The structural characteristics associated with the binding of beneficial metals (i.e. - Mg2+, Zn2+ and Ca2+) to natural proteins has typically received more attention than competitive binding by toxic metals (e.g. – Pb2+, Hg2+, Cd2+, La3+, etc.). In this thesis, a statistical analysis of Pb2+-binding in crystallized protein structures indicates that Pb2+ does not bind preferentially with nitrogen, as generally assumed, but binds predominantly with oxygen, and to a lesser degree, sulfur. A comparison of Ca2+ and Pb2+ indicates that Pb2+ binds with a wider range of coordination numbers, with less formal change, and with less defined structure than Ca2+. The Pb2+ ion also appears to displace Ca2+ with little conformational stress in calcium binding proteins (CaBP’s). Experimental data from the binding of metals with engineered fluorescent proteins indicate that both Pb2+ and Gd3+ will occupy grafted calcium-binding sites with greater affinity than Ca2+, and strong evidence is presented to support the hypothesis that Pb2+ and Gd3+ will bind non-specifically on the protein surface. These results suggest that toxicity is associated with two binding mechanisms: displacement of the metal cofactor which disrupts protein function, and non-specific binding which maintains higher solubility of the metal.

Genesis of life begins with the fusion of female and male haploid gametes through a process of fertilization leading to the formation of a diploid cell, the zygote. This undergoes successive cleavage divisions forming 2-, 4- and 8- cell embryos and their individual cells (blastomeres) are totipotent. As development proceeds, there is a gradual restriction in their totipotency, resulting in the generation of two distinct cell lineages i.e., the differentiated trophectoderm (TE) cells and the undifferentiated, inner cell mass (ICM) during blastocyst morphogenesis (Rossant and Tam 2009). During the course of development, the ICM cells can give rise to all cell types of an organism and can also provide embryonic stem (ES)-cells when cultured in vitro (Evan and Kaufman 1981). ES-cells are pluripotent cells, having the ability to self-renew indefinitely and differentiate into all the three primary germ layers (ectoderm, mesoderm and endoderm) derived-cell types. ES-cells are an excellent developmental model system to understand basic mechanisms of self-renewal, cell differentiation and function of various genes in vitro and in vivo (Capecchi 2001). Importantly, their cell derivatives could potentially be used for experimental cell-based therapy for a number of diseases. Although, human ES-cell lines have been successfully derived and differentiated to various cell types (Thomson et al., 1998; Odorico et al., 2001), their cell-therapeutic potential is far from being tested, in view of the lack of our understanding of lineage-specific differentiation, homing and structural-functional integration of differentiated cell types in the host environment. To understand these mechanisms, it is desirable to have fluorescently-marked ES-cells and their differentiated cell-types, which could facilitate experimental cell transplantation studies. In this regard, our laboratory has earlier generated enhanced green fluorescent protein (EGFP)-expressing FVB/N transgenic ‘green’ mouse, under the control of ubiquitous chicken -actin promoter (Devgan et al., 2003). This transgenic mouse has been an excellent source of intrinsically green fluorescent cell types. We have been attempting to derive ES-cell line from this transgenic mouse. Because the derivation of ES-cell line is genetic strain-dependent, with some strains being relatively permissible for ES-cell derivation while others are quite resistant (non permissive), it has been extremely difficult to derive ES-cell line from the FVB/N mouse strain. There is a need to evolve experimental strategies to derive ES-cell line from FVB/N mouse, a strain extensively used for transgenesis. Thus, the aims of the study described in the thesis are to: (1) develop an experimental