Water stress affects vine productivity, disease tolerance, and enological characteristics of grape. Florida Hybrid Bunch grape are developed through hybridization of local grape spp with Vitis vinifera. These cultivars are mostly grown in southeast region of United States. Water deficit conditions resulted due to failure of rains in the region has developed concern among Florida grape growers to increase water use efficiency of grape. The goal of this research is to identify genes and proteins differentially expressed in response to water stress and to correlate these changes with enological characteristics. Investigating transcripts and proteins will allow us to correlate them and confirm the involvement of specific genes responding to stress. Florida hybrid bunch ‘Suwannee’ grape plants were maintained under green house conditions. Water stress was induced by withholding irrigation. The leaf samples were collected from both irrigated and stressed plants at 5, 10, 15 and 20 day interval. We generated over 200 Subtractive Hybridization PCR products from control and water stressed leaf tissues. Cloning, sequencing and transcript analysis revealed that, 54 genes related to drought and defense regulated pathways out of 125 characterized transcripts. Proteins were extracted from leaf tissue with trichloroacetic acid /acetone and separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The proteins were sequenced in LC/Mass Spectrophotometer. The most important differentially expressed genes include sucrose synthase, actin, isoprene synthase, ABF3, SNF1 related protein kinase, WRKY type transcription factors, AP2, ASR2, glyoxalase I and, cytochrome b which play significant role in cell permeability, transportation, photosynthesis and, maintenance in osmotic stress. We have found that ribulose bisphosphate carboxylase and phosphoribulokinase, which play major role in photosynthesis, were suppressed in response to water stress in Florida hybrid bunch. The results suggested that water stress affects expression of cDNAs associated with defense and drought regulated functions. Such profiling studies will be used to explicate specific pathways disconcerted by water deficit treatments, and in the identification of varietal differences.

La doxorubicine (DXR) est l’un des médicaments les plus efficaces en chimiothérapie, mais sonapplication clinique est limitée par ses effets cardiotoxiques. Malgré des décennies de recherche, sesmécanismes pathogéniques ne sont toujours pas entièrement compris. Il s’ensuit qu’aucun traitementsatisfaisant, curatif ou préventif, n’existe. Dans cette étude, nous recherchons les mécanismes designalisation cellulaire impliqués. Deux modèles expérimentaux de toxicités, aigue d’une part (coeurisolé et perfusé de rat avec la DXR), et chronique d’autre part (rat traité à la DXR), ont permis deréaliser une étude ciblée (sur les voies de signalisation énergétiques) et deux études systémiques(phosphoprotéomique et transcriptomique). Les résultats combinés de ces travaux ont montré que laDXR modifiait le niveau de phosphorylation (activation) ou l’expression génique de protéinesimpliquées dans trois domaines fonctionnels distincts : métabolisme énergétique, réponses au stress,et structure/fonction du sarcomère. (i) Métabolisme énergétique : nous avons confirmé la surprenanteinhibition de l’AMPK, probablement provoquée par un contrôle négatif exercé par des partenaires designalisation (Akt et ERK), plutôt que par une modification des kinases activatrices en amont. Nousavons également montré l’augmentation du niveau de phosphorylation de la PDH, ce qui, en inhibantl’enzyme, ralentit le cycle de Krebs. Cependant, nous avons également observé un phénomènecompensatoire de surexpression de gènes codant pour des enzymes de la glycolyse et du cycle deKrebs ; (ii) Réponses au stress : dans nos modèles, la DXR génère des stress énergétique,génotoxique et oxydatif. Cependant, seuls quelques mécanismes compensatoires sont activés (lesvoies de signalisation de DNA-PK–Akt–GSK3, diverses