Dissertations / Theses on the topic '2D-PAGE'

Orientador: Pedro de Magalhães Padilha
Resumo: Este trabalho apresenta os resultados de proteínas associadas ao mercúrio em amostras de tecido muscular e hepático de pirarucu (Arapaima gigas) e piranha preta (Serrasalmus rhombeus) oriundos do complexo hidrelétrico de Jirau, na Bacia do Rio Madeira, região amazônica do Brasil. O proteôma do músculo e fígado dessas espécies foi obtido por eletroforese bidimensional em gel de poliacrilamida (2D PAGE). O mercúrio presente nos spots proteicos foi determinado por espectrometria de absorção atômica em forno de grafite (GFAAS) após mineralização ácida assistida por banho de ultrassom. Os spots proteicos que apresentaram mercúrio foram caracterizados por espectrometria de massas por ionização com eletrospray em sequência (ESI- MS/MS) após digestão tríptica. As determinações GFAAS indicaram que a maior parte do mercúrio está ligada a fração proteica com massa molar (Mm) inferior a 90 kDa. As concentrações de mercúrio nos spots apresentaram-se na faixa de 4,07 – 164,63 µg g-1 no tecido muscular de pirarucu; 0,86 – 25,34 µg g-1 no tecido hepático de pirarucu; 7,67 – 156,18 µg g-1 no tecido muscular de piranha preta e 2,17 – 31,42 µg g-1 no tecido hepático de piranha preta. A análise por ESI-MS/MS permitiu caracterizar em dezenove spots proteicos as seguintes proteínas e/ou enzimas: Triosephosphate isomerase, Fructose-bisphosphate aldolase, Ckmb protein,, Cofilin 2 (Muscle), Actin_ alpha_ cardiac muscle 1a, Actin_ alpha 1_ skeletal muscle, Novel protein similar to zebrafish hemoglobin... (Resumo completo, clicar acesso eletrônico abaixo)
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Mercury is a potentially toxic element with a wide distribution on the Amazonian environment. This metal is dangerous and responsible for environmental contaminations and human intoxications as it is capable to biomagnifications and bioaccumulate throughout the food chains becoming the main way of exposing the riverine Amazonian communities to methylmercury to whom the main diet is fish. Therefore, studies related to mercury toxicity are of fundamental importance to health and life quality of the Amazonian communities. This study aimed to detect and evaluate possible proteic biomarkers of mercury toxicity in samples of human milk collected in riverine populations of Madeira and Negro rivers in the Brazilian Amazon. Initially total mercury was determined in the hair of breast feeding women to identify who were contaminated with mercury followed by the obtaining of the proteome of milk samples by two-dimensional electrophoresis (2D PAGE) after precipitation of proteins in half acetone. On the proteic spots obtained in the process of protein fractionation of milk samples, detection of mercury was carried out by atomic absorption spectrometry in graphite furnace(GFAAS), where the results showed that mercury was bonded in proteins of molecular weight around 14-26 kDa. The concentration determination of total mercury by GFAAS was also carried out with milk in natura, lyophilized milk and the proteic pellets aiming a mass balance of mercury related to the concentration of this element on milk and pellets. The measurements of mass balance permitted to observe that, in relation to the milk samples from Madeira River, about 85 to 95%of the mercury present in the lyophilized milk is on the proteic fraction. In relation to the breastfeeding women of the Negro River, about 50% of the total mercury is bound in the proteic fraction and the difference of 51% might be bound to the lipidic fraction. However, more studies in this line of research need to be pursued to achieve more robust conclusions.
O mercúrio é um elemento potencialmente tóxico com ampla distribuição no ambiente amazônico. Este metal é perigoso e responsável por contaminações ambientais e intoxicações humanas, já que é capaz de biomagnificar e bioacumular através das cadeias alimentares, tornando-se assim a principal via de exposição às comunidades amazônicas ribeirinhas do metilmercúrio, cuja dieta é baseada em peixes. Sendo assim estudos relacionados à toxicidade do mercúrio são de fundamental importância para a saúde e a qualidade de vida das comunidades amazônicas. Este estudo buscou detectar e avaliar possíveis biomarcadores proteicos da toxicidade do mercúrio em amostras de leite materno coletadas de populações ribeirinhas do rio Madeira e do rio Negro, na Amazônia brasileira. Inicialmente, determinou-se mercúrio total no cabelo das lactantes para identificar quais estavam contaminadas com mercúrio, em seguida obteve-se o proteoma das amostras de leite por eletroforese bidimensional (2D-PAGE) após precipitação das proteínas em meio acetônico. Nos spotsproteicos obtidos no processo de fracionamento das proteínas, nas amostras leite, foram feitas determinações de mercúrio por espectrometria de absorção atômica em forno de grafite (GFAAS), onde os resultados mostraram que o mercúrio se encontra ligado em proteínas de massa molecular na faixa de 14-26 kDa. A determinação da concentração de mercúrio total por GFAAS foi feita também no leite in natura, leite liofilizado e nos pelletsproteicos, com o objetivo de se fazer um balanço de massa de mercúrio em relação à concentração deste elemento no leite e pellets. As medidas de balanço de massa permitiram observar que, em relação às amostras de leite do rio Madeira, cerca de 85 a 95% do mercúrio presente no leite liofilizado encontrase na fração proteica. Em relação às lactantes do rio Negro, cerca de 50% do mercúrio total está ligado na fração proteica e a diferença de 51% pode estar ligado na fração lipídica. Contudo, mais estudos nesta linha de pesquisa devem ser desenvolvidos, para que se possam ter conclusões mais robustas.

