenatos de pulmão. EA reduziu a acumulação de neutrófilos no parênquima pulmonar, tanto LPS it e LPS ip (p <0,01) e no LBA (p <0,01). EA atenuou os níveis de citocinas pró-inflamatórias no LBA e soro (p <0,05), avaliada por ELISA. EA teve os níveis da citocina anti-inflamatória IL-10 aumentados no soro e LBA (p <0,05). EA também reduziu a expressão de TLR4 nos homogenatos de pulmão (p <0,05) e aumentou a expressão de TLR7 no grupo de LPS + EA avaliada por western blotting (p <0,05). Os resultados para a enzima antioxidante SOD apresentou aumento significante nos grupos submetidos ao EA. Conclui-se que EA reduziu o impacto de SDRA induzida por LPS, independente da etiologia e tais efeitos parecem estar mediados pela modulação do EA na secreção de citocinas anti-inflamatórias, principalmente da IL-10, na modulação de TLR4 e TLR7 e no aumento da expressão de SOD (p<0,05).

Electrodeposition is a novel method for fabrication of diamond-like carbon (DLC) films on metal substrates. In this work, DLC was electrochemically deposited on different substrates based on an anodic oxidation cyclization of acetylene in liquid ammonia. Successfully anodic deposition was carried out for DLC onto nickel substrate at temperatures below -40°C. Comparative studies were performed on a series of different carbon sources (acetylene, sodium acetylide, and a mixture of acetylene and sodium acetylide). The films were characterized using a variety of methods including Raman spectroscopy, Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), XPS valence band spectra, and/or scanning electron microscopy (SEM). Raman spectroscopy is used as a bench mark technique to verify the presence of deposited DLC films, to access the films homogeneities, and to provide the ratio of the different carbon phases, mainly disordered graphite (D) and graphite (G) phases in the films. A combination of the Raman with FTIR and valence band spectra analysis allowed the distinction between hydrogenated DLC and unhydrogenated DLC films. Three different kinds of DLC [(1) hydrogenated DLC (a-C:H); (2) tetrahedral hydrogenated DLC (ta-C:H); and (3) graphitic-like DLC] were deposited depending upon the deposition conditions and substrates. Temperature and current density are the most important parameters to govern the quality of the deposited films, where adding of acetylide into the electrolyte led to films with a higher degree of graphitic phases. The proposed mechanism for acetylene anodic oxidation does not involve direct electron transfer but electrochemical cyclization of acetylene radical cations and hydrogen abstraction at the termination steps. Sodium acetylide, however, dissociates to an acetylenic ion, C2H-, in liquid ammonia. The electrochemistry heterogeneity also leads to island and two-dimensional (2D) nucleation growth of DLC films. Different bond formations of metal to carbon and different chemisorptions of acetylene on metal play important roles in governing the film properties. Using mixed C2HNa and C2H2 as electrolyte, polycrystalline diamond and hexagonal diamond are formed on Mo and stainless steel, respectively. This is the first time to report that polycrystalline diamond can be grown electrochemically at temperature below -40ºC. The preliminary studies on substrate pretreatment with diamond powder and SiC 600 are studied. The effect of the substrate on the film quality for the electrodeposited DLC films described herein is similar to that for diamond deposition via chemical vapor deposition (CVD).

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22143号 / 医博第4534号 / 新制||医||1039(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 妻木 範行, 教授 森本 尚樹, 教授 別所 和久 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22350号 / 医博第4591号 / 新制||医||1042(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 斎藤 通紀, 教授 浅野 雅秀, 教授 安達 泰治 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Multicellular organisms typically depend on O₂ for energy production to maintain normal cellular function, and even brief periods of O₂ deprivation may have fatal consequences. The aqueous environment is prone to changes in ambient water O₂ tension (PO₂) and thus the ability of fish to sense changes in water PO₂ and to elicit appropriate physiological responses is essential for their survival. Studies on fish O₂ chemoreception have identified neuroepithelial cells (NECs), which are characterized as having dense-cored vesicles containing serotonin (5-HT), as peripheral O₂ chemoreceptors. Upon exposure to hypoxia, isolated and cultured NECs in vitro depolarize, likely resulting in neurotransmitter release. However, to date there is no evidence that NECs are activated by hypoxia in vivo to initiate physiological responses such as the hypoxic ventilatory response (HVR), which is the focus of this thesis. Initial findings demonstrated that larval zebrafish fine-tune the HVR as early as 4 days post fertilization (dpf) and by 7 dpf, the HVR aids in O₂ uptake under hypoxic conditions. In addition, the HVR is multiphasic, with an initiation phase followed by a decline phase that gradually stabilizes above normoxic baseline values (Chapter 2). In the absence of tools to probe the hypoxia sensitivity of NECs in vivo, research focused on Merkel-like cells (MLCs), a newly proposed O₂ chemoreceptor in larval zebrafish. Using in vivo calcium imaging it was shown that MLCs are stimulated by hypoxia. Data suggest that MLCs are responsible for the initiation phase of the HVR, while peripheral sensory neurons (PSNs)/peripheral sensory ganglia (PSG) that innervate MLCs play a more important role in reducing ventilation during the decline phase of the HVR (Chapter 4). Attempts at identifying the putative neurotransmitter(s) involved in the O₂ signal transduction pathway revealed that adrenaline (AD), serotonin (5-HT), and dopamine (DA) are probable candidates (Chapter 4), though the presence of AD and DA within MLCs is yet to be confirmed. In addition, 5-HT likely plays a role in the central nervous system (CNS), integrating peripheral signals resulting in the final HVR (Chapter 3). Taken together, this thesis provides the first evidence of putative O₂ chemoreceptors responding to hypoxia in vivo and thus significantly advances models for O₂ signal transduction in larval zebrafish.