chaperonnes). Les autres semblent êtreinhibées suggérant que l’amoindricement des réponses au stress serait un des mécanismes de lacardiotoxicité de la DXR; (iii) Structure/fonction du sarcomère: L’augmentation de la phosphorylationde la desmine ainsi que la réduction du nombre de transcrits codant pour des protéines essentiellesau développement cardiaque normal pourraient être la cause de la désorganisation du réseaumyofibrillaire. En conclusion, ces résultats révèlent potentiellement de nouveaux mécanismes de lacardiotoxicité induite par la DXR et permettent d’envisager de nouvelles cibles moléculaires pour ledéveloppement de stratégies protectrices. / Doxorubicin (DXR) is an efficient anticancer drug, the use of which is limited by seriouscardiotoxicity. Despite decades of research, its pathogenic mechanisms are not fully understood, andefficient preventive or curative strategies are not available. Here we address the question whethermechanisms in cardiac cell signaling contribute to the toxicity phenotype. Using experimental modelsfor acute (DXR-perfused, isolated rat hearts) or chronic toxicity (rats injected with DXR), we conducteda targeted study (focusing on energy signaling pathways) and two non-biased studies(phosphoproteomics and transcriptomics). The combined data reveal DXR-induced alterations inphosphorylation (activation) status or gene expression of proteins in mainly three functional domains:energy metabolism, stress responses, and sarcomere structure. (i) Energy metabolism: We confirm aparadox inhibition of AMPK signaling, that is rather due to inhibitory cross-talk with related signalingpartners (Akt, ERK) than impaired AMPK upstream signaling. We also show, among others, theincrease of inhibitory phosphorylation of pyruvate dehydrogenase, slowing down Krebs cycle, but alsoa compensating upregulation of glycolysis and Krebs cycle enzyme transcripts. (ii) Stress-responses:In our models, DXR generates energetic, oxidative and genotoxic stress, but only some compensatorystress responses are activated (DNA-PK–Akt–GSK3 pathway, chaperones). Many others seem to beinhibited, suggesting a blunted response to stress as component of DXR toxicity. (iii) Sarcomerestructure/function: We detect increased phosphorylation of desmin and reduced transcripts essentialfor e.g. normal heart development as potential causes for a disorganized myofibrillar network. Inconclusion, these results reveal some novel potential mechanisms of DXR-induced cardiotoxicity andsuggest new targets for protective strategies.

Living conditions of every organism are influenced by various factors at this time. Some of them have positive effect on organism, some negative. Basic condition for surviving is the ability to resist and adapt to changing metabolic and living conditions. Every single stress effect can lead to changes in metabolism but organisms have ability to develope sufficient mechanisms for stress response. Some of them are similar for all living organisms (enzyme production, endogenous primary stress metabolites) some of them are specific for certain organism or stress type. Cell stress response can be observed on different levels (proteomic, genomic, metabolomic). In proper conditions it can be used indrustrially. In this work, influences of various stress factors were studied. These factors were applied on selected organisms – carotenogenic yeast and plant materials. Yeast stress response was induced by osmotic and oxidation stress factors. Changes on proteomic level and in production of selected secondary metabolites were observed. Proteome was analyzed by 1D and 2D electrophoresis with subsequent analysis of proteins by mass spectrometry. Yeast strain Rhodotorula glutinis CCY 20-2-26 showed the best adaptation to stress factors, which was moreover accompanied by overproduction of carotenoids. This finding can be premise for next industrial production of carotenoids. In plant samples predominantly enzymes and metabolites involved in antioxidant response were studied.

