The diploma thesis engages in the reconstruction of railway tunnel Domasovsky that is situated at the track section Olomouc – Krnov. It illustrates the current state of the construction and the geotechnical conditons of the area of interest, it also presents the potencial solutions. The design is verified by stuructural analysis, the design documentation and technical report are also enclosed.

Plant development is complex and highly regulated. Tens of thousands of genes have been sequenced for the model plant <em>Arabidopsis thaliana</em>, yet few have been functionally annotated and characterized. This thesis describes the expression analysis and characterization of four genes in <em>Arabidopsis</em>. Three of these belong to the eukaryotic translation initiation factor 5A (eIF5A) gene family, and the fourth encodes diacylglycerol acyltransferase 1 (DGAT1). Putative roles for these genes in the development of <em>Arabidopsis thaliana</em> are described. <br /><br /> eIF5A is the only known protein to contain the amino acid hypusine. It has been demonstrated previously that eIF5A acts as a shuttle protein, moving specific mRNAs from the nucleus to the cytoplasm for translation. In <em>Arabidopsis thaliana</em> (At), there are three isoforms of eIF5A, and it is clear from the present study that they each have a unique temporal and spatial expression pattern. AteIF5A-1 and -2 are up-regulated during natural senescence and wounding/pathogenesis, respectively, and it is proposed that they regulate the onset of programmed cell death during these events. AteIF5A-3 is up-regulated in elongating meristem of the root, and it is proposed that this isoform is involved in cell growth. <br /><br /> Over-expression of the individual <em>AteIF5A</em> isoforms <em>in planta</em> resulted in pleiotropic phenotypes. When <em>AteIF5A-1</em> or <em>AteIF5A-2</em> was over-expressed, the phenotypes observed were indicative of their putative roles in the translation of proteins required for programmed cell death. When <em>AteIF5A-3</em> was over-expressed, the phenotypes were indicative of a role for this protein in the regulation of cell and tissue elongation. <br /><br /> Lipid analysis of rosette leaves from <em>Arabidopsis thaliana</em> revealed an accumulation of triacylglycerol with advancing leaf senescence coincident with an increase in the abundance and size of plastoglobuli. The terminal step in the biosynthesis of triacylglycerol in <em>Arabidopsis</em> is catalyzed by DGAT1. When gel blots of RNA isolated from rosette leaves at various stages of development were probed with the <em>Arabidopsis</em> EST clone, E6B2T7, which has been annotated as DGAT1, a steep increase in DGAT1 transcript levels was evident in the senescing leaves coincident with the accumulation of triacylglycerol. The increase in DGAT1 transcript correlated temporally with enhanced levels of DGAT1 protein detected immunologically. Two lines of evidence indicated that the triacylglycerol of senescing leaves is synthesized in chloroplasts and sequesters fatty acids released from the catabolism of thylakoid galactolipids. First, triacylglycerol isolated from senescing leaves proved to be enriched in hexadecatrienoic acid (16:3) and linolenic acid (18:3), which are normally present in thylakoid galactolipids. Second, DGAT1 protein in senescing leaves was found to be associated with chloroplast membranes. These findings collectively indicate that DGAT1 plays a role in senescence by sequestering fatty acids de-esterified from galactolipids into triacylglycerol.

