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Background: Globally, there are 10 million new cases of tuberculosis (TB) disease annually and 95% of cases occur in low- and middle-income countries (LMIC). More than 1 billion people use tobacco, and 80% of tobacco users reside in LMIC. Smoking approximately doubles the risk of TB disease and is associated with excess mortality during TB treatment. We aimed to estimate the proportion of annual incident TB cases and TB mortality attributable to tobacco smoking in high burden TB countries. Methods: To estimate population attributable fractions (PAF), we obtained country specific estimates of TB incidence and TB mortality rates from the WHO 2015 Global TB Report. Country specific smoking prevalence was estimated from WHO 2015 tobacco surveillance reports and the Tobacco Atlas. Risk ratios for the effect of smoking on TB incidence and TB mortality were obtained from previously published meta-analyses. Country specific PAF of smoking for TB disease were age and sex adjusted. Results: In high burden countries during 2014, an estimated 4.5 million adults developed TB disease and 163,000 people died from TB. An estimated 740 million adult smokers lived in those high burden countries in 2014. We estimated that tobacco smoking was attributable for 17.7% (95% confidence interval [CI] 8.6-21.9%) of TB cases and 15.0% (95% CI 1.9-31.6%) of TB mortality. Of the high burden countries, Russia had the highest proportion of smoking attributable TB disease (31.8%, 95% CI 16.0-37.8%) and death (28.1%, 95% CI 3.8-51.3%). India had the greatest absolute number of TB cases (233,000) and TB deaths (7,400) attributable to smoking. Men (30.5%, 95% CI 14.9%-36.9%) had a greater proportion of TB cases attributable to smoking than women (4.7%, 95% CI 1.9%-6.2%). Conclusion: In high-burden TB countries, nearly one-sixth of all TB cases and TB deaths were attributable to smoking. Our findings highlight the need for tobacco control in high TB burden regions and specifically among patients with TB. Reaching key populations and integrating smoking cessation efforts into TB programs will be essential to achieve global TB control goals.

A dissertation submitted in partial compliance with the requirements for the Masters Degree in Technology: Homoeopathy, Technikon Natal, 1997. / Most economically important crop plants may become infected with viruses. Several of these virus diseases are limiting factors in agricultural production and have contributed to serious economic and social hardship in many countries, especially in tropical and subtropical regions. Homoeopathic microdoses have been investigated for their role in the control of virus diseases, with good results. However, few of the studies contain statistical analyses. The object of this study was to assess the effect of a homoeopathic preparation of a leaf infected with tobacco mosaic virus (TM Viricum) in the contol of tobacco mosaic virus (TMV). The potencies used were 6CH, 12CH, 30CH and 200CH. iv Trays of 24 tomato seedlings per tray were the subjects of this study. Tomato plants were systemically infected with TMV. Four trays were used per treatment. There was an uninoculated and an inoculated control group. The rest of the test population was divided into two groups. The / M

Nitrogen (N) belongs among necessary elements for plant growth and development. In the past attention was paid mostly to the inorganic forms - nitrate and ammonium. In soil N is also present in organic forms, including proteins, for which plants could compete with soil microorganisms. Recently two ways have been considered - the hydrolysis of proteins by secreted proteases and endocytosis of native proteins, possibly their confluence. Tobacco plants were grown in vitro under sterile conditions in modified Murashige-Skoog medium with casein as the only source of N (CAS), decreased concentration of inorganic forms of N (AD) or in complete Murashige-Skoog medium as control plants (MS). After the 12 weeks growth, the standard growth parameters were measured. The CAS plants were able to grow without inorganic N, and protein content in the leaves was higher than in other experimental plants. Proteomic analysis documented differences in protein expression in plant roots in the dependence on the form of N. In total 185 proteins were identified, 75% of proteins were less and 14% more abundant in the CAS plants. The uptake of casein conjugated with fluorescein was followed and the proteolytic activity was analyzed by confocal microscopy. Among proteins secreted from roots to the medium aspartic protease...