Paclitaxel is one of the most successful drugs for the treatment of cancer because of its ability to target tubulin, block cell cycle progression at mitosis, and induce apoptosis. Despite the success of Paclitaxel, the development of drug resistance hampers its clinical applicability. Paclitaxel is used in malignant tumors in the present research, including non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), head-neck scale epitheliomatous, urinary bladder cancer, tumor of the reproduction organ and so on. Clinical treatment of paclitaxel be injected 250mg/m2 to previously non-treated patients of small lung cancer its effect about 34% and previously non-treated patients of non-small lung cancer its effect about 21% to 24% . On the other hand we had established a taxol-resistant human lung carcinoma subline A549R by paclitaxel to compare the different proteins with A549 wild type and treat the different concention of paclitaxel with MTT assay so as to observe the tolerance dosage of subline growth.We obtained the patient¡¦s specimens of lung cancer to treat with paclitaxel some of resistant and some of non-resistant to compare differentially expressed proteins between normal and tumor. When we cultured taxol-resistant human lung carcinoma subline in which paclitaxel and calcium regulate growth, owing to the proteins of changes result to resistance. The extraction cell subline and specimens were analyzed by 2-D electrophoresis patterns and we found that interact paclitaxel with calcium it is the important factor of drug resistant. Verified from clinical treatment might have hypercalcemia in malignant tumors and calcium ion may increase paclitaxel drug resistance and hypercalcemia patient will be more insensitive to paclitaxel treatment.

Die Therapie von disseminierten malignen Tumoren durch Chemotherapie allein oder in Kombination mit Bestrahlung oder Hyperthermie führt nur in 20-50% zu einem Ansprechen. Dieser Behandlungserfolg wird häufig dadurch limitiert, daß im Verlauf der Therapie zur Entwicklung einer Chemoresistenz kommt. Ziel der Arbeit war es, eine generelle Analyse der Proteinexpression in der in vitro herbeigeführten Chemoresistenz durchzuführen. Dafür wurden Zellkulturen von Magen-, Kolon-, Pankreas, Mammakarzinom und Fibrosarkom die eine Resistenz gegen Daunorubicin und Mitoxantron besitzen benutzt und mit der zweidimensionalen Gelelektrophorese im immobilisierten pH-Gradienten (pH 4,0-8,0) in der ersten Dimension und einen linearen Polyarcylamidgel (12%) in der zweiten Dimension analysiert. Nach Färbung in Coomassie blau wurde eine rechnergestützte Imageanalyse mit dem PDQuest System durchgeführt. Proteinspots die eine auffällige Änderung zeigten wurden isoliert, und nach enzymatischer Gelhydrolyse mikrosequenziert bzw. durch Massenspektrometrie und anschließender micropore HPLC Analyse identifiziert. Acht Proteine, die in den chemoresistenten Zellinien überexprimiert waren, konnten identifiziert werden: Thioredoxin, Annexin 1; Cofilin, Stratifin(14-3-3sigma), Rho-GDP-Dissoziations Inhibitor, Fettsäurebindendes Protein(E-FABP), Adenin Phosphoribosyl Transferase, und BCSG-1 / The therapy of advanced cancer using chemotherapy alone or in combination with radiation or hyperthermia yields an overall response rate of about 20-50%. This success is often marred by the development of resistance to cytostatic drugs. The aim was to study the global analysis of protein expression in the development of chemoresistance in vitro. We therefore used a cell culture model derived from the gastric, colorectal, pancreatic, mamma carcinoma and fibrosarcoma cell line selected to daunorubicin and mitoxantrone. These cell lines were analysed using two-dimensional electrophoresis in immobilized pH-gradients (pH 4.0-8.0) in the first dimension and linear polyacrylamide gels (12%) in the second dimension. After staining with coomassie brilliant blue, image analysis was performed using the PDQuest system. Spots of interest were isolated using preparative two-dimensional electrophoresis and subjected to microsequencing after enzymatic hydrolysis in gel, mass spectrometric data and sequencing of the peptides after their fractionation using microbore HPLC identified. Eight proteins were identified that were overexpressed in chemoresistant cell lines: Thioredoxin, Annexin 1, Cofilin, Stratifin(14-3-3sigma), Rho-GDP-dissoziation Inhibitor, fatty acid binding protein(E-FABP), adenin phosphoribosyl Transferase, and BCSG-1.

