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.

No description available.

Micro- and nanostructured polymer brushes on diamond can be directly prepared by carbon templating and amplification of the latent structures by photografting of a broad variety of vinyl monomers such as styrenes, acrylates and methacrylates. Even template structures with lateral dimensions as small as 5 nm can be selectively amplified and defined polymer brush gradients of a variety of functional polymers are realizable by this technique. Furthermore, conjugation with a model protein (GFP) results in protein density gradients of high loading and improved chemical stability. The effective functionalization of chemically and biologically inert diamond surfaces with stable functional polymer brushes, the possibility of structuring by the carbon templating technique and the direct biofunctionalization are crucial steps for the development of diamond based biosensors. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

O objetivo desse trabalho foi construir o fago recombinante D29::gfp e testar a sua utilização como um agente revelador da viabilidade bacilar na determinação da concentração inibitória mínima (GIM) aos principais fármacos administrados no tratamento da tuberculose. O fago recombinante contém o promotor hsp70 e o gene da proteína verde fluorescente (gfp) e foi construído através da restrição pela Spe I em uma região intergênica próxima a extremidade coesiva direita no genoma do fago D29. O promotor hsp70 e gfp clonados no pYL GFP foram amplificados pela PCR utilizando iniciadores com sítios para Spe I. O DNA do fago D29 digerido pela Spe I foi ligado com o fragmento hsp 70- gfp empregando a T 4 DNA ligase e os produtos da reação de ligação foram transformados de acordo com o protocolo de encapsulamento. A infecção do M.smegmatis com esse fago recombinante induziu a expressão da proteína verde fluorescente (GFP). Para avaliar o uso do fago recombinante em teste de sensibilidade aos fármacos anti-tuberculose, 100 isolados clínicos foram testados quanto ao perfil de sensibilidade a isoniazida (H), rifampicina (R), estreptomicina (S) e etambutol (E), utilizando o método das proporções em Lowenstein-Jensen (L-J), técnica em microplaca com a resazurina (REMA) e técnica em microplaca com D29::gfp. Os resultados do REMA demonstraram que 30 isolados clínicos foram sensíveis à H e 58 (66 %) isolados clínicos foram resistentes, dentre os quais a CIM foi 1 µg/mL ou maior para 41 (71 %). A CIM da R para 49 (56%) dos isolados clínicos resistentes foi de 0,5 µg/mL para 17 (35%). A CIM da S para 33 (37%) dos isolados clínicos resistentes foi de 2 µg/mL para 13 (40%) e CIM do E para 34 (39%) dos isolados clínicos resistentes foi de 16 µg/mL ou maior para 19 (56%). A caracterização molecular pela PCR IS6110 identificou 88 isolados clínicos como M.tuberculosis e pelo PRA hsp65, sete isolados clínicos foram M.kansasii, quatro foram M.abscessus e um M.szulgai. Após empregar o fago recombinante como um agente indicador da viabilidade bacilar para testar a atividade dos fármacos anti-tuberculose conclui-se que a expressão da proteína verde fluorescente foi inespecífica e não reprodutiva, não justificando o seu uso para determinar a CIM para os principais fármacos administrados no tratamento da tuberculose. / The objective of this work was to construct the recombinant phage D29::gfp and to use this phage as an indicator agent of cell viability in a minimal inhibitory concentration (MIC) assay for the mains drugs used for tuberculosis treatment. The recombinant phage contains the mycobacteria-specific hsp70 promoter controlling the green fluorescent protein gene (gfp) and was constructed by Spe I restriction in the intergenic region next to the right cohesive termini of the D29 phage genome. An hsp 70 promoter and gfp previously cloned in p YL GFP was amplified by PCR using primers with Spe I sites. The Spe I-restricted D29 phage DNA was ligated with the hsp 70-gfp fragment using T4 DNA ligase and ligated product was transformed using the packing protocol. Infection of M.smegmatis with this recombinant phage indicated the expression of green f1uorescent protein (GFP). To use the recombinant phage for assaying the activity of anti-TB drugs, 100 clinical isolates was tested for susceptibility to isoniazid (H), rifampicin (R), streptomycin (S), and ethambutol (E) using both the proportion method on Lowenstein-Jensen (L-J) medium, resazurin microtiter assay plate (REMA), as well as a microplate assay using D29::gfp. The REMA plate method showed that 30 clinical isolates were susceptible to H and 58 (66%) clinical isolates were resistant, where the MICs were 1 µg/mL or higher for 41 (71%). The R MICs for 49 (56%) resistant clinical isolates were 0,5 µg/mL for 17 (35%). The S MICs for 33 (37%) resistant clinical isolates were 2 µg/mL for 13 (40%) and E MICs for 34 (39%) resistant clinical isolates were 16 µg/mL or higher for 19 (56%). Molecular characterization by PCR IS6110 showed that 88 clinical isolates were M.tuberculosis and by PRA hsp65 were seven clinical isolates were M.kansasii and four was M.abscessus, and one M.zulgai. After using the recombinant phage as an indicator agent of cell viability for assaying the activitity of anti-TB drugs we can conclude that the expression of green fluorescent protein was non-specific and not reproducible, rendering it not useful for the determination of the MIC of the principal drugs used for the treatment of tb.