Oxalate oxidase is a water soluble, thermolabile, homo-oligomeric glycoprotein the synthesis of which marks the onset of germination In wheat and barley embryos. The protein Is also highly abundant In barley roots. The enzyme has an average oligomer molecular mass of about 115 kDa and about 22.8 kDa for the monomers, as determined by mass spectrometry. The ollgomeric cereal oxalate oxidases are resistant to dissociation In SDS containing media and to digestion by pepsin. The cereal organs produce two oxalate oxidase Isoforms (G and G') which possess the same apoprotein but are differentially glycosylated. The oligosaccharide side chain(s) has a molecular mass of about 2-3 kDa. Barley root also contains a third active oxalate oxidase isoform with a mass of about 22.5 kDa, which was not detected in germinating embryos of the same cultlvar. All of the cereal oxalate oxidases were shown to have identical N-terminal amino acid sequences and almost identical kinetic properties This thesis describes the characterisation of oxalate oxidases Isolated from three transgenic plants lines, expressing chimeric CaMV 35S-oxalate oxidase genes. SGS5 tobacco was expressing a gene with the native oxalate oxidase signal peptide and 3S1 oilseed rape and C26 tobacco were expressing a gene containing a foreign extensin signal peptide. Transgenic SGS5 tobacco produced an oxalate oxidase which was almost indistinguishable from the native cereal protein, in terms of Its structure, stability, enzyme activity and resistance to dissociation In SDS containing media and digestion by pepsin. This work Illustrated the ability of a dicotyledonous plant (tobacco) to recognised and correctly process a transgenic monocotyledon protein (wheat).Transgenic 3S1 oilseed rape and C26 tobacco were shown to produce active oligomeric oxalate oxidases, which did not exhibit any of the unusual resistance properties normally associated with these proteins. Instead the 3S1 and C26 oxalate oxidases were unstable and exhibited significantly altered kinetic properties compared with the native cereal and transgenic SGS5 enzymes. The instability was thought to have arisen from the Incorrect processing of the 3S1 and C26 oxalate oxidases, resulting in the partial cleavage of the extensin signal peptide, which in turn gave rise to a mature oxalate oxidase with an altered N- terminal sequence compared with the native cereal enzyme. The use of vacuum infiltration confirmed the association of the transgenic enzymes with the extracellular spaces, although the majority of the enzyme was shown to be intracellular. The main objective for producing the transgenic oilseed rape expressing oxalate oxidase was to Improve fungal pathogen resistance against oxalic acid secreting pathogens. The results described in this thesis are concerned with a direct comparison of the structure, stability and kinetics between the native cereal and transgenic oxalate oxidases and the possible consequences for pathogen resistance In plants expressing unstable yet active transgenic enzymes.

RESUMO Introdução. O tabaco é, mundialmente, uma relevante causa prevenível de morte. O hábito de fumar, na maioria das vezes, estabelece-se na adolescência. Considerando-se a prevalência de tabagismo e o potencial de seu crescimento, entre os jovens brasileiros, justifica-se o presente estudo que visa a embasar programas abrangentes de controle do tabagismo. Objetivo. Identificar fatores associados ao tabagismo em estudantes de 13 a 15 anos de idade, nas capitais dos três estados da Região Sul do Brasil. Métodos. Foram utilizados dados secundários provenientes do Inquérito de Tabagismo em Escolares, relativos a Curitiba, Florianópolis e Porto Alegre, em 2002 e 2004. A população compreendeu adolescentes de 13 a 15 anos, cursando as 7a. e 8a. séries, do ensino fundamental, e primeira, do ensino médio, de escolas públicas e privadas. Coletou-se a informação por meio de um questionário auto-aplicável e anônimo. Consideraram-se tabagistas os que informaram ter fumado em um ou mais dias, nos últimos trinta dias. Para análise, foram estimados proporções ponderadas e os respectivos intervalos com 95% de confiança e aplicadas técnicas de regressão logística múltipla por meio do programa computacional SPSS?, para detectar os principais fatores associados ao vício de fumar. O nível de significância adotado foi de 10% (? <= 0,10). Resultados. A prevalência de fumantes entre esses escolares variou de 10,7% em Florianópolis a 17,7% em Porto Alegre e foi sempre mais elevada, entre as meninas. Observou-se, nas três capitais, que as proporções entre estudantes fumantes foram maiores na presença de pai fumante, mãe fumante ou ambos fumantes, amigo fumante, exposição à fumaça ambiental em casa e fora de casa, de possuidores de objetos com o logotipo de marca de cigarros e que receberam mais freqüentemente oferta gratuita de cigarros, comparativamente às dos não fumantes. Conclusões. Entre escolares residentes nas capitais do Sul do Brasil, a prevalência de tabagismo é elevada, e os fatores comuns associados ao tabagismo, estatisticamente