The role of cyclic guanosine monophosphate (cGMP) dependent protein kinase (PKG) in necrotic and apoptotic pathways of many cell types is well established; however its role in the ischemic preconditioning (IPC) of cardiomyocytes is not clearly defined. In the current study, we assessed the hypothesis that PKG protects against cell death following ischemidreperfusion injury in myocytes subjected to IPC. Freshly isolated adult rat ventricular myocytes were subjected to IPC by incubating in ischemic buffer for 30 minutes (min) followed by incubation in normal medium for 30 min. Prolonged simulated ischemia (SI) was created by incubating myocytes in the ischemic buffer for 90 min and reoxygenation (RO) for 120 min in the normal medium. Necrosis was determined by trypan blue exclusion and apoptosis was assessed by TUNEL assay. IPC reduced necrosis as shown by significant decrease in trypan blue positive cells as compared to virgin non-preconditioned myocytes subjected to SI and RO alone (p<.01). Similarly, the number of TUNEL positive myocytes following SI and 18 hrs of RO were significantly reduced in the IPC group. Treatment with PKG inhibitor, KT5832 (2pM) completely abolished the protection against necrosis by IPC. However, KT5832 failed to abolish the protective affect of IPC against apoptosis. Furthermore, myocytes infected with an adenoviral construct of PKG-la (1 x 1 o4 particles/cell) significantly reduced the number of trypan blue and TUNEL positive cells. These results suggest that the PKG signaling pathway plays an essential role in the preconditioning of myocytes against necrosis following SI / RO injury. Furthermore, while the overexpression of PKG protects myocytes against necrosis, as well as apoptosis, IPC may not induce a sufficient level of PKG during 18 hours of RO to induce protection against apoptosis.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disease in the world. The most accepted hypothesis for the cause of this disease is the amyloid cascade hypothesis, which postulates that the formation of extracellular neurotoxic amyloid-beta binds specific receptors on the surface of neuronal and glial cells to increase cell stress leading to cell death. Our laboratory previously showed that treatment of cultured human SHSY neuronal cells with amyloid beta increases the cellular levels of two key components (caspases-2 and -3) of the extrinsic apoptotic pathway, leading to cell death. The amyloid beta induced caspase elevation was blocked by simultaneously treating the cells with a short mimetic of human ependymin neurotrophic factor, hEPN-1, and the hEPN-1 treatment also blocked cell death. This thesis extends the AD investigation to show that treatment of SHSY cells with amyloid beta may also activate an intrinsic apoptotic mitochondrial stress pathway (assaying caspase-9 as a marker enzyme), and that hEPN-1 treatment significantly lowers this activation. In addition, our laboratory previously showed that treating SHSY cells with amyloid beta increases TUNEL staining, an assay for DNA fragmentation (a hallmark of end stage of apoptosis, and a different apoptotic marker than caspase activation). Treatment with hEPN-1 simultaneously with the amyloid beta, or 6 hrs post amyloid beta, significantly lowered the amyloid beta induced TUNEL signal. This thesis extended the earlier TUNEL experiments to show that hEPN-1 treatment can significantly lower the amyloid beta induced TUNEL staining even when added 18 hrs post amyloid beta. With respect to caspase-8, an initiator caspase in the extrinsic pathway, immunoblot assays of brain lysates from 8 month old transgenic AD mice showed that a 2 week oral delivery of hEPN-1 (conjugated to a carrier to deliver it across the blood brain barrier) significantly lowered caspase-8 levels. Finally, an assay of cellular inhibitors of apoptosis (cIAP) showed a significant increase in their cellular levels in SHSY cells, and in transgenic AD mice treated with hEPN-1, showing for the first time that hEPN-1 may aid cell survival by upregulating proteins known to directly bind specific caspases to block their activity leading to their degradation. The cIAP upregulation occurred in the presence or absence of amyloid beta, indicating that hEPN-1 likely does not block cell death by directly interfering with the interaction of amyloid beta with its cell surface receptors, but instead hEPN-1 may activate an independent cell survival signal transduction pathway in neuronal cells.

Spermatogenesis is a complex process of male germ cell development from diploid spermatogonia to haploid fertile spermatozoa. Apoptosis plays a vital role in limiting cell numbers and eliminating defective germ cells. This requires novel gene products, and precise and well-coordinated programmes of gene expression. It is therefore possible that a disruption of transcription factor function would significantly impact germ cell development. The present work was undertaken to use Staput separation followed by culture of purified germ cells of rodent testis since mammalian spermatogenesis cannot yet be recreated in vitro. Specificity of separation was assessed using immunocytochemistry to identify spermatogonia, spermatocytes and spermatids. The genotoxins H2O2, doxorubicin, N-ethyl-N-nitrosourea, N-methyl-N-nitrosourea, 6-mercaptopurine, 5-bromodeoxyuridine, methyl methanesulphonate and ethyl methanesulphonate were investigated. Cells were cultured and treated with different concentrations for each agent. DNA damage and apoptosis were measured by Comet and TUNEL assay respectively. Up-regulation of expression of the transcription factors Tbpl1, FHL5 and Gtf2a1l that are important post-meiotically, were examined using RT- PCR and qPCR. Protein production was evaluated using Western blotting. Tbpl1, FHL5 and Gtf2a1l were cloned in-frame into the inducible expression vector pET/100-TOPO. The recombinant clones were induced and successful expression of the proteins in E. coli was confirmed by SDS-PAGE and Western blotting. The recombinant clones obtained were used to demonstrate genotoxin induced impairment of gene expression. Thus, Staput-isolated rodent testicular germ cells seem to be a suitable model to study genotoxicity in vitro yielding result comparable to those reported in vivo. Furthermore, the work shows that genotoxins can impair gene expression.