This diploma thesis was focused on the study of lysozyme and lysozyme-like proteins, either of similar function (antibacterial) or molecular weight (14 - 17 kDa), of synanthropic acaroid mites. In general, animals utilize lysozymes for defensive (antimicrobial) or digestive purposes but also as a digestive enzyme. Some chitinases or other enzymes that act similarly to lysozyme can be utilized for similar purposes. Chitinases belong to house dust mite allergens. One of major mite are historically named lysozyme-like proteins which name relates to their size similar to lysozyme. Bacteriolytic activity has also 14.5 kDa (UniprotKB Q8MWR6) protein. The species selected for the study were domestic mites Dermatophagoides farinae, D. pteronyssinus and Lepidoglyphus destructor. Presence of lysozyme was detected by direct detection with polyclonal antibody using immunohistochemistry and dot blots. Immunohistochemistry proved presence of lysozyme epitopes in the feces of D. farinae, D pteronyssinus a L. destructor. Dot blot analysis demonstrated the presence of imunoreactivity of antibody in spent growth medium extracts (SGME) of all three species. This implies that lysozyme is synthesized in the midgut. The presence of lysozyme and lysozyme-like proteins was proved using 2D electrophoresis and MALDI TOF/TOF...

Biofilms represent universal strategy for bacterial survival. Living in form of biofilms, bacteria acquire wide range of advantages over planktonically growing cultures. It can be assumed that nearly 99% of world bacterial population is living in form of biofilms. There are benefits and drawbacks associated with bacterial biofilms for mankind. Life in biofilms makes pathogens more effective and persistent through higher antibiotic resistance and helps them to hide before immune system of the host. Mycobacteria, which are capable of forming biofilms on variety of surfaces, differ from most of other bacteria by unique composition of their cell wall. It provides them with high resistance against physical or chemical damage. This is one of the reasons for considering Mycobacterium tuberculosis as a highly potent pathogen. The studies of mycobacterial biofilms are motivated by effort to improve or find new therapeutic methods. This work is aimed at morphological and proteomic comparative analyses of biofilms obtained from Mycobacterium smegmatis grown on surface of glass and silica/zirconium beads, on liquid medium surface or grown submerged in shaken planktonic culture. We have developed technique for preparation of "floating" biofilm sample to be observed in SEM. We have shown that the growth of...

Streptomyces, filamentous Gram-positive bacteria are producers of more than 70% of antibiotics used in human therapy and agriculture. They are remarkable because of their complex life cycle (morphological differentiation) which leads to a formation of dormant spores able to survive unfavorable living conditions and allowing long-term survival of the organism. Soil represents their mostly natural living environment. In laboratory conditions they are cultivated in liquid media or on agar. We have developed in our laboratory two phase cultivation system which allows quantitative and reproducible preparation of samples for proteomic, transcriptomic and metabolomic analyses of Streptomycetes differentiation. The system is composed of inert micro- beads submerged in liquid medium. We used two types of micro-beads in our studies, glass and zirconia/silica beads. We followed the surface growth and differentiation of Streptomycetes on both types of beads using optical and electron microscopy (SEM) techniques. We observed major growth and higher antibiotic production on glass beads. Another difference we observed was in size and shape of colonies. In further research, using comparative proteomics, we attempted to identify proteins which might be responsible for recognition and adhesion of Streptomycetes to...

Microorganisms grow in planktonic form, but more often they adhere to a number of surfaces and create three-dimensional structures called biofilms. Floating biofilms, which are formed at the water-air interface, are one of the life strategies, which the bacteria can take. Non-pathogenic Mycobacterium smegmatis was used as a laboratory model for the study of this kind of biofilm. The understanding of mechanisms of their formation of this species may be applicable to the pathogenic species of the genus Mycobacterium, study of which in the laboratory brings a number of disadvantages. This diploma thesis focuses on the morphological and proteome analysis of the M. smegmatis floating biofilm. Using a stereo microscope and scanning electron microscopy was observed that bacteria clump and create the "nucleation centres" at the beginning of the biofilm development. This centers grow to the surroundings and connect afterwards. In the later stages of the development the centers fuse in compact layer, which then grows into the compact and multilayer biofilm. The key method in this study was two-dimensional electrophoresis of proteins. The proteome analysis of floating biofilm was performed with this method. The preparation of protein samples and the method for protein concentration measurement was optimized....