significantes, foram possuir indivíduos fumantes como melhores amigos e estar exposto à fumaça ambiental, fora de casa. / ABSTRACT Introduction. Tobacco use is the leading preventable cause of death worldwide and adolescents are at a great risk to initiate the smoking habit. The prevalence of tobacco use and its potential growth among Brazilian school youth justify this work, which intends to subside a comprehensive tobacco control program. Objective. To identify relevant factors associated with the tobacco use among students aged 13 to 15 years, in the capital cities of the three States of the Brazilian South Region. Methods. Sample data was obtained in the Global Youth Tobacco Survey, related to Curitiba, PR, Florianópolis, SC, and Porto Alegre, RS, in 2002 and 2004. Adolescents 13 to 15 years, attending the 7th, the 8th grades and the 1st grade of highschool of private and public schools, have composed the study population. Data was collected through an anonymous and self-administered questionnaire. Those who smoked at least one day within the last 30 days were considered smokers. For the statistical analysis of the results, weighted proportions and their respective confidence intervals of 95%, as well as multinomial logistic regression model were applied through the SPSS?, a computer statistical program. The level of significance adopted was 10% (? <= 0.10). The smoking prevalence among the students varied from 10.7% in Florianópolis, SC, to 17.7% in Porto Alegre, RS, and was higher among girls. In the three capitals, the proportion of smokers was higher among those whose mother, father, both parents or best friends had the smoking habit; also, the occurrence of smokers was higher among students exposed to tobacco smoke environment (at home or outside); the same situation was detected among the students who owned objects with a cigarette brand logo, or if more often were offered free cigarettes. Conclusions. Among school youths living in the three capitals of the states of the South of Brazil, it was estimated high prevalence of smokers and the factors statistically associated with the tobacco use were presence of best peer friends addicted to the smoking habit and environmental exposition to the smoke outside home.

Tobacco addiction is a leading preventable cause of death worldwide and places a heavy social and financial burden on society. There exists a substantial genetic variability in smoking behavior, the mechanisms of which are largely unknown. Despite significant advances in sequencing power, progress in the identification of genetic variants affecting smoking behavior based on human genome wide association studies has been slow. Thus this thesis investigates the utility of zebrafish as a model species in which to search for genetic variants affecting nicotine seeking. The work is based on the premise that as zebrafish are vertebrate with conserved neurochemical pathways and circuitry with humans, and the pathways involved in drug mediated reward and addiction are evolutionarily ancient, homologues of genes affecting zebrafish nicotine-seeking behavior will likely affect human smoking behavior. Thus results in zebrafish can be used to direct human genetic studies. The first result chapter addresses the hypothesis that zebrafish show conserved reward responses to common drugs of abuse. A conditioned place preference assay is used to assess zebrafish reward responses to stimulants, opioids, benzodiazepines and alcohol. The results indicate that, with the exception of benzodiazepines, reward responses are conserved, supporting the use of this model in a screen for genetic variants affecting nicotine preference. The second and third results chapters describe the findings of a pilot screen of ENU-mutagenized zebrafish provided by the Sanger Institute, Cambridge. I demonstrate that nicotine preference is heritable in fish as in Abstract 5 humans and identify 3 mutant lines that show increased or decreased nicotine place preference. Genotyping indicated that one of the families showing increased nicotine preference carries a predicted loss of function mutation in the slit3 gene. The involvement of this gene in nicotine preference was confirmed in a separate line. Further characterization of this line using qPCR showed slit3 mutants to have altered developmental expression of key nicotinic and dopaminergic genes. Having identified the slit3 gene as a locus affecting nicotine seeking in fish, I then tested the hypothesis that results in fish could be used to predict loci that affect human smoking behavior. Cohorts of patients were genotyped for 20 SNPs within the slit3 locus. Results of this analysis identified 1 novel SNP in the slit3 gene associated with smoking behavior in a cohort of individuals that were heavy smokers. This result was validated in cohorts of low and normal smoking prevalence. These data demonstrate the utility of behavioral assays in zebrafish to identify genes affecting human behavior and pave the way for the use of zebrafish to inform human studies exploring the genetic basis of drug seeking and behavioral disease.