La compréhension des mécanismes cellulaires et moléculaires qui sont mis en jeu au moment de la croissance et la maturation pré-ovulatoire induite par la LH, permettra de définir des marqueurs de qualité et de maturité du follicule destiné à ovuler, et ainsi de mieux anticiper le moment de l’ovulation. L’objectif majeur de cette thèse était d’identifier certain des facteurs régulateur impliqués dans la maturation folliculaire par deux approches globales d’analyse protéomique et transcriptomique. La première étude a permis d’établir pour la première fois les cartes protéiques du liquide folliculaire équin et canin. Les analyses comparatives des liquides folliculaires provenant de différents stades physiologiques n’ont montré, dans nos conditions expérimentales, que peu de différences. Nos résultats obtenus dans la deuxième étude indiquent que les différentes méthodes enrichissement du liquide folliculaire peuvent améliorer, pour certaines de manière conséquente, la résolution des gels 2D-PAGE. Notre étude transcriptomique globale a révélé un groupe de gènes différentiellement exprimés dans les cellules folliculaires aux différents stades étudiés. Ces gènes sont potentiellement impliqués pendant le développement folliculaire dans l’espèce équine. Les deux approches (protéomique et transcriptomique) que nous avons utilisées au cours de ce travail sont complémentaires car la connaissance des gènes exprimés par les cellules folliculaires peuvent permettre d’identifier certains gènes codant pour des protéines sécrétoires retrouvées dans le liquide folliculaire
An understanding of the cellular and molecular mechanisms involved in the growth and maturation of the preovulatory follicle induced by LH, will help us to understand and identify the markers of quality and maturity of the follicle destined to ovulate, and better anticipate the time of ovulation. The main objective of this thesis was to identify some regulatory factors involved in follicle maturation using two global approaches: proteomic and transcriptomic analysis. The first study established for the first time the protein map of equine and canine follicular fluids. The comparative analyses of follicular fluid from different physiological stages were shown little or no difference in our experimental conditions. Results obtained with the second study suggested that between depletion and enrichement methods, the enriched follicular fluid can improve for some consistent manner, the resolution of 2D-PAGE gels. Our global transcriptomic study revealed a group of genes differentially expressed in follicular cells at different physiological stages. These genes are potentially involved during follicle development in the equine species. The two approaches (proteomic and transcriptomic) that we used in this work are complementary, as the knowledge of genes expressed by follicle cells can help to identify some genes coding for secretory proteins secreted in follicular fluid

A mandioca (Manihot esculenta Crantz) é uma das principais culturas do mundo, havendo grande variabilidade genética. As variedades são classificadas com base na palatabilidade e toxicidade das raízes, em mansas ou doces e bravas ou amargas. Apesar da importância, o potencial da mandioca é pouco explorado, não sendo conhecidos, em nível molecular, os elementos determinantes para as suas características. Assim, pretendeu-se identificar, empregando a 2D-PAGE, proteínas que possam estar associadas com as diferenças físico-químicas das raízes tuberosas de variedades de mesa (IAC 576-70 e IAC 06-01), indústria (Cigana Preta, IAC 12 e IAC 90) e de uso misto (Vassourinha Paulista). Após extração de proteínas e separação por 2D-PAGE, as imagens dos géis foram analisadas no programa Delta2D (DECODON), sendo realizada análise estatística utilizando-se ANOVA (p<0,01), Heat Map e Análises de Componentes Principais (ACP) e de Agrupamentos. Os 146 spots de interesse foram removidos dos géis e suas proteínas digeridas e sequenciadas por espectrometria de massas. Algumas proteínas refletiram as características fenotípicas das variedades em estudo, especialmente entre as de mesa e indústria. Pela ACP, foram explicados 54,54% da variabilidade entre as amostras. A primeira componente separou as variedades exclusivamente de mesa de todas as demais, enquanto a segunda separou a IAC 90 de todas as outras, sendo esta caracterizada por um perfil proteico diferente das demais amostras de uso industrial. A IAC 576-70 e a IAC 12 apresentaram alta correlação positiva, assim como, a Vassourinha e a Cigana. A Análise de Agrupamentos corroborou as informações da ACP, revelando que o proteoma das raízes tuberosas refletiu diferenças fenotípicas entre as variedades.
Cassava (Manihot esculenta Crantz) is a main crop with large genetic variability. The varieties are classified according palatability and toxicity of the roots as sweet or bitter cassavas. Despite its importance, little is known about the molecular basis of phenotypic characteristics. Therefore, this study aimed to identify proteins associated to the differences between the sweet (\'IAC 576-70\' e \'IAC 06-01\'), bitter (\'Cigana Preta\', \'IAC 12\' e \'IAC 90\') and the mixed-use (\'Vassourinha Paulista\') varieties by 2D-PAGE. After the protein extraction and separation by 2D-PAGE, the gel images were analyzed through the software Delta 2D (DECODON), and the statistical analysis were performed with ANOVA (p<0,01), Heat Map, Principal Component Analysis (PCA) and Cluster Analysis. The 146 significant spots were removed from the gels, digested and sequenced by mass spectrometry. Some proteins were related to the physico-chemical characteristics of the varieties, especially between the sweet and the bitter. Variability of the samples was explained at the level of 54,54% by the PCA. The first component separated the sweet varieties from all others while the second one separated the \'IAC 90\' from all others. This variety was characterized by a different protein profile among the bitter cassavas. The \'IAC 576-70\' and the \'IAC 12\' were positively correlated, as well as, \'Vassourinha\' and the \'Cigana\'. Cluster Analysis agreed the PCA information, revealing that the proteomes of the tuberous roots reflected phenotypic differences among the varieties.