system to derive EGFP-expressing fluorescently-marked ES-cell line from a non-permissive FVB/N mouse strain; (2) characterize the established ES-cell line; (3) achieve differentiation of various cell types from EGFP-expressing ES-cell line and (4) understand role of FGF signaling in cardiac differentiation from the established ES-cell line. In order to have an appropriate and relevant literature background, the 1st chapter in this thesis describes a comprehensive up-to-date review of literature, pertaining to the early mammalian development and differentiation of blastocyst, followed by origin and properties of ES-cells. Various ES-cell derivation strategies from genetically permissive and non-permissive mouse strains are described and also the ES-cell differentiation potential to various progenitors and differentiated cell types. Subsequently, details on molecular basis of cardiac differentiation and the therapeutic potential of ES-cell derived differentiated cell types to treat disease(s) are described. This chapter is followed by three data chapters (II-IV). Chapter-II describes the issues related to non-permissiveness of FVB/N strain for ES-cell derivation and strategies to overcome this hurdle. This is followed by detailed results pertaining to generation of homozygous EGFP-expressing transgenic mice and development of a two-pronged ES-cell derivation approach to successfully establish a permanent ES-cell line (named ‘GS-2’ ES-cell line) from the EGFP-transgenic ‘green’ mouse. This chapter also provides results pertaining to detailed characterization of the ‘GS-2’ ES-cell line which includes colony morphology, expansion efficiency, alkaline phosphatase staining, expression analysis of pluripotent markers by RT-PCR and immunostaining approaches and karyotyping. Following this, the outcome of results and significance in the context of reported information are discussed in detail. Having successfully derived the ‘GS-2’ ES-cell line, it is necessary to thoroughly assess the differentiation competence of the ‘GS-2’ ES-cell line. Therefore, the Chapter-III describes detailed assessment of the in vitro and in vivo differentiation potential of the ‘GS-2’ ES-cell line. For in vitro differentiation, results pertaining to ES-cell derived embryoid body (EB) formation and their differentiation to ectodermal, mesodermal and endodermal cell types, expressing nestin, BMP-4 and α-fetoprotein, respectively, are described. Besides, the robustness of adaptability of ‘GS-2’ ES-cells to various culture conditions for their maintenance and differentiation are described. Also shown in the chapter is the relatively greater propensity of this cell line to cardiac differentiation. For in vivo differentiation, the ‘GS-2’ ES-cell derived teratoma formation in nude mice and its detailed histological analysis showing three germ layer cell types and their derivatives are described. Last part of the data described in this chapter, pertains to generation of chimeric blastocysts by aggregation method. Because the ‘GS-2’ ES-cell line exhibited a robust differentiation potential, including an efficient cardiomyocyte differentiation, it is of interest to enhance the efficiency of cardiomyocyte differentiation by exogenous addition of one of the key growth factors i.e., FGF8b since this has been implicated to be critical for cardiogenesis in non-mammalian verterbrate species. Therefore, Chapter-IV is focused on assessing the ability of ‘GS-2’ ES-cell line for its cardiomyocyte differentiation property with particular emphasis on the FGF-induced cardiac differentiation. Results pertaining to the expressions of various FGF ligands and their receptors during differentiation of ES-cells are described. Besides, increases in the cardiac efficiency, following FGF8b treatment and the associated up-regulation of cardiac-specific markers such as GATA-4, ISL-1 and α-MHC are shown. At the end of data chapters, separate sections are devoted for ‘Summary and Conclusion’ and for ‘Bibliography’.

No description available.