Cheung Siu Chung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 86-91). / Abstracts in English and Chinese. / Thesis Committee --- p.ii / Statement --- p.iii / Acknowledgements --- p.iv / Abstract --- p.v / 摘要 --- p.vii / Table of Contents --- p.viii / List of Tables --- p.xiii / List of Figures --- p.xiv / Lists of Abbreviations --- p.xvii / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- The plant secretory pathways --- p.2 / Chapter 1.1.1 --- Three different protein sorting pathways to plant vacuoles --- p.3 / Chapter 1.1.2 --- VSD and VSR --- p.6 / Chapter 1.2 --- Prevacuolar compartments --- p.7 / Chapter 1.2.1 --- Lytic PVC --- p.7 / Chapter 1.2.2 --- BP-80 reporter as a lytic PVC marker --- p.8 / Chapter 1.2.3 --- PVC of PSV --- p.9 / Chapter 1.2.4 --- α-TIP CT reporter as a PVC of PSV marker --- p.10 / Chapter 1.3 --- Project objectives --- p.11 / Chapter Chapter 2 --- Development of Transgenic Tobacco BY-2 Cell Lines Expressing Fluorescent Reporters for Golgi and Prevacuolar Compartments / Chapter 2.1 --- Introduction --- p.13 / Chapter 2.2 --- Materials and Methods --- p.15 / Chapter 2.2.1 --- Chemicals --- p.15 / Chapter 2.2.2 --- Oligonucleotides: Primers and Adapters --- p.15 / Chapter 2.2.3 --- Bacterial Strains --- p.17 / Chapter 2.2.4 --- "Preparation of single-reporter constructs (GONST1 -CFP, CFP-BP-80 and CFP-a-TIP CT reporters)" --- p.17 / Chapter 2.2.4.1 --- "Cloning of pGONSTl-CFPK, a Golgi marker" --- p.17 / Chapter 2.2.4.2 --- "Cloning of pCFP-BP-80K, a lytic PVC marker" --- p.20 / Chapter 2.2.4.3 --- "Cloning of pCFP-α-TIP CTK, a putative marker for PVC of PSV" --- p.22 / Chapter 2.2.5 --- "Preparation of double-reporter constructs (CFP-BP-80-GONST1 - YFP, CFP-α-TIP CT-GONST1-YFP, CFP-BP-80-YFP-α-TIP CT and CFP-α-TIP CT-YFP-BP-80 reporters)" --- p.24 / Chapter 2.2.5.1 --- Insertion ofAdapter-XH to pCFP-BP-80K and pCFP-α-TIP CTK --- p.24 / Chapter 2.2.5.2 --- "Cloning of pCFP-BP-80-GONST 1 -YFPK, pCFP-α-TIP CT- GONST 1-YFPK, pCFP-BP-80-YFP-α-TIP CTK and pCFP- α-TIP CT-YFP-BP-80K" --- p.26 / Chapter 2.2.6 --- Agrobacterium electroporation --- p.30 / Chapter 2.2.7 --- Agrobacterium-mediated transformation of tobacco BY-2 cells --- p.30 / Chapter 2.2.8 --- Selection and screening of transformed BY-2 cells --- p.31 / Chapter 2.2.8.1 --- Antibiotic selection --- p.31 / Chapter 2.2.8.2 --- Fluorescence microscopic screening --- p.31 / Chapter 2.2.9 --- Detection of CFP and YFP reporter genes and their expressions --- p.32 / Chapter 2.2.9.1 --- CTAB genomic DNA extraction --- p.32 / Chapter 2.2.9.2 --- PCR test for CFP (and YFP) transgene in genomic DNA --- p.33 / Chapter 2.2.9.3 --- Subcellular fractionation and protein extraction --- p.33 / Chapter 2.2.9.4 --- Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis --- p.34 / Chapter 2.2.9.5 --- Confocal microscopic study --- p.35 / Chapter 2.3 --- Results --- p.36 / Chapter 2.3.1 --- Establishment of kanamycin-resistant BY-2 cells expressing CFP (and YFP) reporters --- p.36 / Chapter 2.3.2 --- Fluorescence microscopic screening of transgenic BY-2 cell lines --- p.37 / Chapter 2.3.3 --- CFP (and YFP) reporter was successfully integrated into transgenic BY-2 cell genome --- p.41 / Chapter 2.3.4 --- CFP (and YFP) reporter was expressed in transgenic BY-2 cell lines --- p.44 / Chapter 