Il progetto di dottorato è stato condotto nel Laboratorio di Proteomica del Dipartimento di Biotecnologie dell’Università di Verona. Per la stesura di tale progetto sono state instaurate collaborazioni con alcuni laboratori, sia interni allo stesso Dipartimento sia appartenenti ad altre Università o Istituti di Ricerca. In particolare, per quanto riguarda lo studio su cellule di cancro colorettale trattate con un nuovo chemiofarmaco, il Laboratorio del Prof. Zunino (Fondazione IRCCS, Istituto Nazionale dei Tumori, Milano) ha fornito tutti i campioni biologici, mentre il gruppo del Prof. Marengo (Dipartimento di Scienze dell’Ambiente e della Vita, Università degli Studi del Piemonte Orientale, Alessandria) ha effettuato l’analisi statistica multivariata dei dati. Per quanto riguarda invece la caratterizzazione microbiologica, biochimica e proteomica del ceppo tannasi-positivo VP08 di Lactobacillus plantarum, il lavoro è stato condotto in collaborazione con il Dottor Zapparoli (Laboratorio di Microbiologia, Dipartimento di Biotecnologie, Università di Verona). L’indagine proteomica su campioni di foglie di Vitis vinifera suscettibile a Plasmopara viticola è stata realizzata in collaborazione col gruppo della Dottoressa Polverari (Laboratorio di Biotecnologie Fitopatologiche, Dipartimento di Biotecnologie, Università di Verona). Infine, il gruppo del Prof. Zolla (Laboratorio di Proteomica, Dipartimento di Scienze Ambientali, Università degli Studi della Tuscia, Viterbo) ha effettuato le analisi di spettrometria di massa per l’identificazione delle proteine di interesse individuate nei diversi studi. Ulteriori analisi di massa sono state svolte, in parte, presso il medesimo Laboratorio di Proteomica del Dipartimento di Biotecnologie dell’Università di Verona e, in parte, presso il Laboratorio di Proteomica del Dottor Vindigni (Centro Internazionale di Ingegneria Genetica e Biotecnologia “ICGEB”, Trieste). In questo progetto di dottorato sono stati applicati i metodi classici della proteomica comparativa basata sull’elettroforesi bidimensionale per l’analisi di campioni umani, microbici e vegetali legati a tre differenti problematiche, riguardanti rispettivamente: 1) la risposta di una linea cellulare di cancro al colon-retto ad un nuovo tipo di inibitore delle istone deacetilasi 2) l’effetto dell’acido tannico sul microrganismo del vino Lactobacillus plantarum in condizioni di carenza di nutrienti 3) i cambiamenti nel proteoma di foglia di Vitis vinifera cultivar Pinot Noir a diversi tempi dopo infezione con Plasmopara viticola
The proteome of a cell or an organelle provides information about the ensemble of proteins and protein isoforms expressed in that cell or organelle under specific physiological conditions and at a specific time. Proteomic approaches provide several novel possibilities to address biological questions. In fact, the large-scale screening approach of proteomics enables protein expression studies that are impossible to perform using classical molecular biology and biochemical techniques, in which the expression of only one or a few proteins is studied at a time. Instead, proteomic techniques allow for the analysis of up to thousands of proteins simultaneously, in any tissue or organelle, under any given physiological condition. Thus, proteomic applications are growing in many areas of research and proteomic approaches are nowadays widely exploited for cancer, microbial, and plant investigations. The present work was focused on three proteomics studies: 1) evaluation of the cell response to a novel histone deacetylase inhibitor in colon cancer cell 2) effect of tannic acid on lactobacillus plantarum wine strain during starvation 3) analysis of grapevine leaves after Plasmopara viticola infection The thesis work was conducted at the Proteomics Laboratory of the Biotechnology Department of the University of Verona, in collaboration with other laboratories: concerning the study on colorectal cancer cells, the Laboratory of Oncology of the IRCCS Foundation “Istituto Nazionale dei Tumori”, Milan, provided all the biological samples, whilst the multivariate analysis of protein profiles was possible thanks to the collaboration with the Department of Environmental and Life Sciences of the University of Eastern Piedmont, Alessandria. The biochemical and proteomic analysis of lactobacillus plantarum wine strain was the result of the collaboration with laboratory of Dr. Zapparoli (Department of Biotechnology of the University of Verona). Proteomic investigations on the Grapevine leaves infected by Plasmopara viticola were performed in collaboration with laboratory of Dr. Polverari (Department of Biotechnology of the University of Verona). Finally, the identification of proteins for all the proteomic analyses performed were possible thanks to the collaboration with the Proteomics laboratories of Department of Environmental Sciences, Tuscia University, Viterbo, and of International Centre for Genetic Engineering and Biotechnology, Trieste. The results thus obtained are here discussed and evaluated.