This thesis tests the hypothesis that increased neuronal cell death in SIDS infants is related to the ability of risk factors, such as prone sleeping, to expose infants to intermittent hypercapnic hypoxia (IHH). Based on the hypothesis that the NMDA system is linked to neuronal death, by way of excitotoxicity, correlations were also sought between cell death and changes in NMDA receptor (NR1) expression in brainstem nuclei controlling cardiorespiratory function. The first aim of this study was to verify that increased neuronal cell death occurs in SIDS infants. To verify a piglet model of SIDS risk factors, brainstem changes were examined in piglets exposed to IHH, and comparisons were made to changes seen in SIDS infants. The NMDA receptor was characterised in controls for both the human infant and the piglet groups. Comparisons of neuronal changes were made with SIDS infants, and piglets exposed to IHH. Non-radioactive in-situ hybridisation and immunohistochemistry were performed on formalin fixed and paraffin embedded brainstem tissue to identify markers of cell death (caspase-3, active caspase-3, and TUNEL), and to examine NR1 mRNA and protein expressions. Staining was quantified using computerised image analysis software. Eight nuclei from the brainstem medulla (caudal in piglets, and mid in infants), and two nuclei from the rostral pons (infants) were studied. The first dataset included human infants aged 1-6 months with a diagnosis of SIDS (n=15) or non-SIDS (n=10). The second dataset comprised developing piglets aged 13-14 days, with controls (n=6), against those exposed to IHH for 2 (n=6) or 4 (n=5) days. Increased neuronal cell death was not verified in the SIDS infants, but abnormalities in NR1 expression were present in selected nuclei of the medulla. Piglets exposed to IHH had increased neuronal cell death and changes in NR1 in selected nuclei of the medulla. There was also a positive correlation between increased cell death and high NR1 levels. Preliminary data showed that SIDS infants who usually slept prone had some differences in NR1 compared to those who did not usually sleep prone. From these findings, it was concluded that IHH may underlie the abnormalities in NMDA receptor expression that are present in the brainstem of SIDS infants. Although IHH can induce an increase in neuronal cell death, its significance in the aetiology of SIDS is not known. In piglets, IHH induced cell death correlated with high NMDA expression in some brainstem nuclei, supporting the hypothesis that excitotoxicity may be involved in the mechanism for cell death. Moreover, this thesis presents for the first time, �preliminary pathological proof� of an association between prone sleeping and abnormal NMDA receptor expression in SIDS infants.