Células transformadas de E.coli DH5-&#945; expressando a proteína verde fluorescente (GFPuv, pico de excitação e emissão de 394nm e 509nm) foram submetidas a extração pelo método de partição de três fases (TPP) e o extrato obtido purificado por cromatografia de interação hidrofóbica (HIC). O objetivo principal deste trabalho foi estudar a termoestabilidade da GFPuv extraída, para avaliar a sua possível utilização como indicador biológico econômico, de resposta rápida e precisa para processos térmicos de esterilização utilizando o calor úmido. A estabilidade térmica da proteína foi estudada em diferentes soluções-tampão (acetato, fosfato e tris-HCI 10mM) no intervalo de valor de pH de 5,O a 9,0 e, em temperaturas entre 75&#176; e 95&#176;C. Os parâmetros de resistência térmica determinados foram: o tempo de redução decimal (Valor D - min), valor z (&#176;C), coeficiente Q10 e valor de energia de ativação (kcal/mol). A termoestabilidade da GFPuv, expressa em valor D, mostrou correlação linear para valores de pH &#8805; 5,50, em tampão acetato. Em tampão fosfato, para valores de pH &#8805; 7,50 a estabilidade térmica da proteína foi independente do valor de pH da solução. Em tampão tris-HCI, o valor D mostrou-se inconstante ao aumento do valor de pH da solução. No intervalo de temperatura estudada, em tampão acetato a GFPuv apresentou melhor termoestabilidade (Ea de 19,27 kcal/mol) do que em tampão fosfato (Ea de 26,18 kcal/mol ao valor de pH 6,S) e em tampão tris-HCI (Ea 28,19 kcallmol ao valor de pH 7,0). Em tampão acetato e tris-HCI ao valor de pH 7,0, a termoestabilidade da proteína mostrou-se equivalente. Entretanto, em tampão fosfato aos valores de pH 7,5 e 8,0 e em tampão tris-HCI aos valores de pH 8,0 e 8,5 a GFPuv apresentou menor estabilidade térmica A GFPuv apresenta potencialidade para ser utilizada como indicador biológico em processos térmicos que utilizam calor úmido às temperaturas inferiores a 100°C. / Transformed cells of Escheríchía coli DH5-&#945; expressing recombinant green fluorescent protein (GFPuv, excitation and emission peaks at 394nm and 509nm), were subjected to the three-phase partitioning (TPP) method and the release extracts were eluted through methyl HIC column with a buffer solution (10 mM Tris-HCI, 10mM EDTA, pH=8.0). The purpose of this work was to study the thermal stability of the TPP-extracted recombinant protein, GFPuv, to determine its utility as a quick, accurate and economical biological indicator for moist heat-treatments. The thermal stability of the extracted GFPuv was studied in different buffer solutions (acetate, phosphate and tris-HCI 10mM) in the range of pH between 5.0 and 9.0 and at temperature between 75-95°C. The thermal resistance parameters determinated were: decimal reduction times (D-values, min), z-value (&#1776C), Q<sub<10 coefficient and Activation Energy (Ea, Kcal/mol). The thermal stability of GFPuv, expressed in D-values, showed linear correlation for pH &#8805; 5.50 in acetate buffer. In phosphate buffer, for pH &#8805; 7.50 the thermal stability was independent of pH value. In tris-HCI buffer the D-value was shown variable with the increase of pH value. In the studied temperature range, the acetate buffer at pH 6.0 presented better thermal stability for GFPuv (Ea 19.27kcal/mol) than phosphate (Ea 26.18 kcal/mol at pH 6.5) and tris-HCI buffer (Ea 28.19 kcal/mol at pH 7.0). In acetate and tris-HCI buffers at pH 7.0, GFPuv showed equivalent thermal stability. However, GFPuv showed lower thermal stability in phosphate buffer at pH 7.5 and 8.0 and in tris-HCI buffer at pH 8.0 and 8.5. The TPP-extracted GFPuv has great potential to be applied as a biological indicator in moist heat processes at temperatures below 100°C.

