Blood vessel mimics (BVMs) are tissue engineered blood vessels that are intended as an intermediate testing environment for intravascular devices, such as stents. Specifically, Cal Poly’s Tissue Engineering Lab hypothesizes that BVMs can be used to test endothelial cell and smooth muscle cell responses to existing and new vascular stents. Characterization techniques are required for BVMs to be accepted as a valid testing model, prior to being employed as an in vitro model to determine the effects of medical treatments. Quantitative real-time polymerase chain reaction (qPCR) is one available option for evaluating gene expression of tissues. qPCR can be performed on DNA synthesized from RNA isolated from cells, and in this application, will provide quantitative information on what proteins where being transcribed within the cells at the time of RNA isolation. qPCR can be used to determine the proteins expressed in BVMs at baseline in order to then characterize changes in protein expression induced by stent deployment within the BVM. The aim of this thesis was to optimize existing qPCR protocols, and implement the optimized protocols to characterize gene expression of stented and unstented blood vessel mimics (BVMs) and cells from a donor with Diabetes grown in Cal Poly’s Tissue Engineering Laboratory. To accomplish this goal, existing qPCR protocols were evaluated and modified to ensure reproducible, valid results were produced. Standard operating procedures were created for RNA isolation, cDNA synthesis, qPCR and qPCR data analysis. Optimized qPCR methods were then applied to BVMs from umbilical and coronary cell sources to compare the models and to study the BVM responses to stent deployment. Additional primers were also identified for potential usage as reference genes and as diabetic markers for diseased BVMs.

This thesis provides evidence for the presence of multiple RNAs in the axons and growth cones of developing thalamic cells, particularly the mRNA for the cell adhesion and Wnt-signalling-related molecule b-catenin. After many decades of effort, mRNAs have been shown to be present in the axons of many different systems in recent years. Furthermore, these mRNAs have been shown to be locally translated at the growth cone, and this local translation is required for axons to turn in response to multiple guidance cues. As studies accumulate, it is becoming clear that different axonal systems contain different complements of mRNAs and have different requirements for local translation. One axonal system which has not been investigated to date is the thalamocortical tract. The nuclei of the thalamus are connected to the areas of the cortex via bundles of axons which travel from the thalamus to the cortex via the ventral telencephalon during embyronic development. These axons make a number of turns and are guided by many cues and other axonal tracts before innervating their cortical target. In this thesis, a quantitative real-time polymerase chain reaction (qRT-PCR) approach is developed to isolate multiple mRNAs from developing thalamic axons in vitro, including b-catenin mRNA, b-actin mRNA, 18S ribosomal RNA and ten other mRNAs. The method used should be suitable for use with other axonal systems and also for testing the effect of guidance cues on mRNA expression in axons. The qRT-PCR results for b-catenin, b-actin and 18S have been validated using in situ hybridisation. Analysis of in situ hybridisation results indicates that b-catenin and 18S, but not b-actin, are upregulated in the growth cone compared to the axon. As b-catenin has been shown to be involved in axon guidance via Slit and ephrin guidance cues in other axonal systems, and these guidance cues act upon thalamocortical axons, the identification of b-catenin mRNA in thalamic axons is an important step towards a full understanding of the thalamocortical system. The results presented here indicate that local protein synthesis is likely to occur in thalamic axons as it does in other axonal systems, and that local translation is likely to be important for thalamic axonal responses to guidance cues and other axonal tracts.