Plant development is complex and highly regulated. Tens of thousands of genes have been sequenced for the model plant <em>Arabidopsis thaliana</em>, yet few have been functionally annotated and characterized. This thesis describes the expression analysis and characterization of four genes in <em>Arabidopsis</em>. Three of these belong to the eukaryotic translation initiation factor 5A (eIF5A) gene family, and the fourth encodes diacylglycerol acyltransferase 1 (DGAT1). Putative roles for these genes in the development of <em>Arabidopsis thaliana</em> are described. <br /><br /> eIF5A is the only known protein to contain the amino acid hypusine. It has been demonstrated previously that eIF5A acts as a shuttle protein, moving specific mRNAs from the nucleus to the cytoplasm for translation. In <em>Arabidopsis thaliana</em> (At), there are three isoforms of eIF5A, and it is clear from the present study that they each have a unique temporal and spatial expression pattern. AteIF5A-1 and -2 are up-regulated during natural senescence and wounding/pathogenesis, respectively, and it is proposed that they regulate the onset of programmed cell death during these events. AteIF5A-3 is up-regulated in elongating meristem of the root, and it is proposed that this isoform is involved in cell growth. <br /><br /> Over-expression of the individual <em>AteIF5A</em> isoforms <em>in planta</em> resulted in pleiotropic phenotypes. When <em>AteIF5A-1</em> or <em>AteIF5A-2</em> was over-expressed, the phenotypes observed were indicative of their putative roles in the translation of proteins required for programmed cell death. When <em>AteIF5A-3</em> was over-expressed, the phenotypes were indicative of a role for this protein in the regulation of cell and tissue elongation. <br /><br /> Lipid analysis of rosette leaves from <em>Arabidopsis thaliana</em> revealed an accumulation of triacylglycerol with advancing leaf senescence coincident with an increase in the abundance and size of plastoglobuli. The terminal step in the biosynthesis of triacylglycerol in <em>Arabidopsis</em> is catalyzed by DGAT1. When gel blots of RNA isolated from rosette leaves at various stages of development were probed with the <em>Arabidopsis</em> EST clone, E6B2T7, which has been annotated as DGAT1, a steep increase in DGAT1 transcript levels was evident in the senescing leaves coincident with the accumulation of triacylglycerol. The increase in DGAT1 transcript correlated temporally with enhanced levels of DGAT1 protein detected immunologically. Two lines of evidence indicated that the triacylglycerol of senescing leaves is synthesized in chloroplasts and sequesters fatty acids released from the catabolism of thylakoid galactolipids. First, triacylglycerol isolated from senescing leaves proved to be enriched in hexadecatrienoic acid (16:3) and linolenic acid (18:3), which are normally present in thylakoid galactolipids. Second, DGAT1 protein in senescing leaves was found to be associated with chloroplast membranes. These findings collectively indicate that DGAT1 plays a role in senescence by sequestering fatty acids de-esterified from galactolipids into triacylglycerol.

This thesis tests the hypothesis that increased neuronal cell death in SIDS infants is related to the ability of risk factors, such as prone sleeping, to expose infants to intermittent hypercapnic hypoxia (IHH). Based on the hypothesis that the NMDA system is linked to neuronal death, by way of excitotoxicity, correlations were also sought between cell death and changes in NMDA receptor (NR1) expression in brainstem nuclei controlling cardiorespiratory function. The first aim of this study was to verify that increased neuronal cell death occurs in SIDS infants. To verify a piglet model of SIDS risk factors, brainstem changes were examined in piglets exposed to IHH, and comparisons were made to changes seen in SIDS infants. The NMDA receptor was characterised in controls for both the human infant and the piglet groups. Comparisons of neuronal changes were made with SIDS infants, and piglets exposed to IHH. Non-radioactive in-situ hybridisation and immunohistochemistry were performed on formalin fixed and paraffin embedded brainstem tissue to identify markers of cell death (caspase-3, active caspase-3, and TUNEL), and to examine NR1 mRNA and protein expressions. Staining was quantified using computerised image analysis software. Eight nuclei from the brainstem medulla (caudal in piglets, and mid in infants), and two nuclei from the rostral pons (infants) were studied. The first dataset included human infants aged 1-6 months with a diagnosis of SIDS (n=15) or non-SIDS (n=10). The second dataset comprised developing piglets aged 13-14 days, with controls (n=6), against those exposed to IHH for 2 (n=6) or 4 (n=5) days. Increased neuronal cell death was not verified in the SIDS infants, but abnormalities in NR1 expression were present in selected nuclei of the medulla. Piglets exposed to IHH had increased neuronal cell death and changes in NR1 in selected nuclei of the medulla. There was also a positive correlation between increased cell death and high NR1 levels. Preliminary data showed that SIDS infants who usually slept prone had some differences in NR1 compared to those who did not usually sleep prone. From these findings, it was concluded that IHH may underlie the abnormalities in NMDA receptor expression that are present in the brainstem of SIDS infants. Although IHH can induce an increase in neuronal cell death, its significance in the aetiology of SIDS is not known. In piglets, IHH induced cell death correlated with high NMDA expression in some brainstem nuclei, supporting the hypothesis that excitotoxicity may be involved in the mechanism for cell death. Moreover, this thesis presents for the first time, �preliminary pathological proof� of an association between prone sleeping and abnormal NMDA receptor expression in SIDS infants.