Orientador: Pedro de Magalhães Padilha
Resumo: Devido a sua grande concentração de nutrientes, tais como proteínas, vitaminas e minerais, o peixe é considerado um dos alimentos mais saudáveis que se pode encontrar na natureza. No entanto, a ingestão de peixes é considerada a forma predominante de via de exposição do ser humano ao mercúrio (Hg), principalmente para as populações que vivem às margens dos rios, onde o peixe constitui a principal fonte de proteína. Na tentativa de elucidar os mecanismos de toxicidade das espécies mercuriais, o teor desse metal tem sido estudado intensamente pela comunidade científica nas últimas décadas em amostras de solo, sedimentos, humanos e peixes na Amazônia brasileira. Sabe-se que as espécies mercuriais bioacumuladas nos tecidos dos seres vivos ligam-se a metaloproteínas, e quando há uma concentração alta de metal tóxico nos organismos, esses passam a expressar proteínas de defesa, denominadas metalotioneínas (MTs) responsáveis pelo transporte e eliminação de metais tóxicos. Apesar de estudos mostrarem o aumento das metalotioneínas em animais expostos a metais potencialmente tóxicos, essas proteínas não foram caracterizadas para confirmação de sua veridicidade, são analisadas por métodos indiretos, esse fato leva a necessidade de técnicas mais precisas na identificação de metalotioneínas. Levando em consideração o exposto esse estudo teve como objetivo otimizar métodos de quantificação de mercúrio e técnica de eletroforese para identificação de possíveis metalotioneínas biomarcadoras d... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Due to its high concentration of nutrients, such as proteins, vitamins and minerals, fish is considered one of the healthiest foods that one can find in nature. However, fish intake is considered to be the predominant human exposure pathway to mercury (Hg), especially for populations living along riverbanks where fish are the main source of protein. In the attempt to elucidate the toxicity mechanisms of mercurial species, the content of this metal has been intensively studied by the scientific community in recent decades in soil, sediment, human and fish samples in the Brazilian Amazon. It is known that mercurial species bioaccumulated in the tissues of living beings bind to metalloproteins, and when there is a high concentration of toxic metal in organisms, they begin to express defense proteins, called metallothioneins (MTs) responsible for the transport and elimination of Toxic metals. Although studies have shown the increase of metallothioneins in animals exposed to potentially toxic metals, these proteins have not been characterized to confirm their veridicity, are analyzed by indirect methods, this fact leads to the need for more precise techniques in the identification of metallothioneins. Taking into account the above, this study aimed to optimize mercury quantification methods and electrophoresis technique for identification of possible mercury biomarkers metallothionein in muscular and hepatic tissue of fish of economic interest, Tucunaré (Cichla spp.), Filhote (Bra... (Complete abstract click electronic access below)
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Medical diagnosis is the process of attempting to determine and/or identify a possible disease or disorder. This process is revealed by biomarkers, defined by The Food and Drug Administration (FDA) as “characteristics that are objectively measured and evaluated as indicators of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention”. The process of biomarker discovery has been boosted in the last years by proteomics, a research discipline that takes a snapshot of the entire wealth of proteins in an organism/ tissue/ cell/ body fluid. An implementation of the analysis methods can help in isolate proteins present in the low range of concentrations, such as biomarkers very often are. An established biomarker can further be measured with the help of biosensors, devices that can be employed in the point-of care diagnostics. This PhD thesis shows and discusses the results of three projects in the field of protein biomarkers discovery and quantification. The first project exploited proteomics techniques to find relevant protein markers for Intrauterine Growth Restriction (IUGR) in cordonal blood serum (UCS) and amniotic fluid (AF). A 14 proteins in UCS and 11 in AF were successfully identified and found to be differentially expressed. Molecularly Imprinted Polymers (MIPs) directed towards proteins and peptides containing phosphotyrosine were then produced, with the final goal of selectively extracting phosphopeptides from a peptide mixture. An alteration of the phosphorylation pattern is in fact often associated to important diseases such as cancer. The polymers were produced as nanoparticles, that were characterised with Dynamic Light Scattering (DLS) and Atomic Force Microscopy (AFM). A recipe was also tested for binding capacity towards phosphotyrosine. A Surface Plasmon Resonance (SPR) biosensor to quantify hepcidin hormone was finally produced. This is the major subject in iron homeostasis in vertebrates and marker of iron unbalance diseases. A calibration curve was made and affinity/kinetic parameters for the ligand employed were measured.

>Magister Scientiae - MSc
Chromosome doubling has become an important tool in breeding programmes as it offers the ability of introducing novel traits into existing plants. Doubled haploid plants are highly valued by both consumers and breeders as these plants usually show larger flower, leaves and fruit, thus making them more marketable. Marianna open pollinated plum rootstocks’ adaptability to different soil types and moisture conditions has been favoured in polyploidy studies as parental material in breeding programmes. The potential of the microtubule depolymerizing herbicide (oryzalin) for in vitro chromosome doubling were investigated by optimizing the concentration and incubation time of plant shoots to the antimitotic agent. Meristem tissues were treated for two time intervals (24 and 48 h) with five different concentrations of oryzalin (50, 75, 100, 150 or 200 μM) in liquid Murashige and Skoog (MS) medium. After treatment, plants were allowed to grow under a 16/8 h light/dark photoperiod at 24±2˚C for 4 weeks. One and two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry (MS) was used to separate, visualise and identify differently expressed proteins. Furthermore, changes in ROS accumulation, photosynthetic pigmentation, lipid peroxidation and antioxidant enzyme activity (SOD, APX and GR) were investigated. Flow cytometry results revealed that treatment of plants with oryzalin concentrations ranging from 75 to 150 μM induced ploidy after 24 h exposure whereas, 200 μM produced mixoploids containing both tetraploid and octoploids plants after 24 h exposure. Longer incubations of 48 h were detrimental to plant tissues as complete mortality was observed in the higher concentration (100 to 200 μM) treatments. Mass spectrometry analysis identified 14 differentially expressed protein spots that were characterized into different functional categories. ROS accumulation, the extent of lipid peroxidation and antioxidant capacity were differentially regulated in response to oryzalin treatment whereas photosynthetic pigments were significantly enhanced. The results suggests that oryzalin-induced proteins may act as potential biomarkers to improve fruit characteristics in future breeding programs whereas antioxidant enzymes play an important role in scavenging ROS in plants to enhance their adaptability to different environmental conditions.