The present study investigated the expression profiles of nine genes involved in immune defense of the Pacific white shrimp Litopenaeus vannamei and their responses to oral administration of beta-1,3-glucan. The nine immune related genes were beta-glucan binding protein-high density lipoprotein (BGBP-HDL), lipopolysaccharide/beta-glucan binding protein (LGBP), hemocyanin, prophenoloxidase (proPO), transglutaminase (TGase), penaeidin-3 (PEN-3), crustin, cytosolic manganese superoxide dismutase (cMnSOD), and lysozyme. A series of experiments were carried out in the study including: (1) cDNA cloning and characterization of proPO and LGBP; (2) tissue mRNA expressions of the nine genes in adult shrimp; (3) expression and localization of the nine genes during larval and postlarval ontogenic development; (4) the effects of dietary beta-1,3-glucan on the expression of the nine genes. The cDNA cloning study showed that the proPO cDNA contains an open reading frame of 2061 bp and encodes a 686 amino-acid peptide. The protein sequence of the proPO has a similarity of 85% with those of Penaeus monodon and P. semisulcatus and has an identity of between 58 and 77% with other crustaceans. Northern blot analysis revealed that proPO was constitutively expressed mainly in hemocytes. Its transcripts were observed in hemocytes and many other tissues when detected with RT-PCR. The results of in situ hybridizations showed that the hemocytes that infiltrated in tissues were responsible for the positive signals. The LGBP cDNA contains an open reading frame of 1104 bp and encodes a 367 amino-acid protein with a 17 a. a. signal peptide. The protein sequence of the LGBP has a similarity of 97% with LGBP of L. stylirostris, >90% identity with BGBP of P. monodon and LGBP of Fenneropenaeus chinensis and has an identity of between 63 and 86% with other crustaceans. Northern blot analysis revealed that LGBP was constitutively expressed mainly in hepatopancreas. The results of in situ hybridizations showed that the hepatopancreatic F cells might be the major cell type for LGBP production. Using the complete cDNAs of proPO and LGBP and partial fragments of the other seven genes, their tissue expressions were analyzed by conventional RT-PCR, quantitative real time PCR (qPCR) and in situ hybridization. The results demonstrated that BGBP-HDL, LGBP and hemocyanin were mainly expressed in the hepatopancreas and their expressions levels were about 10 to 30% those of

Magister Scientiae (Biodiversity and Conservation Biology) / Land-based abalone aquaculture in South Africa started in the early 1990s and is based on the local species Haliotis midae. This industry expanded with great success over the last decade. In 2006 abalone exhibiting typical clinical signs of tubercle mycosis was discovered for the first time in South African abalone culture facilities,posing a significant threat to the industry. Halioticida noduliformans, a fungus belonging to the Peronosporomycetes (formerly Oomycetes), has been identified as the causative agent of abalone tubercle mycosis (ATM). While diagnoses of this disease are currently done by gross observation and histopathology, these methods fail to be sensitive enough to identify the causative agent accurately and reliably.Molecular confirmation could provide for quicker more accurate diagnostic information. The aim of this study was to develop a DNA based molecular diagnostic test. Polymerase chain reaction (PCR) has been used to rapidly detect, characterise and identify a variety of organisms. Nucleotide sequences of the smalland large-subunit ribosomal ribonucleic acid (rRNA) and mitochondrial cytochrome oxidase subunit II (cox2) genes of H. noduliformans were compared with closely related Peronosporomycete gene sequences to identify potential PCR primer sites. H. noduliformans specific real-time quantitative PCR (Q-PCR) primer sets were designed and optimised for each of the selected genes. Results indicate that, although all tested primers sets could amplify fungal DNA, only the LSU and cox2 primer sets - v -demonstrated no cross-amplification with the closely related Peronosporomycete and non-fungal DNA tested in the present study. The H. noduliformans specific LSU primer set was chosen for further analysis and used for all subsequent real-time PCR assays. The lowest detection limit for the LSU primer set was evaluated by running Q-PCR on serial dilutions of known quantities of extracted H. noduliformans DNA.Serial dilutions were made in PCR grade water as well as in an abalone tissue matrix.The sensitivity of the Q-PCR reaction was determined to be 266 pg of H.noduliformans DNA per 25 μL reaction volume. However, inclusion of a nested PCR step, utilising universal fungal outer primers, followed by Q-PCR with the H.noduliformans LSU specific primers improved sensitivity to 0.266 pg of H.noduliformans DNA per 25 μL reaction volume. This equates to approximately 2.4spores per 25 μL reaction volume. DNA extraction protocols were optimised to ensure efficient and repeatable extraction of high quality fungal DNA from pure fungus and tissue samples spiked with known quantities of fungal DNA. PCR amplification efficiency and potential inhibition were examined for each extraction method. Results suggest that real-time PCR has great potential in monitoring and quantifying H. noduliformans on abalone culture facilities in South Africa.