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / O presente trabalho teve por objetivo estudar as respostas fisiolÃgicas e bioquÃmicas de plÃntulas de cajueiro anÃo-precoce (Anacardium occidentale L.) à salinidade em duas condiÃÃes ambientais de cultivo. Para isso, as plÃntulas foram cultivadas em vasos de polietileno contendo somente soluÃÃo nutritiva (tratamento controle) ou soluÃÃo nutritiva com NaCl a 50, 100, 150 e 200 mM (tratamentos salinos), sendo mantidas em duas condiÃÃes ambientais: casa de vegetaÃÃo e sala de germinaÃÃo. Os efeitos do estresse salino foram avaliados atravÃs de medidas de crescimento, trocas gasosas, teores de clorofila, potencial osmÃtico foliar e teores de solutos orgÃnicos (prolina, N-aminossolÃveis e carboidratos solÃveis) e inorgÃnicos (Na+, Cl- e K+) nas folhas e raÃzes. TambÃm foram estudadas as alteraÃÃes na expressÃo gÃnica com a salinidade, o que foi feito atravÃs da comparaÃÃo dos padrÃes eletroforÃticos 2D das proteÃnas de folhas e raÃzes. A salinidade reduziu o crescimento das plÃntulas em ambas as condiÃÃes ambientais de cultivo, sendo que nas plÃntulas da casa de vegetaÃÃo, a inibiÃÃo do crescimento foi mais acentuada do que naquelas da sala de germinaÃÃo. Este fato correlacionou-se com as maiores reduÃÃes na fotossÃntese lÃquida, na transpiraÃÃo e na condutÃncia estomÃtica das plÃntulas da casa de vegetaÃÃo em relaÃÃo Ãs da sala de germinaÃÃo. Nas duas condiÃÃes de cultivo, os efeitos inibitÃrios do NaCl foram mais conspÃcuos nas raÃzes do que na parte aÃrea. A salinidade nÃo causou grandes mudanÃas nas concentraÃÃes internas de CO2 das plÃntulas de cajueiro, sugerindo a participaÃÃo de fatores nÃo-estomÃticos na inibiÃÃo das taxas fotossintÃticas. Os teores foliares de clorofila a, b e total foram influenciados pela salinidade e pelas condiÃÃes de cultivo das plÃntulas, sendo que as da sala de germinaÃÃo apresentaram os maiores conteÃdos e as menores reduÃÃes desses pigmentos devido à salinidade. As leituras feitas com o medidor portÃtil de clorofila, SPAD-502, correlacionaram-se positivamente com os teores foliares de clorofila, expressos em g.cm-2, tanto nas plÃntulas da casa de vegetaÃÃo quanto nas da sala de germinaÃÃo. As maiores reduÃÃes no potencial osmÃtico e os maiores acÃmulos de Na+ e Cl- nas folhas pela salinidade, em relaÃÃo ao controle, foram observados nas plÃntulas da casa de vegetaÃÃo. Por outro lado, os teores de K+ nesse ÃrgÃo nÃo diferiram muito entre as duas condiÃÃes de cultivo empregadas. As raÃzes acumularam grandes quantidades de Na+ e Cl- em seus tecidos, as quais foram acompanhadas de grandes decrÃscimos nos teores de K+, em ambas as condiÃÃes de cultivo. Com o aumento da salinidade, os teores de prolina foram aumentados, principalmente nas folhas, sendo os maiores incrementos observados nas plÃntulas da casa de vegetaÃÃo. Os teores de carboidratos solÃveis foram aumentados e reduzidos, devido à salinidade, somente nas folhas das plÃntulas da sala de germinaÃÃo e nas raÃzes das plÃntulas da casa de vegetaÃÃo, respectivamente. Nas duas condiÃÃes de cultivo, a salinidade aumentou os teores de N-aminossolÃveis nas folhas e nas raÃzes das plÃntulas de cajueiro. O padrÃo de expressÃo gÃnica das folhas e das raÃzes foi alterado pelo estresse salino em ambas as condiÃÃes ambientais. A salinidade causou aumentos e diminuiÃÃes nas taxas de expressÃo de vÃrias proteÃnas, sendo que algumas desapareceram completamente e outras foram aparentemente sintetizadas de novo nas plÃntulas estressadas. As proteÃnas diferencialmente reguladas pelo estresse salino foram bastante diferentes nas duas condiÃÃes ambientais empregadas. Faz-se