The development of metal-tolerance and antibiotic resistance in bacteria may be caused by metals polluting a particular environment. During mining and mineral processing activities, large quantities of metals are deposited into the soil. These high concentrations of metals are evolutionary pressures selecting for microorganisms tolerant to these metals. Metaltolerance maybe conferred to these organisms by mobile genetic elements such as plasmids. This study describes the characteristics of plasmids isolated from various bacteria that displayed an ability to withstand high metal concentrations. The isolated plasmids were individually transformed into Escherichia coli JM109. Transformants were then evaluated for metal-tolerant capabilities using a microdilution approach. Plasmids were then isolated from the transformants and the concentration of the plasmid DNA ranged between 11.75 – 118.06 ng/μl. These plasmids were of the same size as the original ones. This demonstrated that successful transformations with plasmid DNA were conducted. In order to determine the compatibility group, plasmids were subjected to PCR amplification using IncQ, IncP-9 and IncW specific primers. Only the IncW provided positive results. To demonstrate that the plasmids were free of genomic DNA, a 16S rDNA PCR test was included. The plasmids that were positive for IncW PCRs were all negative for the rDNA PCRs. Plasmids were stably inherited and at least three, isolated from three different Gram positive species, belonged to the Inc W group of plasmids. These were originally isolated from Paenibacillus ginsingari, Paenibacillus lautus and Bacillus cereus. Minimum inhibition concentrations (MICs) were carried out to determine the ability of transformed E. coli JM109 to tolerate metals at varying concentrations. Results indicated that transformed E. coli JM109 developed ability to grow in the presence of several heavy metals. Some strains were resistant to high concentrations (+10 mM) of Ni2+/Al3+, Pb2+ and Ba2+. The order of metal resistance was Ni/Al=Pb>Ba>Mn>Cr>Cu>Co=Hg. All the x transformants were sensitive to 1 mM of Co2+ and Hg2+. Moreover, protein profiling was used to determine the impact of plasmids on E. coli JM109. Proteins were extracted from both transformed and un-transformed E. coli JM109 using acetone-SDS protocol and subjected to one-dimensional (1D) and two-dimensional (2D) Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS- PAGE). Transformed E. coli JM109 were grown under the metal stress. One dimension SDS-PAGE illustrated general similarity of the profiles except for two banding positions in the 30 to 35 kDa region where bands were present in the transformants that were grown in the Ni/Al alloy containing media. Twodimensional electrophoresis PAGE analysis showed that some of the proteins were upregulated while others were down-regulated. The largest numbers of proteins were from 15 – 75 kDa. The majority of these proteins had isoelectric points (pI) between 5 and 6. It was concluded that plasmids isolated from various heavy metal-tolerant bacterial species were successfully transformed into E. coli JM109 rendering various new metal-tolerant E. coli JM109 strains. Furthermore, the study showed that metal resistance was due to the presence of the plasmids. Two-dimensional SDS-PAGE resolved more differences in the protein expression profiles. Since the plasmids rendered the E. coli JM109 tolerant to metals tested, it also can be concluded that the change in the protein profiles was due to the effects of the plasmids. Furthermore, plasmids were also re-isolated from the transformants and these plasmids were of the same size as the original ones.. All the plasmids in this study were also stably inherited, a feature associated with IncW plasmids. More detailed genetic characterization of these plasmids is required. Plasmids isolated and characterized in this study may hold biotechnology potential. Such features should be exploited in follow-up experiments.
Thesis (Master of Environmental Sciences)--North-West University, Potchefstroom Campus, 2013.

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Conselho Nacional de Desenvolvimento Científico e Tecnológico
O aumento da produção de cana-de-açúcar, decorrente da crescente demanda por etanol e biocombustíveis, implica na necessidade de obter variedades mais produtivas e adaptadas a fatores ambientais limitantes, como a seca. Assim, torna-se necessário o conhecimento dos genes e proteínas cuja expressão pode ser útil ao melhoramento genético. Este trabalho objetivou a identificação de peptídeos produzidos diferencialmente em resposta potencial ao estresse hídrico nos híbridos comerciais: RB867515 (tolerante à seca) e RB72454 (sensível à seca), através de análise proteômica. Proteínas totais de folha e entrenó imaturo das variedades sob irrigação ou seca foram separadas por 2D-PAGE e analisadas via espectrometria de massas. Foram identificadas várias proteínas associadas a mecanismos de resposta ao estresse hídrico, entre elas: proteínas associadas à regulação da transcrição (proteína de ligação ao RNA rica em glicina) e à tradução (fator de enlongação 1 alfa e proteína ribossômica 60S L30); componentes estruturais (histona H2A.2.2); proteínas relacionadas aos componentes do citoesqueleto (fator de despolimerização da actina 3); quinases (nucleosídeo-difosfato quinase I); enzimas envolvidas no metabolismo da glicose (gliceraldeído-3-fosfato desidrogenase, citossólica); várias proteínas do choque térmico (HSP70kDa, 17.5kDa classe II, 81-1 e 18.0kDa); e proteínas associadas à fotossíntese (complexo de evolução do oxigênio 1-1; subunidade N do centro de reação do FSI). Estas proteínas podem ser úteis como alvos potenciais para o melhoramento genético e desenvolvimento de marcadores funcionais para seleção de genótipos de cana-de-açúcar tolerantes à seca

Enterohepatic Helicobacter species (EHS) are emerging infectious disease agents. Infection of the enterohepatobiliary tract of several mammals by this group of bacteria results in various pathological disorders. The availability of the Helicobacter hepaticus sequenced and annotated genome, allowed molecular characterisation of the responses of H. hepaticus to host factors such as bile. The adaptation/responses of the bacterium to bovine, porcine and human bile were investigated using proteomics and transcriptomics. Ninety-one different proteins were identified in the responses of H. hepaticus response to the three types of bile. These proteins participate in several key cellular processes including DNA replication; protein transcription, translation and folding; oxidative stress response; motility; virulence; and metabolism. In particular, the bacteria deployed several strategies such as inhibition of the TCA cycle and the electron transport chain as well as iron sequestration to ensure control of the levels of hydroxyl radicals. The results of this study revealed also the modulation by bile of the expression of H. hepaticus genes involved in response to oxidative stress and virulence. The responses of human HEp-2 and Huh7-derived cell-lines to H. hepaticus and Helicobacter bilis, respectively, were investigated employing proteomics and transcriptomics. One-hundred and twenty different proteins were differentially expressed in the responses of the human cells to the presence of Helicobacter spp. in the cell cultures. These proteins are involved in regulation of cell proliferation and structure; metabolism; protein transcription, translation and modification; stress response; and tumour induction. For example, in co-cultures of Huh7-derived cells and H. bilis, the activation of several mitochondrial and endoplasmic reticulum stress-related proteins and the dysregulation of several apoptosis effectors were suggested as mechanisms that could result in the death of the liver cells. Importantly, the differential expression of several tumour-related proteins by the Huh7 cells supported a possible role for Helicobacter spp. in liver cancer.