The immune system is essential for keeping the integrity of multicellular organisms. We were able to make a step forward in studying the complex immune reactions in mammals in vivo and/ or in situ using the major histocompatibility complex (MHC) class II/ enhanced green fluorescent protein (EGFP) knock-in mouse model. Due to the EGFP visualization of MHC II expressing cells we were able to observe antigen presenting cells, which are essential for the onset of immune responses, in their natural environment. Thus, we report some original features of the immune system. We have identified MHC II+ cell clusters with unknown, probably unique function, in the intestine. We have also described MHC II+ cell migration to the lactating mammary gland and tested few hypotheses about the role of this phenomenon for the development of the mammary gland, milk secretion or infant immune system establishment. Lastly, we observed residential macrophages in the cornea. The presence of APCs in the cornea is a very contradictory issue due to the fact that cornea is an immunologically privileged tissue and therefore harbors special immune features. key words: antigen presenting cells (APC), major histocompatibility complex class II (MHC II), enhanced green fluorescent protein (EGFP), immune system, knock-in mouse model

Células transformadas de E.coli DH5-&#945; expressando a proteína verde fluorescente (GFPuv, pico de excitação e emissão de 394nm e 509nm) foram submetidas a extração pelo método de partição de três fases (TPP) e o extrato obtido purificado por cromatografia de interação hidrofóbica (HIC). O objetivo principal deste trabalho foi estudar a termoestabilidade da GFPuv extraída, para avaliar a sua possível utilização como indicador biológico econômico, de resposta rápida e precisa para processos térmicos de esterilização utilizando o calor úmido. A estabilidade térmica da proteína foi estudada em diferentes soluções-tampão (acetato, fosfato e tris-HCI 10mM) no intervalo de valor de pH de 5,O a 9,0 e, em temperaturas entre 75&#176; e 95&#176;C. Os parâmetros de resistência térmica determinados foram: o tempo de redução decimal (Valor D - min), valor z (&#176;C), coeficiente Q10 e valor de energia de ativação (kcal/mol). A termoestabilidade da GFPuv, expressa em valor D, mostrou correlação linear para valores de pH &#8805; 5,50, em tampão acetato. Em tampão fosfato, para valores de pH &#8805; 7,50 a estabilidade térmica da proteína foi independente do valor de pH da solução. Em tampão tris-HCI, o valor D mostrou-se inconstante ao aumento do valor de pH da solução. No intervalo de temperatura estudada, em tampão acetato a GFPuv apresentou melhor termoestabilidade (Ea de 19,27 kcal/mol) do que em tampão fosfato (Ea de 26,18 kcal/mol ao valor de pH 6,S) e em tampão tris-HCI (Ea 28,19 kcallmol ao valor de pH 7,0). Em tampão acetato e tris-HCI ao valor de pH 7,0, a termoestabilidade da proteína mostrou-se equivalente. Entretanto, em tampão fosfato aos valores de pH 7,5 e 8,0 e em tampão tris-HCI aos valores de pH 8,0 e 8,5 a GFPuv apresentou menor estabilidade térmica A GFPuv apresenta potencialidade para ser utilizada como indicador biológico em processos térmicos que utilizam calor úmido às temperaturas inferiores a 100°C. / Transformed cells of Escheríchía coli DH5-&#945; expressing recombinant green fluorescent protein (GFPuv, excitation and emission peaks at 394nm and 509nm), were subjected to the three-phase partitioning (TPP) method and the release extracts were eluted through