Made available in DSpace on 2014-06-11T19:31:33Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-07Bitstream added on 2014-06-13T19:20:42Z : No. of bitstreams: 1 oliveira_rb_dr_botfca.pdf: 2430018 bytes, checksum: 83351333d3d9c8a67f6e396d9780730d (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / A alta demanda por agrotóxicos pelos sistemas de produção agrícola, aliada ao possível uso inadequado desses produtos, torna a deriva um dos maiores problemas potenciais da agricultura atual, e a escolha correta de adjuvantes pode ser uma das alternativas para minimizar os impactos negativos causados pela deriva nas pulverizações. Este trabalho foi desenvolvido com o objetivo de avaliar o efeito proporcionado por diferentes grupos de adjuvantes nas propriedades físicas e químicas das soluções, no espectro de gotas, bem como no potencial do risco de deriva quantificado em túnel de vento, como forma de auxiliar na escolha correta desses produtos para melhor utilização nas aplicações de agrotóxicos. Foram selecionados 18 adjuvantes comumente utilizados em misturas com agrotóxicos em pulverizações agrícolas, avaliando-se as propriedades físicas e químicas dos mesmos em soluções aquosas (viscosidade, densidade, tensão superficial e condutividade elétrica), o espectro de gotas e o potencial de deriva medido em túnel de vento. O experimento foi composto de 33 tratamentos, tendo a água como testemunha adicional, resultantes da diluição dos adjuvantes em diferentes concentrações, com três repetições. Para os ensaios em túnel de vento e análise de espectro de gotas foi utilizada uma ponta de pulverização XR8003 VK na pressão de 200 kPa, gerando um padrão de gotas médias. A deriva foi coletada no túnel de vento com fluxo de ar na velocidade de 2 m s-1. O corante Azul Brilhante a 0,6% (m v-1) foi utilizado como marcador para todas as soluções pulverizadas visando a quantificação da deriva por espectrofotometria. Os resultados mostraram que a adição de adjuvantes alterou as propriedades físicas e químicas das soluções aquosas em diferentes magnitudes, dependendo da concentração utilizada. Os surfatantes organosilicones... / Due to the high demand for pesticide by the agricultural systems and the possible inadequate use of the products, drift became one of the biggest concerns of the modern agriculture, and the correct use of adjuvants may be one of the possible alternatives to minimize the negative impact of spray operations. This study was developed to evaluate the effect of different adjuvants on physical and chemical properties of spray solutions, droplets spectra as well as drift potential measured in wind tunnel, aiming the correct choice of adjuvants to improve efficiency in pesticide application. For this purpose 18 commercially available agricultural spray adjuvants were selected including products usually mixed up with pesticides in the sprayer tank. The evaluation included physical and chemical properties of spray solutions (viscosity, density, surface tension and electric conductivity), droplets spectra and drift risk potential measured in wind tunnel. The experiment was set up with 33 treatments (solutions) obtained from 18 adjuvants used in different concentrations with 3 replications, including pure water as a standard. For wind tunnel tests and droplet spectra analysis it was used a Teejet XR8003 flat fan nozzles at 200 kPa (medium droplets) and all solutions were mixed up with a food color dye (Blue FDC) at 0,6% m v-1 for spectrophotometry analysis of drift deposits on the wind tunnel. The results showed that the addition of adjuvants changed physical and chemical properties of spray solutions in different magnitudes according to the product concentration. The organosilicon surfactants (Silwet 0.1% e 0.2% and BreakThru 0,1%) had the lowest values for surface tension, while the drift retardant based polymers (Define 0,06% e 0,12%) had the highest values for viscosity and density. There was high correlation between the Volume Median Diameter (VMD) and percentage of droplets smaller... (Completo abstract click electronic access below)

The master's thesis deals with the aerodynamic interaction od two vehicles, modelling in CFD software, how the vehicle behaves in leeward.