Obesity is a globally increasing disease particularly in developing countries and among children. It is mainly caused by intake of diets high in fat and the lack of physical activity. Obesity is a risk factor for diseases such as type II diabetes, high blood pressure, high cholesterol and certain cancers. Prediabetes is a condition where blood glucose levels are above normal but have not 
reached those of diabetes. It is difficult to diagnose, as there are no signs or symptoms. Some type II diabetes patients bear no symptoms at all and the disease is discovered late. Proteomics is a field that can provide opportunities for early diagnosis of diseases through biomarker discovery. The early diagnosis of diabetes can assist in the prevention and treatment of diabetes. Therefore there is a need for the early diagnosis of diabetes. Twenty Wistar rats were used. The rats were initially fed a CHOW diet, which is the standard balanced diet for rats, for 4 weeks. The rats were then divided into 2 groups of 10 where 1 group was fed CHOW and another was fed a high fat (HF) diet in order to induce obesity. The two groups were fed their respective diets for 18 weeks. Rats were weighed. Rats were placed in metabolic chambers and 24 hour urine samples were collected. Ketone levels were measured by Ketostix. Urine proteins were precipitated by acetone, quantified and separated on both the 1D SDS-PAGE and the 2D SDS-PAGE. Protein expression changes between CHOW and HF fed rats were determined and identified using MALDI-TOF mass spectrometry. Protein spots intensities increased and decreased between the CHOW and HF fed rats. Transducin (beta)-like 3 was identified as the only differentially expressed protein, which might serve as a potential biomarker for prediabetes.

In this study, a halophilic archaea Halobacterium salinarium TG13 which is isolated from Tuz Lake in Central Anatolia was characterized biochemically and genetically. Halobacterium salinarium DSM3754 and Halobacterium salinarium S9 strains were used as a reference strain through the experiments. In biochemical characterization
total protein profiles of strains was compared by using 1D SDS PAGE. Total protein profile of the isolated strain has shown differences. The SDS-PAGE profile of the purified purple membrane showed only single band by coomassie staining. Molecular weight and pI values of the protein isolated from Halobacterium salinarium TG13 and Halobacterium salinarium S9 were estimated by 2D SDS-PAGE as 22 kD and 5.4, respectively. Photoactivity of purple membrane of the strains was investigated. pH change of the purple membranes were observed upon illumination. This protein might be corresponded to bacteriorhodopsin. In genetical characterization
polymorphism of genomic DNA of strains was scanned with RAPD-PCR. Plasmid DNA profiles of strains was determined to make use of RFLP technique. RAPD-PCR and RFLP analyses have shown that Halobacterium salinarium TG13 is different strain from reference Halobacterium salinarium strains (H.s. S9 and H.s. DSM3754).

O líquido folicular é o microambiente do oócito durante sua maturação in vivo que é, em parte constituído por exsudato do soro sanguíneo e por substâncias produzidas localmente, que estão relacionados com a atividade metabólica das células ovarianas. Tais substâncias podem ser essenciais para a proliferação e diferenciação das células somáticas bem como na maturação e posterior fertilização de um oócito competente. A busca por biomarcadores capazes de predizer a saúde de um folículo ou a capacidade do oócito em se tornar um embrião saudável é objeto de estudo na medicina reprodutiva humana e veterinária. Para tanto é essencial o conhecimento a nível molecular dos constituintes do liquido folicular e suas funções. O objetivo do presente estudo foi avaliar o perfil proteico do líquido folicular de éguas submetidas à indução de ovulação, com dois diferentes protocolos usuais na prática clínica, utilizando eletroforese bidimensional em gel de poliacrilamida. Para tanto 19 amostras de liquido folicular de éguas que tiveram sua ovulação induzida por dois diferentes protocolos (1000UI hCG IV, Grupo H, ou 1000UI hCG IV + 1,5mg de acetato de deslorelina IM, Grupo H + G) foram submetidos a eletroforese 2D e posterior análise dos géis no PDQuest. Os valores de proteína total foram significativamente diferente nos Grupo H e Grupo H+G, 63,97 ± 6,97 e 73,07 ± 6,42, respectivamente. O número máximo de spots em um mesmo gel foi de 157 e o mínimo de 34, com média de 90 spots para o Grupo H e 83 spots para o Grupo H+G. Os 19 géis foram avaliados e a porcentagem máxima de spots relacionados foi de 52% e a mínima de 0%. Com média de 37,8% de similaridade entre spots para o Grupo H e 22% para o Grupo H+G. Estes resultados são de grande importância devido à escassez de trabalhos com proteômica de liquido folicular de éguas induzidas a ovulação e demonstram que a associação entre hCG e acetato de deslorelina aumenta a concentração de proteínas no líquido folicular em folículos pré-ovulatórios (>35 mm).
Follicular fluid is the oocyte microenvironment during its in vivo maturation. It is partly composed by blood serum exudate, and also by locally produced substances, related to ovarian cells metabolic activity. These substances may be essential for somatic cells proliferation and differentiation, as well as on the oocyte maturation and fertilization. The search for biomarkers able to predict oocyte ability to grow into a healthy embryo are targets on human and veterinary reproductive medicine. It is essential to know the components of follicular fluid and their functions. The aim of the present study was to evaluate protein profile of the follicular fluid in mares with inducted ovulation, in two different protocols, using 2D electrophoresis in polyacrylamide gel. 19 follicular fluid samples from mares in which ovulation induction was performed with two different protocols (1000UI hCG IV or 1000UI hCG IV + 1,5mg deslorelin acetate IM), submitted to 2D electrophoresis, and gel analysis on PDQuest. Total protein values were significantly different in Group H and Group H+G, 63,97 ± 6,97 and 73,07 ± 6,42, respectively. The highest number of spots on a same gel was 157 and the minimum was 34, with a mean of 90 spots to Group H and 83 spots for Group H+G. All of the19 gels were evaluated according to MasterGel and the highest percent of related spots was 52% and the lowest, 0%, with mean similarity between spots 37,8% to Group H and 22% to Group H+G. These results are of great importance, due to lack of works on follicular fluid proteomics using fluid from mares with induced ovulation, and demonstrate that the association hCG + deslorelin acetate increase proteins concentration on pre-ovulatory follicles fluid.