methyl HIC column with a buffer solution (10 mM Tris-HCI, 10mM EDTA, pH=8.0). The purpose of this work was to study the thermal stability of the TPP-extracted recombinant protein, GFPuv, to determine its utility as a quick, accurate and economical biological indicator for moist heat-treatments. The thermal stability of the extracted GFPuv was studied in different buffer solutions (acetate, phosphate and tris-HCI 10mM) in the range of pH between 5.0 and 9.0 and at temperature between 75-95°C. The thermal resistance parameters determinated were: decimal reduction times (D-values, min), z-value (&#1776C), Q<sub<10 coefficient and Activation Energy (Ea, Kcal/mol). The thermal stability of GFPuv, expressed in D-values, showed linear correlation for pH &#8805; 5.50 in acetate buffer. In phosphate buffer, for pH &#8805; 7.50 the thermal stability was independent of pH value. In tris-HCI buffer the D-value was shown variable with the increase of pH value. In the studied temperature range, the acetate buffer at pH 6.0 presented better thermal stability for GFPuv (Ea 19.27kcal/mol) than phosphate (Ea 26.18 kcal/mol at pH 6.5) and tris-HCI buffer (Ea 28.19 kcal/mol at pH 7.0). In acetate and tris-HCI buffers at pH 7.0, GFPuv showed equivalent thermal stability. However, GFPuv showed lower thermal stability in phosphate buffer at pH 7.5 and 8.0 and in tris-HCI buffer at pH 8.0 and 8.5. The TPP-extracted GFPuv has great potential to be applied as a biological indicator in moist heat processes at temperatures below 100°C.

L'action de la dopamine dans les cellules est transmise par sa fixation sur des récepteurs qui appartiennent à deux classes, D1 et D2. Quatre sous-types du récepteur D1 (D1A, D1B/D5, D1C et D1D) ont été clonés jusqu'à présent chez les vertébrés. L'analyse évolutive montre que les sous-types D1A et D1B sont les plus conservés alors que les sous-types D1C et D1D sont absents chez les mammifères. Malgré cette diversité, les fonctions de la dopamine dans l'organisme ne peuvent pas être rapportées à l'action d'un sous-type précis. C'est pourquoi au cours de ce travail nous avons identifié des caractères fonctionnels capables de distinguer chacun des sous-types et de comprendre pour quelle raison ils ont été conservés chez les vertébrés. La désensibilisation est un des paramètres fonctionnels les plus important. Le récepteur D1A se caractérise par une baisse d'activité forte et biphasique, le récepteur D1B par un profil proche avec une amplitude plus faible conséquence de son activité constitutive. Enfin, le récepteur D1C ne semble pas être capable de se désensibiliser. La construction de chimères entre chacun des sous-types du récepteur D1 et la protéine GFP ont permis la visualisation des récepteurs au cours de la désensibilisation. Elles montrent que l'internalisation ne semble pas, pour les récepteurs D1 de la dopamine, intervenir dans le processus de désensibilisation fonctionnelle. L'activation simultanée des récepteurs A1 de l'adénosine bloque l'activité des récepteurs D1. L'analyse des voies de signalisation MAPK a montré que l'activation de la voie ERK était rapide et forte, et différente selon les sous-types. La voie p38 n'est que faiblement activée et la voie JNK semble ne pas l'être du tout. Il semble donc que les mécanismes d'activation et de régulation des voies de signalisation différencient les sous-types du récepteur D1 chez les vertébrés. Ces paramètres participent de façon majeure à la transmission régulée des fonctions de la dopamine dans l'organisme.