Millions of years of evolution have resulted in an immense number of different proteins, which participate in virtually every process within cells and thus are of utmost importance for allknown forms of life. In addition, there are several examples of natural proteins which have found use in applications outside their natural environment, such as the use of enzymes infood industry and washing powders or the use of antibodies in diagnostic, bioseparation or therapeutic applications. To improve the performance of proteins in such applications, anumber of techniques, all collectively referred to as ‘protein engineering’, are performed in thelaboratory.Traditionally, methods involving ‘rational design’, where a few alterations are introduced atspecific protein locations to hopefully result in expected improvements have been applied.However, the use of more recent techniques involving a simultaneous construction of a large number of candidate variants (protein libraries) by various diversification principles, fromwhich rare clones showing enhanced properties can be isolated have contributed greatly to thefield of protein engineering.In the present thesis, different protein traits of biotechnological importance have beenaddressed for improvements by the use of such methods, in which there is a crucial need tomaintain a clonal link between the genotype and the phenotype to allow an identification of protein library members isolated by virtue of their functional properties. In all protein library investigations included in this thesis this coupling has been obtained by Escherichia coli bacterialcell-membrane compartmental confinement.In a first study, a combination of error prone PCR and gene-shuffling was applied to the Tobacco Etch Virus (TEV)-protease gene in order to produce collections from which genesencoding variants showing an enhanced soluble expression of the enzyme frequently used inbiotechnology to cleave fusion proteins were identified. Using Green Fluorescence Protein(GFP)-based cell fluorescence analysis, a clone with a five-fold increase in the yield of solubly produced protein was successfully isolated. In a second study, a novel and different GFPbased selection system, in addition also involving targeted in vivo protein degradation principles,was employed for investigations of the substrate sequence space of the same protease. In two additional studies, a selection system denoted Protein Fragment Complementation Assay(PCA), based on the affinity driven structural complementation of a genetically split β-lactamase enzyme was used to identify variants having desired target protein binding abilities,including both specificity and affinity. Using Darwinian principles concerning clonal growth advantages, affibody binding proteins showing sub-nanomolar dissociation constants to thehuman cytokine TNF-α were isolated. Taken together, these studies have shown that the bacterial format is very well suited for use in various aspects of protein library selection. / QC 20100729

Orientador: Alessandra Alves de Souza / Texto em português e inglês / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-25T17:07:39Z (GMT). No. of bitstreams: 1 Niza_Barbara_M.pdf: 2368175 bytes, checksum: 2c97ffad74e7a3e1c33ae99c78818b99 (MD5) Previous issue date: 2014 / Resumo: A citricultura brasileira é um importante setor para a economia do país, contribuindo com superávits comerciais e geração de empregos, entretanto, o setor passa por uma grave crise econômica em decorrência do alto custo de produção e do baixo valor pago pela caixa de laranja no Brasil. O principal motivo do alto custo de produção é a alta incidência de pragas e doenças que atingem essa cultura. Dentre as doenças, a Clorose Variegada do Citros (CVC) causada pela bactéria Xylella fastidiosa e transmitida a seus hospedeiros por cigarrinhas vetoras, é a que até hoje causou mais danos à citricultura brasileira. O mecanismo de patogenicidade da X. fastidiosa permanece não conclusivo porém a hipótese mais aceita está relacionada à facilidade da bactéria em colonizar o hospedeiro, ou seja, em se movimentar e multiplicar dentro dos vasos do xilema da planta infectada, seguido da formação do biofilme. O conhecimento da doença bem como das interações planta-patógeno e vetor-patógeno estão muito avançados para a doença de Pierce (PD), doença causada pela X. fastidiosa em videiras nos Estados Unidos. Esse avanço no conhecimento para PD ocorreu principalmente devido à obtenção de estirpes geneticamente modificadas da bactéria, permitindo a execução de estudos funcionais e de colonização. A implementação da aquisição de X. fastidiosa em sistema de dieta artificial para