>Magister Scientiae - MSc / Abiotic stress, mainly in the form of extreme temperatures, drought and salinity has caused major crop losses worldwide, putting a severe strain on agriculture. Salinity severely limits plant growth and productivity and affects all aspects of the plant’s development including the most crucial stage; germination. This study investigated the effect of salt (NaCl) stress on Sorghum bicolor seedlings and the role of exogenously applied calcium (Ca2+) to ameliorate the effects of salt stress during germination. Sorghum seeds were germinated in the presence and absence of various NaCl (100, 200 and 300 mM) and Ca2+ (5, 15 and 35 mM) concentrations. Several assays including physiological (germination and growth assays), biochemical (osmolytes and oxidative stress markers), anatomical (epidermal and xylem layers) and expression profiles of key genes [antioxidant (SbSOD, SbAPX2 and SbCAT3), Salt Overly Sensitive (SbSOS1, 2 and 3) pathway enzymes and the vacuolar Na+/H+ exchanger antiporter2 (SbNHX2)] were investigated. Salt stress delayed germination and negatively affected growth as observed by the reduced root and shoot length and decreased fresh and dry weight. There was an increase in proline content and oxidative stress markers (H2O2 and MDA) under salt stress. Oxidative stress resulted in damage to the epidermal and xylem layers as observed on Scanning Electron Microscopy (SEM) images. Quantitative real-time polymerase chain reaction revealed that salt stress induced the expression of SbAPX2, SbCAT3 and SbSOS1 genes, whereas SbSOD4A, SbSOS2, SbSOS3 and SbNHX2 genes were not affected by salt. Exogenous application of Ca2+ counteracted the harmful effects of salt stress by improving germination efficiency, promoting seedling growth, reducing oxidative damage and the Na+/K+ ratio, indicating the protective effect. Ca2+ also effectively protected the epidermis and xylem layers from the severe damage caused by salt stress. In the presence of Ca2+ the expression of SbAPX2 and SbCAT3 was reduced except for the SbNHX2 gene, which increased by 65-fold compared to the control. The results obtained suggests that sorghum is able to respond to salt stress by inducing osmolytes, the antioxidant defence system as well as the SOS pathway. Furthermore, 5 mM Ca2+ was determined as the optimum Ca2+ concentration required to enhance sorghum’s tolerance to salt stress.

Plant alkaloids are secondary metabolites that may be synthesized in an inducible defense response to herbivory (Baldwin 1999). Genetic engineering of secondary metabolic pathways in plants to enhance or reduce metabolite production is limited by the current understanding of these pathways and their regulation in response to environmental conditions. This study was intended to provide new insights into the mechanism and regulation of alkaloid biosynthesis in N. tabacum by identifying genes that are coordinately regulated during conditions that induce alkaloid biosynthesis and by comparing their expression in regulatory mutant backgrounds that differ at two quantitative alkaloid loci, A and B. In order to identify novel genes that are differentially expressed during alkaloid biosynthesis, the transcriptional profiling procedure, fluorescent differential display (FDD), was used to screen total RNA isolated from Burley 21 (WT, AABB) and LA21 (low alkaloid regulatory mutant, aabb) tobacco root cultures that were induced for alkaloid synthesis. Four of thirteen cloned FDD fragments showed sequence homology to genes with defense-related functions. The differential expression of genes represented by selected FDD gene fragments was confirmed by comparing Northern blots of transcripts of those genes to known alkaloid biosynthetic genes, putrescine methyl transferase (PMT3), ornithine decarboxylase (ODC3), arginine decarboxylase (ADC1), and quinolinate phosphoribosyltransferase (QPRT). The role of the A and B loci in differential expression of genes represented by FDD clones and of known nicotine biosynthetic genes was examined using quantitative real time polymerase chain reaction (QRT-PCR) to measure transcript levels of these genes in four tobacco genotypes differing in alkaloid content, Burley 21(AABB), HI21 (AAbb), LI21(aaBB), and LA21 (aabb). Results of this study suggest that the A/B regulon is not limited to alkaloid biosynthetic genes, but includes multiple genes with defense-related functions. QRT-PCR analysis of nicotine biosynthetic genes and genes represented by confirmed differentially expressed FDD clones showed increased mRNA accumulation in response to alkaloid induction in all the tested genotypes, which suggests that the A and B mutations affect overall mRNA accumulation levels, rather than gene inducibility, per se. Baldwin, I.T. 1999. Inducible nicotine production in native Nicotiana as an example of adaptive phenotypic plasticity. Journal of Chem. Ecol. 25: 3-30. / Master of Science