Au cours de ce travail expérimental, nous nous sommes intéressés aux propriétés photo-physiques d'un mutant de la protéine Fuorescente verte (Green Fluorescent Protein, GFP). Plus particulièrement, nous avons caractérisé par spectroscopie femtoseconde résolue spectralement la dynamique des états excités de ce mutant. Nous avons tout d'abord comparé les résultats obtenus avec ceux de la protéine naturelle. Cela nous a permis de montrer que la dynamique des états excités s'avère très sensible aux modifcations de l'environnement proche de la partie optiquement active de la protéine. Dans une deuxième partie qui concerne l'étude de fusion génétique fragment d'anticorps-GFP, nous avons utilisé la sensibilité offerte par la dynamique des états excités de la protéine pour caractériser l'état de repliement de l'anticorps fusionné. Une autre partie de ce travail a été consacrée à l'étude des propriétés dynamiques de certains dérivés de la coumarine dont la nature de rotors moléculaires a été évoquée dans la littérature. Nous avons montré que la dynamique des états excités ne présente pas de signatures d'un tel comportement. Parallèlement aux études dynamiques, nous avons étudié le vieillissement ou diminution du nombre de molécules fluorescentes d'un ensemble bien déterminé de GFP sous illumination continue. Cette étude a mis en évidence le fait que l'évolution temporelle du système est gouvernée par une statistique de Lévy. L'une des conséquences principales étant l'existence d'un état instable à durée de vie moyenne infnie.

Mathematical models of signal transduction pathways are characterized by a large number of proteins and uncertain parameters, yet only a limited amount of quantitative data is available. The dissertation addresses this problem using two different approaches: the first approach deals with a model simplification procedure for signaling pathways that reduces the model size but retains the physical interpretation of the remaining states, while the second approach deals with creating rich data sets by computing transcription factor profiles from fluorescent images of green-fluorescent-protein (GFP) reporter cells. For the first approach a model simplification procedure for signaling pathway models is presented. The technique makes use of sensitivity and observability analysis to select the retained proteins for the simplified model. The presented technique is applied to an IL-6 signaling pathway model. It is found that the model size can be significantly reduced and the simplified model is able to adequately predict the dynamics of key proteins of the signaling pathway. An approach for quantitatively determining transcription factor profiles from GFP reporter data is developed as the second major contribution of this work. The procedure analyzes fluorescent images to determine fluorescence intensity profiles using principal component analysis and K-means clustering, and then computes the transcription factor concentration from the fluorescence intensity profiles by solving an inverse problem involving a model describing transcription, translation, and activation of green fluorescent proteins. Activation profiles of the transcription factors NF-κB, nuclear STAT3, and C/EBPβ are obtained using the presented approach. The data for NF-κB is used to develop a model for TNF-α signal transduction while the data for nuclear STAT3 and C/EBPβ is used to verify the simplified IL-6 model. Finally, an approach is developed to compute the distribution of transcription factor profiles among a population of cells. This approach consists of an algorithm for identifying individual fluorescent cells from fluorescent images, and an algorithm to compute the distribution of transcription factor profiles from the fluorescence intensity distribution by solving an inverse problem. The technique is applied to experimental data to derive the distribution of NF-κB concentrations from fluorescent images of a NF-κB GFP reporter system.