estudos com o vetor também foi de grande contribuição para esse avanço, uma vez que esse sistema elimina a utilização de plantas fonte para aquisição da bactéria. Em citros, sabe-se que existem fontes de resistência natural à CVC como tangerinas, tangors, limas e limões, entretanto, todas as variedades de laranja doce plantadas no Brasil são suscetíveis a essa doença. Sabe-se também que há uma resposta genética diferente entre um genótipo resistente e o suscetível quando inoculados com X. fastidiosa, porém, não se conhece como se dá a colonização in planta, e se existe uma correlação entre a resposta genética da planta e o comportamento da bactéria. Buscando melhorar o entendimento dos fatores envolvidos no patossistema CVC este trabalho teve como objetivo a obtenção de uma estirpe patogênica de X. fastidiosa de citros transformada com a proteína verde fluorescente (GFP) afim de avaliar sua colonização in planta em genótipos parentais e híbridos de citros, resistentes e suscetíveis, além da implementação da aquisição de X. fastidiosa por cigarrinhas vetores por meio do sistema de dieta artificial. A obtenção do transformante de X. fastidiosa expressando GFP permitiu o acompanhamento da colonização da bactéria nos vasos do xilema de plantas suscetíveis e resistentes e a avaliação mostrou uma colonização diferenciada entre caule e pecíolo. Também foi verificado um padrão diferencial de colonização dos caules de genótipos suscetíveis em relação aos resistentes, no qual a bactéria parece não capaz de se mover em genótipos resistentes, permanecendo aprisionada no xilema primário dessas plantas, sugerindo um possível mecanismo de resistência. A implementação da aquisição de células bacterianas em sistema de dieta artificial foi estabelecida com sucesso para vetor e estirpe de X. fastidiosa em citros, abrindo perspectivas para vários estudos na área de interação vetor-patógeno e transmissão da CVC / Abstract: The citrus agribusiness is an important segment for the country economy, contributing to employment and trade surpluses, however, it is passing through an economic crisis on behalf of the high cost of production and the low price paid by the orange box in Brazil. The main reason for the high cost of production is the high incidence of pests and diseases affecting this crop. Among the diseases, the Citrus Variegated Chlorosis (CVC) is caused by the bacterium Xylella fastidiosa and transmitted to its hosts by sharpshooters, and it is the disease that more damage have been causing to the citrus agribusiness in Brazil. The X. fastidiosa pathogenicity mechanism still not clear but the currently accept hypothesis is related to its facility to colonize the host, in other words, on move and multiply within the xylem vessels of an infected host, followed by the biofilm formation. The knowledge about the disease and the interactions between plant-pathogen and vector-pathogen are advanced for the Pierce disease, which is caused by X. fastidiosa on grapes in the United States. This occurs mainly on behalf of bacterial mutant¿s obtainment, allowing functional and colonization studies, besides the artificial diet system establishment for insect vectors studies, once this system eliminates the use of source plant acquisition, a limiting factor for X. fastidiosa acquisition since its colonization in host plants is random. In citrus, is known that natural resistant sources against CVC exists like tangerine, tangors, limes and lemons, while all the sweet orange varieties grown in Brazil are susceptible. Also, was verified a different genetic response between a resistant and a susceptible genotype when inoculated with X. fastidiosa, although, the colonization process in planta still unkown as well as if there is a correlation between the plant genetic response and the bacterial behavior. In order to better understand the factors involved in the CVC pathosystem, this work had the goal of obtain a pathogenic X. fastidiosa strain from citrus transformed with the green fluorescent protein (GFP) to evaluate its colonization in planta on parent and hybrid citrus genotypes, resistant and susceptible, besides de X. fastidiosa artificial acquisition by the insect vector using the artificial diet system. Was obtained a X. fastidiosa strain transformed with the GFP which allowed the bacterial colonization monitoring within the xylem vessels of resistant and susceptible plants and this evaluation showed a differential colonization of stems and petioles. Was also verified a different stem colonization pattern between resistant and susceptible genotypes on which the bacteria seems to be not able to move in resistant ones, staying contained into the primary xylem of these plants, suggesting a possible mechanism of resistance. The bacterial cells acquisition on artificial diet system was successfully established using a citrus insect vector and bacterial strain, opening perspectives for various studies on vector-pathogen interaction and transmission of CVC / Mestrado / Genetica de Microorganismos / Mestra em Genética e Biologia Molecular