Orientador: Marcos Silveira Buckeridge / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-15T21:23:11Z (GMT). No. of bitstreams: 1 Brandao_AndreaDias_D.pdf: 9019715 bytes, checksum: 89d92b9413ee5bda07f54d0245d9d040 (MD5) Previous issue date: 2010 / Resumo: A cana-de-açúcar pertence a família Poaceae e ao gênero Saccharum. Espécies pertencentes a essa família apresentam a via de fotossíntese C4, mais eficiente para a produção de biomassa quando comparadas com as plantas com metabolismo fotossintético C3 em condições de temperaturas elevadas. A cana-de-açúcar transformou-se em um importante potencial econômico e fonte de energia no mundo, devido a sua capacidade de estocar sacarose (cerca de 50% de seu peso seco) e produzir bioetanol. Nos últimos anos tornou-se alvo prioritário para diversos estudos através do melhoramento genético, biologia molecular, bioquímica e estudos fisiológicos. Os produtos provenientes da cana são amplamente utilizados pela população mundial e representam uma fonte alternativa para a geração de energia. O Brasil ocupa uma posição de destaque entre os países produtores de cana-de-açúcar (34% da produção mundial). Devido a sua origem interespecífica a cana possui um dos genomas mais complexos entre as espécies vegetais tornando-se um importante objeto de estudo para a obtenção de variedades produtivas e ou eficientes, melhor adaptadas às condições climáticas. A propagação clonal através do cultivo in vitro possibilita a obtenção mais rápida de indivíduos da espécie. A utilização de métodos de assepsia para a desinfestação e desinfecção sem causar danos aos tecidos que levam a morte da planta tornou-se um grande desafio para a obtenção de novas plântulas que permitam os estudos de biotecnologia. E o grande interesse em se estudar plantas de cana-de-açúcar se dá pelo acúmulo da sacarose, que ocorre na região do entrenó durante o desenvolvimento da planta. A genética clássica busca a melhora dessa característica, principalmente através do aumento da biomassa realizada pela fixação de carbono, no entanto, há um limitado aumento do conteúdo de sacarose. A giberelina é um fitormônio vegetal, largamente utilizada na agricultura e desempenha uma variedade de funções fisiológicas em plantas. O GA3 produzido industrialmente tem sido aplicado para estimular o crescimento da cana-de-açúcar, para auxiliar a germinação de cevada, na produção de frutas e verduras, entre outras. As giberelinas são extremamente ativas na indução do alongamento do caule. Estudos mostram que a aplicação de GAs provoca aumento no tamanho da célula e no número de células, indicando que as GAs atuam tanto no alongamento da célula como na divisão celular, o que potencializa um aumento na produtividade de sacarose. Já o paclobutrazol (PBZ) atua inibindo a biossíntese de giberelinas. Ele bloqueia a biossíntese de GA, pois interfere nos primeiros passos da rota de oxidação do caureno, impedindo a formação das GAs, e por isso funciona como um controle negativo dos mecanismos de ação das giberelinas. Tanto a presença do paclobutrazol quanto da GA3 induzem alterações da expressão de genes específicos e a ativação de vias de sinalização que agem cooperativamente na tentativa de aliviar o efeito do estresse na tentativa de estabelecer o retorno à homeostasia celular. Nosso maior objetivo nesse estudo é tentar identificar o mecanismo de ação das GAs, para permitir uma melhor compreensão das alterações tanto morfológicas e fisiológicas sofrida pelas plântulas. Para isso em nossos estudos foram selecionados genes que pudessem apresentar relação com metabolismo de carboidratos, com respostas hormonais, com metabolismo de ácidos nucleícos, com a fotossíntese, com o desenvolvimento, com divisão celular, com metabolismo de proteínas, além de diversos fatores de transcrição