Chlamydia trachomatis is an obligate intracellular bacterium and the most prevalent cause of bacterial sexually transmitted disease. Genital chlamydial infections, marked by chronic, intense inflammation, can lead to genital tissue scarring and infertility and is a contributing factor to development of pelvic inflammatory disease and ectopic pregnancy. Iron is required as a cofactor for numerous highly conserved pathways, and nearly all studied organisms rely on iron for growth. In response to iron restriction, the chlamydial developmental cycle arrests at the intracellular reticulate body stage, resulting in a phenomenon termed persistence. Persistence likely plays a role in chlamydial pathogenesis through the expression of virulence factors and antigens in addition to sustaining chronic infection; however, little is known concerning how chlamydiae respond to iron limitation at the molecular level, and no systems for iron acquisition have been identified in Chlamydia. This dissertation presents an investigation into the chlamydial response to iron restriction. Chlamydial heat shock protein 60 (cHsp60) has been implicated in development of the more severe disease sequelae and has been found to increase in expression following iron restriction; however, three cHsp60 homologues were identified following the sequencing of the chlamydial genome. Here, iron restriction is shown to increase expression of cHsp60-2 but not the two other homologs, cHsp60-1 or -3. Next, in order to investigate an alternate model for restricting iron availability to chlamydiae, a cell line with inducible expression of recombinant ferroportin, a eukaryotic iron efflux protein, was examined. Lastly, 10 chlamydial proteins differentially expressed during growth in iron-restricted host cells were identified by proteomic analysis of radiolabeled proteins followed by mass spectrometry analysis; transcripts encoding 5 iron responsive proteins were examined across a timecourse of infection and revealed increased transcript levels at 18 and/or 24 hours post infection. Together, these studies have examined the molecular response of chlamydiae to reduced iron availability and have underlined the importance for pathways involved in protection against oxidative damage and adaptation to stress.

Orientador: Pedro de Magalhães Padilha
Resumo: O mercúrio é um metal tóxico, de distribuição ubíqua, com capacidade para bioacumulação e biomagnificação, que provoca alterações em biomoléculas importantes no metabolismo e contribui para o estabelecimento do estresse oxidativo em organismos aquáticos. Logo, o presente estudo teve por objetivo identificar e avaliar possíveis biomarcadores proteicos e/ou enzimáticos da toxicidade do mercúrio em peixes da região amazônica, por meio do estudo metaloproteômico e avaliação do estresse oxidativo. Foram utilizadas metodologias de fracionamento e identificação de proteínas por eletroforese bidimensional (2D PAGE) associada à espectrometria de massas (MS), mapeamento do mercúrio, em spots proteicos, por espectrometria de absorção atômica em forno de grafite (GFAAS) e avaliação de marcadores de estresse oxidativo. As espécies utilizadas foram o Plagioscion squamosissimus (corvina) e Colossoma macropomum (tambaqui), coletados na área da Usina Hidrelétrica de Jirau (rio Madeira-RO), que foram selecionadas em função da abundância populacional, interesse para a pesca e posição diferente na cadeia trófica (carnívoro e onívoro, respectivamente). Os tecidos amostrados foram o hepático, renal e muscular. Os resultados obtidos demonstraram maior concentração de mercúrio total no P. squamosissimus, espécie carnívora, e padrão de distribuição deste elemento igual para ambas as espécies (fígado>rim>músculo). Há tendência para maior atividade enzimática nos tecidos hepático e renal da espécie com... (Resumo completo, clicar acesso eletrônico abaixo)
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Na cana-de-açúcar, a conversão de meristema vegetativo para reprodutivo é uma etapa importante para o melhoramento genético, no entanto, é indesejável na produção comercial por consumir sacarose para o desenvolvimento da inflorescência. Este estudo teve como objetivo identificar proteínas diferencialmente acumuladas no meristema apical da variedade de cana RB867515 sob aplicação foliar de sulfato de cálcio, através de eletroforese 2D e espectrometria de massas. Foi testado o efeito do cálcio em parâmetros morfofisiológicos, concentração de macronutrientes e anatomia do ápice meristemático por microscopia ótica. Adicionalmente, foi avaliado o efeito do cálcio no proteoma do ápice meristemático durante as fases de pré e pós-indução do florescimento. A aplicação foliar de cálcio aumentou a altura de colmos, e a abertura estomática também foi alterada. A composição dos macronutrientes mostrou maiores níveis de cálcio e menores teores de potássio e magnésio. A aplicação foliar de cálcio reduziu em cerca de 20% o florescimento da cana planta e 35% na cana soca. Na análise proteômica, um total de 60 DEPs foram identificados por PMF a partir de perfis 2D de meristemas em pré ou pós indução floral, dos quais 14 foram observadas exclusivamente em meristemas tratados com cálcio, 11 foram identificadas exclusivamente após a indução floral, e 29 foram comuns em ambas as situações, porém mais abundantes em meristemas sem aplicação de cálcio antes da indução floral. A aplicação foliar de cálcio alterou significativamente o proteoma meristemático da cana-de-açúcar. O cálcio parece também melhorar a sinalização celular e a atividade antioxidante em meristemas. O efeito da aplicação foliar de cálcio no proteoma parece ser atenuado pelo tempo. As proteínas identificadas são fortes candidatas a estudos futuros envolvendo o controle do florescimento da cana, visando sua aplicação como marcador molecular funcional associadas ao cálcio, podendo auxiliar os programas de melhoramento genético desta cultura.
In sugarcane (Saccharum spp.), the conversion of apical meristem to breeding is an important step for the genetic improvement, however, it is undesirable in commercial production due to consuming sucrose to develop inflorescence. This study aimed to identify differentially accumulated proteins in the apical meristem of the RB867515 variety under foliar application of calcium sulfate through 2D electrophoresis and mass spectrometry. It has been tested the calcium effect on morphophysiological parameters, macronutrient contents and anatomy of the shoot apical meristem by optical microscopy. Additionally, we evaluated the effect of calcium on the proteome of sugarcane apical meristem during the phases of pre and post-induction of flowering. Foliar applications of calcium increased the stalk height, and stomatal opening was also measured. The macronutrient composition showed higher calcium levels and lower levels of potassium and magnesium. Leaf applications of calcium reduces flowering by 20% in sugarcane plant, and 35% reduction in the ratoon cane. In the proteome analysis, a total of 60 DEPs have been identified by PMF from 2D meristems profiles in pre and post-floral induction, of which 14 were found exclusively in meristems treated with calcium, 11 were identified only after floral induction, and 29 were common in both cases, but more abundant in meristems without application of calcium before floral induction. Foliar applications of calcium significantly altered the meristematic proteome of sugarcane. Calcium also appears to enhance cell signaling and antioxidant activity in meristems. It was observed that the effect of foliar calcium application in proteome appears to be attenuated by time. The proteins identified are strong candidates for future studies aiming its use as a functional molecular marker involving control of flowering of sugarcane associated with calcium, can help breeding programs of this culture.