Les protéines fluorescentes sont très largement utilisées dans les études de biologie moléculaire depuis maintenant une vingtaine d’année. Pour autant, l’origine de leurs propriétés photophysiques n’est pas totalement élucidée. Dans cette thèse, nous avons essayé d’améliorer la compréhension de la photophysique de deux protéines fluorescentes particulières : Padron et EosFP.Dans la protéine Padron, nous avons étudié l’isomérisation du chromophore et cherché à déterminer si la protonation et l’isomérisation sont simultanées ou successives. Pendant l’isomérisation, le donneur de proton potentiel est le résidu Tyr159. Nous avons d’abord montré que dans le vide, le transfert de proton est peu probable quelle que soit la géométrie du chromophore. Dans la protéine (où l’effet de l’environnement n’est pas négligeable) nous avons mis en évidence par dynamique moléculaire que, durant l’isomérisation, le transfert de proton n’est presque jamais favorable et reste donc un marginal.Par ailleurs, ces mêmes dynamiques ont montré que, à la fin de l’isomérisation, il apparaît de nombreux chemins de molécules d’eau reliant le chromophore au solvant et pouvant permettre un transfert de proton. On conclut doncque l’isomérisation et la protonation ne sont pas simultanées mais successives.Dans le cas de la protéine EosFP, nous avons analysé l’effet d’une molécule d’eau présente dans une partie des structures cristallines. Les dynamiques avec le chromophore à l’état fondamental ont montré que cette molécule ne joue pas de rôle, que ce soit sur le réseau de liaison hydrogène ou sur le spectre d’absorption. Par contre, à l’état excité, les dynamiques ont montré que l’extinction de fluorescence est beaucoup plus rapide sans la molécule d’eau qu’en sa présence.Par ailleurs, ces dynamiques ont mis en évidence que la protéine bloque souvent le chromophore dans des géométries où il ne peut pas retourner à l’état fondamental ni par fluorescence, ni par conversion interne. Ces géométries « noires» jouent un rôle important dans la photophysique.Pour tenir compte de ces géométries, nous avons calculé le rendement quantique et le temps de vie de fluorescence par intégration directe le long des trajectoires et par cinétique chimique. Dans les deux cas, nous avons obtenu un accord qualitatif avec l’expérience. / Fluorescent proteins are widely used in biology studies since 20 years. Yet, the origin of their photophysical properties aren’t totally explained. Here, we try to improve the understanding of two particular fluorescent proteins: Padron and EosFP.In the protein Padron, we work on the isomerization of chromophore and try to determine whether isomerization and protonation are simultaneous or successive processes. During the isomerization, the potential donor is Tyr159.First, we show that, in vacuum, the proton transfer is quite unlikely whatever the chromophore geometry.In the protein (where the environment effect isn’t negligible) we evidence with molecular dynamics that, during isomerization, proton transfer stays marginal.In addition, these dynamics shown the appearance, at the end of isomerization, of a lot of water molecules channel between the chromophore and the solvent allowing a proton transfer. We conclude that isomerization and protonation are successive processes.In the case of the protein EosFP, we first analyze the effect of a water molecule which is found only in some of the crystallographic structures.Molecular dynamics of the protein with the chromophore in the ground state show that the water molecule doesn’t play any role neither in the hydrogen bond network nor in the absorption spectra.On the contrary, in the excited state, dynamics without this water show a significant faster decay of fluorescence that those with the molecule.In addition, those dynamics have demonstrate that during long period, the protein retains the chromophore in geometries in which it is unable to convert to the ground state, neither by fluorescence nor by internal conversion. Those “dark” geometries play a crucial role in the photophysics.To take them into account, we calculate the quantum yield and the fluorescence lifetime by direct integration along trajectories and by a kinetic scheme. We obtain a good qualitative agreement with the two methods.