que possam estar envolvidos nesses processos, baseados em resultados do metabolismo de carboidratos encontrados nas analises bioquímicas das plântulas, assim como nos cortes anatômicos. O resultados mostraram interferência do GA3 no acúmulo de carboidratos, no alongamento celular, em genes relacionados com a via de transdução de sinal das AUX, biossíntese de AUX, GA, além de genes e fatores de transcrição relacionados com o ciclo celular, fotossíntese, fixação de carbono e diversos estresses, entre eles o osmótico. / Abstract: The sugarcane belongs to the grasses's family and the Saccharum genus. Species belonging to this family have the C4 photosynthesis patway, more efficient for biomass production when compared the C3 photosynthetic metabolism plants, in high temperature condicions. The sugarcane became an important economic potential and energy in the world due to its ability to store sucrose (about 50% of its dry weight) and production of bioethanol. In recent years it has become priority for several studies through breeding, molecular biology, biochemistry and physiological studies. Products from sugarcane is widely used by the world's population and represent an alternative source for energy generation. Brazil occupies an outstanding position among the countries producing sugarcane (34% of world production). Because of its interspecific origin, the sugarcane has one of the more complex genomes of plant species became an important object of study for plant breeding and productive or efficient, better adapted to climatic conditions. The clonal propagation through in vitro possible to obtain faster plants copies. The use of aseptic methods for disinfestation and disinfection without causing tissue damage leading to death of the plant has become a major challenge for the procurement of new seedlings to allow the biotechnology study. And the great interest in studying sugarcane plant is caused by the accumulation of sucrose, which occurs in the internode region during the plant development. Classical genetics search to improve this feature, mainly by increasing the biomass held by sequestration, however, there is a limited increase in sucrose content. The gibberellin is a plant phytohormone widely used in the agriculture and plays a variety of physiological functions in the plants. The sintetic GA3 has been applied to stimulate the sugarcane growth, to assist the germination of barley, the production of fruits and vegetables, among others. The gibberellins are extremely active in inducing the elongation of the stem. Studies show that the application of GAs causes an increase in cell size and cell number, indicating that GAs act both in cell elongation and cell division, which leverage an increase in sucrose yield. Since the PBZ acts by inhibiting the biosynthesis of gibberellins. It blocks the biosynthesis of GA, because it interferes in the first steps of the kaurene oxidation patway, preventing the GAs formation, and therefore acts as a negative control mechanisms of action of gibberellins. Both the presence of paclobutrazol and the GA3 induced changes in gene expression and activation of specific signaling pathways that act cooperatively in trying to alleviate the effect of stress in trying to establish a return to cellular homeostasis. Our objectivity in this study is to try to identify the mechanism of action of GAs to allow a understanding of both morphological and physiological changes experienced by seedlings. To do this in our studies we selected genes that could present relationship with carbohydrate metabolism, hormonal responses, with the metabolism of nucleic acids, through photosynthesis, with the development, with cell division, with protein metabolism, and several transcription factors that may be involved in these processes, based on results of the metabolism of carbohydrates found in the biochemical analysis of the seedlings, as well as in anatomical cuts. The results showed interference of GA3 in the accumulation of carbohydrates in cell elongation in genes related to the route of signal transduction of AUX, AUX biosynthesis, GA, in addition to genes and transcription factors related to cell cycle, photosynthesis, fixing carbon and many stresses, including the osmotic. / Doutorado / Biologia Vegetal / Doutor em Biologia Vegetal

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