Polioviruses have been around for a long time in man's history. Before the development of killed and live virus vaccines in the 1960's, poliomyelitis was a serious problem in public health. Since then paralytic poliomyelitis remains a threat in certain underdeveloped countries but has been considered a conquered disease in the developed world. The molecular epidemiology of wild-type 1 polioviruses (WPV1) isolated in Angola in 2005, the Democratic Republic of the Congo (DRC) in 2006-2008 and Namibia in 2006 were investigated by sequence analysis of the complete VP1 gene of all isolates. All outbreak viruses clustered with the Indian type 1 genotype (SOAS) which was unique to India circulating endemically in the Uttar Pradesh (UP) and Bihar provinces in Northern India. Epidemiological and virological analyses suggested that the Namibia outbreak virus had been circulating without detection for at least one year in Angola. Four cases of acute flaccid paralysis (AFP) occurred in children in Madagascar in 2005. Molecular analysis confirmed cVDPV type 2 and 3 in affected patients. The first case, occurred in Toliara II district, on 09 April 2005. The last two cases were in the Toliara I and Beloha districts and paralysis onset on 26 June and 13 July 2005 respectively. Partial genomic sequencing of the poliovirus isolates revealed considerable divergence from the prototype Sabin strain in all cases. This is the second time that type 2 cVDPV is associated with an outbreak of AFP in Madagascar, and to our knowledge the first time that a type 3 cVDPV is identified in Madagascar. A total of fifty-six children with AFP were found to excrete VDPVs of serotype 2 in the DRC between 2005 and 2010. These viruses represent at least three emergences and at least two outbreaks. Partial genomic sequencing of the poliovirus isolates revealed considerable nucleotide sequence divergence of between 1.1% to 2o/o from the prototype Sabin strain in the VP1 region of the viral genome. This was the first time that a type 2 cVDPV outbreak was detected in the DRC. In total, 89 viral isolates obtained from Ethiopia during 2007 to 2010 and partial sequencing analysis confirmed that 13 isolates were VDPV's. Seven AFP cases were type 3, 4 AFP cases were type 2 and 2 contacts for type 3. Partial genomic sequencing of the poliovirus isolates revealed considerable divergence from the prototype Sabin strain in all cases. Finally, cases of AFP where only Sabin-like viruses were identified were investigated in South Africa with 11 possible VAPP cases identified with recombinant events in the 30 region and also revealing a mutation that restore the original stem-loop structure in the internal ribosomal entry site (IRES) in the 5' Non-Translated Region (NTR). In this study, the molecular epidemiology of poliovirus outbreaks that occurred in Angola, Namibia, and the DRC is described that were associated with wild polio 1 and 3. Investigation of Sabin-like vaccine strains in the DRC, Madagascar and Ethiopia identified vaccine-derived polioviruses in AFP cases as well as possible vaccineassociated paralytic poliovirus in South Africa. Copyright / Thesis (PhD)--University of Pretoria, 2012. / Medical Virology / Unrestricted

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Objective: To determine whether there was a correlation between induction of polymicrobial sepsis, modulation of tissue Toll-like receptor (TLR) gene, and protein expression and survival outcome. Design: Prospective, randomized animal study. Setting: University medical school research laboratory. Subjects: Age- and weight-matched ICR/HSD mice. Interventions: Sepsis was induced by cecal ligation and puncture (CLP). No-surgery and sham (laparotomy)-operated mice were controls. We also examined tissue TLR2 and TLR4 messenger RNA and TLR4 protein levels in mice treated with an immunomodulator that increases survival in polymicrobial sepsis. In the immunomodulator study, mice were treated with glucan phosphate (50 mg/kg, intraperitoneally) 1 hr before CLP. No-surgery, sham surgery, glucan + no-surgery, sham surgery + glucan, and CLP groups were employed as controls. Measurements and Main Results: Total RNA was isolated from liver, lung, and spleen at 0, 1, 3, 6, 8, and 24 hrs after CLP. TLR gene expression was assessed by reverse transcription-polymerase chain reaction. Tissue TLR4 protein levels were evaluated at 24 hrs by Western blot and immunohistochemistry. CLP sepsis increased (p < .05) liver and lung TLR2 and TLR4 gene expression compared with controls. TLR4 protein concentrations also were increased. Increased TLR2/4 gene and TLR4 protein expression correlated with mortality. Immunoprophylaxis with glucan phosphate increased (p < .001) long-term survival (20% vs. 70%) but inhibited (p < .05) CLP-induced increases in tissue TLR2 and TLR4 messenger RNA expression as well as TLR4 protein expression. Conclusions: Early increases in TLR2/4 gene and TLR4 protein expression correlated with mortality, whereas blunting TLR gene and protein expression correlated with improved long-term survival. This suggests that early up-regulation of tissue TLR2/4 may play a role in the proinflammatory response and pathophysiology of polymicrobial sepsis.