Les protéines fluorescentes (PF) homologues d’AvGFP (Green Fluorescent Protein de la méduse Aequorea victoria) sont des outils incontournables de l’imagerie des processus de la cellule vivante. Leurs performances conditionnent la précision de l’analyse quantitative des signaux de fluorescence. Le développement de nouvelles PF demande donc à la fois de parvenir à une forte brillance tout en contrôlant la réponse de la protéine aux variations des paramètres physico-chimiques de la cellule en fonction de la question biologique étudiée. A ce jour, les PF jaunes disponibles montrent une forte sensibilité au pH. Afin d’élaborer des mutants moins sensibles, deux approches ont été considérées : une première consiste à mieux appréhender l’incidence de la dynamique du réseau de liaisons hydrogène entourant le chromophore sur son équilibre acido-basique. La seconde vise à identifier les facteurs structuraux à l’origine de la brillance particulièrement élevée de nouvelles PF jaunes et jaune-vert provenant d’un ver marin, Branchiostoma lanceolatum.J’ai d’abord mis au point un algorithme recherchant l’ensemble des liaisons hydrogène présentes au sein d’une protéine et qui étudie leur dynamique au cours de simulations par dynamique moléculaire. Il permet leur agrégation en réseaux, l’identification des réseaux connectés à un atome d’intérêt ainsi que le suivi de leur dynamique. Pour validation, cet algorithme a été appliqué à la recherche des réseaux de liaisons hydrogène présents au sein de différents mutants d’AvGFP pour lesquels un transfert de proton à l'état excité a été étudié expérimentalement. Cet algorithme pourra également servir à comprendre de façon dynamique le mécanisme d’autres systèmes biologiques dont la fonction repose sur le transfert de protons.D’autre part, j’ai résolu la structure de la protéine fluorescente jaune naturelle lanYFP de Branchiostoma lanceolatum, particulièrement brillante mais à la structure quaternaire tétramérique. Cette protéine a été rendue monomérique par évolution dirigée, ce qui a donné la protéine mNeonGreen à la fluorescence jaune-vert, protéine désormais étalon dans cette gamme spectrale, et dont j’ai également résolu la structure. Mon étude a permis de rationaliser a postériori l’ensemble des mutations introduites au cours de l’évolution. Enfin, j’ai réalisé une étude du dégât d’irradiation spécifique des rayons X permettant de comprendre le changement remarquable de couleur observé sur les cristaux de mNeonGreen après collecte de données de diffraction.L’ensemble des résultats obtenus au cours de ma thèse permet de proposer un cadre de compréhension à la fois théorique et expérimental des déterminants contrôlant les propriétés de fluorescence des PF jaunes. / Fluorescent Proteins (FPs) homologous to AvGFP (Green Fluorescent Protein from the jellyfish Aequoria victoria) are versatile tools used in live cell imaging. The amount of information that can be derived from the fluorescence signals depends on the spectroscopic performances of the FP. The development of new FPs should focus on both brightness increase and control of the protein response to physicochemical parameter variations within the cell. Current yellow FPs exhibit a strong sensitivity to pH. In order to engineer less sensitive variants, two complementary approaches have been used: the first one consists in studying the influence of the hydrogen bond network dynamics around the chromophore on its protonation state. In the second one, I have sought to identify the structural determinants of the particularly high brightness of newly discovered yellow FPs from a sea worm, Branchiostoma lanceolatum.First, I wrote an algorithm that can identify all hydrogen bonds within a protein and analyse their dynamics along molecular dynamics simulations. It allows for their clustering in networks, the identification of networks connected to a given atom and the monitoring of their dynamics. The method was validated by using the algorithm on various AvGFP mutants for which excited state proton transfer has been experimentally studied. This algorithm should also be useful for the study of other biological systems whose function is based on proton transfer.Besides, I solved the structure of the natural yellow FP lanYFP from Branchiostoma lanceolatum, which is particularly bright, but presents a tetrameric arrangement. This protein was monomerized by directed evolution, which led to the yellow-green FP mNeonGreen, now a benchmark in this spectral range. I also solved the structure of mNeonGreen, which allowed me to rationalize a posteriori the mutations that have been introduced during the evolution process. Finally, I performed a specific radiation damage study in order to explain the remarkable change in colour of mNeonGreen crystals upon X-ray data collection. Altogether, the results of my PhD work provides a theoretical and experimental framework of the determinants that drive the fluorescence properties of yellow FPs.