2.3.5 --- Punctate CFP (and YFP) signals were detected in transgenic BY-2 cell lines expressing single (or double) reporter --- p.48 / Chapter 2.4 --- Discussion --- p.53 / Chapter 2.4.1 --- "Transgenic BY-2 cell lines expressing single reporter marking Golgi, lytic PVC and putative PVC of PSV have been developed" --- p.53 / Chapter 2.4.2 --- "Golgi, lytic PVC and putative PVC of PSV were separate and distinct organelles" --- p.53 / Chapter 2.4.3 --- Transgenic BY-2 cell lines expressing double reporter were not yet suitable for subsequent study --- p.55 / Chapter Chapter 3 --- Characterization of Transgenic Tobacco BY-2 Cell Lines Expressing Fluorescent Reporters for Prevacuolar Compartments / Chapter 3.1 --- Introduction --- p.58 / Chapter 3.2 --- Materials and Methods --- p.60 / Chapter 3.2.1 --- Confocal immunofluorescence study --- p.60 / Chapter 3.2.2 --- Drug treatment study (for single-reporter transgenic tobacco BY-2 cell line) --- p.62 / Chapter 3.2.2.1 --- Wortmannin treatment --- p.62 / Chapter 3.2.2.1.1 --- Dosage effect --- p.62 / Chapter 3.2.2.1.2 --- Time-course study --- p.62 / Chapter 3.2.2.2 --- Brefeldin A treatment --- p.63 / Chapter 3.2.2.1.1 --- Dosage effect --- p.63 / Chapter 3.2.2.1.2 --- Time-course study --- p.63 / Chapter 3.2.3 --- Drug treatment study (for double-reporter transgenic tobacco BY-2 cell line) --- p.64 / Chapter 3.2.3.1 --- Wortmannin treatment --- p.64 / Chapter 3.2.3.2 --- Brefeldin A treatment --- p.64 / Chapter 3.3 --- Results --- p.65 / Chapter 3.3.1 --- CFP-α-TIP CT reporter-marked compartment was not Golgi apparatus --- p.65 / Chapter 3.3.2 --- Wortmannin induced CFP-α-TIP CT reporter-marked compartment to vacuolate --- p.69 / Chapter 3.3.3 --- BFA induced CFP-α-TIP CT reporter-marked compartment to form aggregates --- p.72 / Chapter 3.3.4 --- Wortmannin and BFA treatment caused lytic PVC to form small vacuole and Golgi to form aggregate respectively in transgenic BY-2 cell lines expressing double-reporter --- p.75 / Chapter 3.4 --- Discussion --- p.77 / Chapter 3.4.1 --- CFP-α-TIP CT reporter-marked compartment was not Golgi apparatus --- p.77 / Chapter 3.4.2 --- CFP-α-TIP CT reporter-marked compartment was not lytic PVC --- p.77 / Chapter 3.4.3 --- Transgenic BY-2 cell lines expressing double reporter could successfully mark two compartments simultaneously in the same cell --- p.78 / Chapter Chapter 4 --- Summary and Future Prospects / Chapter 4.1 --- Summary --- p.80 / Chapter 4.1.1 --- Hypothesis --- p.80 / Chapter 4.1.2 --- Development of transgenic tobacco BY-2 cell lines --- p.81 / Chapter 4.1.3 --- Characterization of α-TIP CT reporter-marked PVC-like compartment --- p.82 / Chapter 4.2 --- Conclusions --- p.84 / Chapter 4.3 --- Future prospects --- p.85 / References --- p.86

Tobacco smoke remains a major public health concern and a factor contributing to the development and progression of various lung diseases world-wide. Smoking cessation can significantly reduce the risk of developing smoking-related diseases, although some smokers remain at an elevated risk despite quitting. Here, I used high-throughput genomic technologies to pave the way for understanding the transcriptomic response in airway epithelium to tobacco exposure, smoking cessation and potentially reduced exposure products (PREP). First, using a longitudinal dataset of nasal airway epithelial cells obtained from active smokers enrolled in smoking cessation programs over 24 weeks, I demonstrated that tobacco-related alterations in the airway gene expression are rapidly reversed within 4 to 8 weeks following smoking cessation. Genes with different biological functions revert towards baseline with different dynamics following smoking cessation. These findings suggest that the nasal-epithelium can serve as a minimally-invasive site to measure the reversible impact of smoking. Secondly, using a dataset of nasal airway epithelial cells from active smokers who switched to potentially reduced exposure products (PREP) for 6 weeks, I showed that gene expression differences induced by switching to PREP may only constitute a partially reduced exposure relative to smoking cessation. My results demonstrate that the nasal-epithelium can also serve as a minimally-invasive site to measure the responses to PREP and may ultimately yield biomarkers to evaluate the potential disease risks associated with these products. Lastly, using small RNA-seq data from bronchial epithelial cells of smokers, I found alterations in airway micro-RNA expression that associate with time since quitting in former smokers. These studies have provided novel insights into the physiologic responses of the airway epithelium to tobacco smoke and PREP and may ultimately serve as a useful approach for evaluating the disease risks associated with changes in smoking behavior. This work sets the stage for additional studies aimed at identifying prevention strategies that decrease the persistent risk of smoking-related lung disease in former smokers and identify biomarkers to assess lung disease risk in former smokers. / 2016-12-31T00:00:00Z

Fung Ka Leung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 83-93). / Abstracts in English and Chinese. / Thesis Committee --- p.ii / Statement --- p.iii / Acknowledgements --- p.iv / Abstract --- p.v / 摘要 --- p.vii / Table of Contents --- p.viii / List of Tables --- p.xii / List of Figures --- p.xiii / List of Abbreviations --- p.xv / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- An overview of the secretory pathway in eukaryotic cells --- p.2 / Chapter 1.2 --- The secretory pathway in plants --- p.4 / Chapter 1.2.1 --- Plant cells contain two functionally distinct vacuoles --- p.4 / Chapter 1.2.2 --- Three vesicular pathways to two vacuole --- p.6 / Chapter 1.2.3 --- Transport vesicles in the three vesicular pathways --- p.9 / Chapter 1.2.4 --- Vacuolar sorting determinants (VSDs) --- p.10 / Chapter 1.2.5 --- Vacuolar sorting receptors (VSRs) --- p.12 / Chapter 1.3 --- The PSVs in mature seeds --- p.15 / Chapter 1.3.1 --- Biogenesis of PSV --- p.15 / Chapter 1.3.2 --- The two chimeric integral membrane reporters --- p.16 / Chapter 1.3.3 --- Subcellular localization of the two chimeric integral membrane reporters in PSVs of mature tobacco seeds --- p.17 / Chapter 1.4 --- Project objectives --- p.19 / Chapter Chapter 2 --- Materials and Methods --- p.20 / Chapter 2.1 --- Construction of the YFP-BP-80 and the YFP- a -TIP reporters --- p.21 / Chapter 2.1.1 --- The pYFP-BP-80-K construct --- p.21 / Chapter 2.1.2 --- The pYFP- a -TIP-K construct --- p.22 / Chapter 2.2 --- Construction of