necessÃrio o seqÃenciamento e a identificaÃÃo dessas proteÃnas para que se possa especular sobre seus possÃveis papÃis no processo de aclimataÃÃo das plÃntulas de cajueiro Ãs condiÃÃes de salinidade. / Early-dwarf cashew seedlings (Anacardium occidentale L.) were used in order to investigate the physiological and biochemical changes induced by salt stress in two environmental conditions. The seedlings were cultivated in plastics pots containing only nutrient solution (control treatment) or nutrient solution with NaCl at 50, 100, 150 and 200 mM (saline treatment). They were kept in two environmental conditions: greenhouse and growth room. The effects of salinity on the growth, gas exchange, chlorophyll content, osmotic potential and organic (proline, soluble amino-N, soluble carbohydrates) and inorganic (Na+, Cl-, K+) solute contents from both leaves and roots were studied. Salt stress induced changes in gene expression were studied both in leaves and roots comparing 2D electrophoretic pattern. Salinity inhibited the growth of seedlings in both environmental conditions, being the reduction in seedlings growth in the greenhouse more conspicuous than those cultivated in the growth room. This fact was correlated with highest reductions in net photosynthetic rate, in transpiration and stomatal conductance of seedlings grown in the greenhouse when compared with those of growth room. In both cultivation conditions, the root growth was affected by NaCl than shoot growth. The salinity stress not caused great changes in CO2 internal concentration, suggesting that the inhibition of photosynthesis also may be attributed to non-stomatal factors. Leaf chlorophyll a, b and total contents were influenced by salinity and environmental conditions, being observed the highest contents and the lowest reductions of these pigments due to salinity in seedlings under growth room conditions. The readings of portable chlorophyll meter, SPAD-502, were positively correlated to leaf chlorophyll contents, expressed in g.cm-2, both in greenhouse and growth room conditions. In the salt stress conditions, the higher reductions of osmotic potential and higher Na+ and Cl- accumulations in leaves were observed in seedlings grown in the greenhouse. On the other hand, leaves K+ contents did not differ much among the cultivation conditions used. The roots accumulated greater amounts of Na+ and Cl- in their tissues, which were accompanied of great decreases in the K+ contents in both cultivation conditions. Proline content increased with the increase in salt stress especially in leaves, being the greater increases observed in seedlings cultivated in the greenhouse. The soluble carbohydrates contents were increased and decreased, due to salinity, only in leaves of seedlings of growth room and roots of those grown in the greenhouse, respectively. In both cultivation conditions, salinity increased the leaf and root soluble amino-N contents of cashew seedlings. The gene expression patterns both leaves and roots were altered by salt stress, in both environmental conditions. Salinity induced increases and decreases in expression of various proteins, being that some proteins disappeared completely and other were apparently synthesized de novo in the seedlings stressed. The proteins differentially regulated by salt stress were enough different among the environmental conditions used. Future studies should be focused on sequencing and identification of proteins whose rate of synthesis varied as a result of salinity, in order to better characterize their possible roles in the process of acclimation of cashew seedlings to salinity conditions