RNA interference (RNAi) is an important mechanism regulating gene expression. In plants, RNAi is triggered by double-stranded RNA (dsRNA) which is processed into small RNAs (sRNAs), usually 21-24 nt long. The sRNAs are loaded into Argonaut (AGO) protein and recognize the target based on sequence complementarity. When the target is mRNA, they can slice it or block translation leading to posttranscriptional gene silencing (PTGS). When the target is DNA, they can induce DNA methylation and chromatin changes, which when present in the promoter can lead to transcriptional gene silencing (TGS). The individual components of RNAi are well described, but less is known about the impact of different types of dsRNA precursors on the dynamics of RNAi. To study these aspects of RNAi, we used tobacco BY-2 cell line expressing GFP reporter and inducible silencers. The silencers used different ways of triggering the dsRNA formation by transcripts from antisense (AS), unterminated sense (UT) and inverted repeat (IR) GFP sequence to initiate PTGS. Additionally, one IR silencer based on the CaMV 35S promoter initiated TGS. This allowed us to study RNAi from the beginning throughout the steady state level and till the recovery phase, all in the highly homogeneous system. Using this system, we described several features...

Upland cotton (Gossypium hirsutum L.) is the world's most prominent fiber crop. Cotton transformation is labor intensive and time consuming, taking 12 to 18 months for rooted T0 plants. One rate limiting step is the necessary production of somatic embryos. In other recalcitrant species, ectopic expression of three genes were shown to promote somatic embryogenesis: WUSCHEL (WUS), SHOOT MERISTEMLESS (STM), and BABY BOOM (BBM). WUS is responsible for maintaining stem-cell fate in shoot and floral meristems. STM is needed to establish and maintain shoot meristems. STM and WUS have similar functions but work in different pathways; overexpression of both together converts somatic cells to meristematic and embryogenic fate. BBM encodes an AP2/ERF transcription factor that is expressed during embryogenesis and ectopic expression of BBM reprograms vegetative tissues to embryonic growth. In prior studies, these genes were constitutively expressed, and cultures did not progress beyond embryogenesis because the embryogenic signal was not turned off. In our study, we set out to use these genes to increase the efficiency of cotton transformation and decrease the time it takes to regenerate a plant. A disarmed cotton leaf crumple virus (dCLCrV) vector delivers WUS, STM, or BBM into cotton tissue cultures through Agrobacterium tumefaciens infection. We propose that virus delivery of embryo-inducing genes is a better approach for transformation because A) inserts more than 800 nucleotides are unstable, and will spontaneously inactivate, B) virus DNA can migrate through plasmodesmata to cells around the infected cell, creating a gradient of embryonic potential, C) the virus DNA does not pass through the germ line and the seed will not contain virus. We propose this method of inducing embryogenesis will facilitate the stable transformation of cotton and will be beneficial to the cotton industry. Ectopic expression of AtBBM, AtSTM, and AtWUS GrWUS:meGFP from a constitutive CaMV 35S promoter produced plants with phenotypes similar to those described in previous studies overexpressing AtBBM, indicating that the AtBBM gene was functional. The cotton cotyledon infiltration of the pART27 constructs showed transformed cells in Coker 312 by GFP localization in the nucleus. Although GFP was detected, no visible embryos appeared from the cotyledon. Cotyledons infiltrated with Agrobacterium harboring overexpression vectors withered and aborted after ~2 weeks. The virus-based vector in tissue culture failed to increase transformation efficiency, resulting in no embryos. The combination of hormone concentration showed no contribution to increasing the transformation efficiency.

In the beginning of this thesis is a short introduction into plasmid DNA which is in the form of a vector used in molecular biology. Plasmids can be used in the form of fluorescent probes to measure changes in membrane potential. Into their structure is added a dye called fluorophore. As an important representative of this thesis is a fluorescent probe ASAP1 which contains green fluorescent protein whose response to the membrane potential change is the decrease in the intensity of emitted light. The aim of this thesis was to make chemical transfection of this plasmid into the HEK293 cell line and carry out its characterization. In the work is also described the design of a method for the analysis of the time course of changes in fluorescence depending on the cell membrane depolarisation. In the end of this thesis is also desribed realized experiment including the discussion of aquired results.