GFP-RMR reporter --- p.23 / Chapter 2.2.1 --- Cloning of pGFP-RMR --- p.23 / Chapter 2.2.2 --- Cloning of pGFP-RMR-K --- p.23 / Chapter 2.3 --- Construction of pGONST1-YFP construct --- p.26 / Chapter 2.3.1 --- The pGONSTl-YFP construct --- p.26 / Chapter 2.4 --- Transformation of Agrobacterium by electroporation --- p.27 / Chapter 2.5 --- Tobacco transformation and selection --- p.28 / Chapter 2.5.1 --- Plant materials --- p.28 / Chapter 2.5.2 --- Tobacco transformation --- p.28 / Chapter 2.6 --- Screening of transgenic tobacco plants expressing YFP fusion proteins --- p.30 / Chapter 2.6.1 --- Kanamycin screening --- p.30 / Chapter 2.6.2 --- Extraction of genomic DNA from leaves --- p.30 / Chapter 2.6.3 --- PCR of genomic DNA --- p.31 / Chapter 2.7 --- Southern blot analysis of genomic DNA --- p.32 / Chapter 2.8 --- Western blot analysis of transgenic tobacco plants --- p.33 / Chapter 2.8.1 --- Extraction of total protein from tobacco leaves or seeds --- p.33 / Chapter 2.8.2 --- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis --- p.34 / Chapter 2.9 --- Confocal immunofluorescence studies --- p.35 / Chapter 2.9.1 --- Preparation of sections --- p.35 / Chapter 2.9.2 --- Single labeling --- p.35 / Chapter 2.9.3 --- Double labeling with one polyclonal and one monoclonal antibodies --- p.36 / Chapter 2.9.4 --- Double labeling with two polyclonal antibodies --- p.36 / Chapter 2.9.5 --- Collection of images --- p.37 / Chapter 2.10 --- Chemicals --- p.38 / Chapter 2.11 --- Primers --- p.38 / Chapter 2.12 --- Bacterial strain --- p.38 / Chapter 2.13 --- Antibodies --- p.39 / Chapter 2.14 --- Growing condition of transgenic plants and determining the developmental stage of tobacco flowers --- p.39 / Chapter Chapter 3 --- Results --- p.41 / Chapter 3.1 --- Generation of transgenic tobacco plants --- p.42 / Chapter 3.2 --- PCR screening of transgenic tobacco plants --- p.46 / Chapter 3.3 --- Southern blot analysis --- p.48 / Chapter 3.4 --- Detection of the YFP fusion proteins in transgenic tobacco plants by western blot analysis --- p.50 / Chapter 3.4.1 --- Detection of the YFP fusion proteins in leaves --- p.50 / Chapter 3.4.2 --- Western blot analysis of vegetative tissues --- p.57 / Chapter 3.4.3 --- Western blot analysis of mature seeds --- p.59 / Chapter 3.5 --- Confocal immunofluorescence studies --- p.61 / Chapter 3.5.1 --- Detection of YFP signals in root tip cells --- p.61 / Chapter 3.5.2 --- Detection of YFP signals in developing seeds --- p.65 / Chapter 3.5.3 --- Subcellular localization of the YFP fusion proteins in mature seeds --- p.67 / Chapter Chapter 4 --- Discussion --- p.72 / Chapter Chapter 5 --- Summary and Future Perspectives --- p.77 / Chapter 5.1 --- Summary --- p.78 / Chapter 5.1.1 --- Generation of transgenic tobacco plants expressing the YFP fusion proteins --- p.78 / Chapter 5.1.2 --- Full-length fusion proteins and cleaved soluble YFP were detected in vegetative tissues --- p.79 / Chapter 5.1.3 --- Only cleaved soluble YFP was detected in mature seeds --- p.79 / Chapter 5.1.4 --- The two fusion proteins might localized in different compartments in developing seeds --- p.79 / Chapter 5.1.5 --- Both fusion proteins were localized within the PSVs of mature seeds --- p.80 / Chapter 5.2 --- Future perspectives --- p.81 / References --- p.83