Many of the cellular mechanisms underlying host responses to pathogens have been well conserved during evolution. Homeostatic interactions between insects and commensal microbes are widespread in nature. Commensal microbes have many roles in the biology and lifecycle of most insect species, affecting different aspects of their life. The recently recognized acetic acid bacteria (AAB) are the most abundant microorganisms in the insects and also metabolically linked to one another. These symbionts establish close interactions with their animal host, including insects and mosquitoes. In particular, gut microbiota establishes strict interactions with its host and, indeed, close interactions are established among the different microbial partners. Fine regulation of the immunity in the host gut is required for homeostasis of gut microbiota. A recent study highlighted the role of bacterium-secreted uracil as a signal molecule controlling immunity in the gut of flies. Moreover, diversity of microbial symbionts suggests that synergistic activity in their role is favored. This gut bacterial symbionts have a role in the fitness of insects that are vectors of the most severe diseases in plant, animal and human affecting the agricultural production and the environment. The mosquitoes Anopheles are vectors of Plasmodium parasites, the causative agents of malaria. Asaia is a useful model for the study of promising tools in the control of disease-transmitting mosquitoes like Anopheles and AAB symbionts. The aim of my Ph.D. research work was to explore the molecular factors of acetic acid bacteria (AAB) involved in the interaction with the host in order to unveil targets addressable for novel strategies of pathogen biocontrol. The purpose was challenged by a comparative proteomic approach considering Asaia SF2.1 as a model AAB-symbiont. Since this approach is based on the analysis of two-dimensional electrophoresis (2D-PAGE) protein profiles, it requires a consistent amount of bacterial-specific protein that cannot be achieved in the gut environment. To this end, hypoxia was applied as a model condition eliciting or suppressing pathways possibly involved in the symbiotic interaction. Hypoxia condition was applied to mimic the insect gut condition, where the oxygen concentration is usually low from 2% to 8%. Microbial population of model host insects (Drosophyla suzukii and Anopheles stephensi) was characterized by using 16S rRNA and ITS-PCR. The analysis showed that Asaia SF2.1 is among the most abundant acetic acid bacteria (AAB) species. Most abundant AAB species were selected from each genus (i.e. Gluconobacter, Acetobacter, Komagataeibacter and Asaia) and the biomass production was defined. Furthermore, for all strains, conditions for protein extraction methods were optimized followed by protein quality assessment and quantification. Asaia SF2.1 was selected for comparative proteomic approach based on the availability of genome sequence, protein quality and quantity. Different sample preparation methods were used for the setup of protein extraction. The best method for protein extraction was selected and was followed by optimization of the 2D-PAGE run conditions. Using bioreactor, hypoxia (6%) and normal (20%) condition (control) of Asaia SF2.1 were setup and growth data evaluated. 2D-PAGE protein profiles of more than 220 spots were obtained by applying the optimized protocol. Total 12 protein spots with altered expression differences were observed and the corresponding proteins were identified by mass spectrometry technique. MS analysis identified different proteins from the selected spots and functional categorization of proteins was carried out according to the annotated information. Protein spots were mostly identified as being involved in transcription, cellular respiratory function, cell wall biogenesis, protein biosynthesis, pentose-phosphate shunt. Gene ontology analysis suggested interesting putative candidate pathways involved in endosymbiosis, thus paving targets for biocontrol strategies.

Aeromonas hydrophila is an opportunistic pathogen that causes a wide range of symptoms and diseases in fish. Development of a commercial vaccine has been problematic due to the heterogenicity between isolates of A. hydrophila. A new approach using immunoproteomics was used in this study to try to develop a vaccine that would protect against a wide range of A. hydrophila strains. The virulence of 14 isolates of A. hydrophila from different geographical regions was determined in common carp (Cyprinus carpio) indicating that 6 isolates were virulent, while 8 isolates were avirulent. Expression of cellular and extracellular products (ECP) of six of these isolates (4 virulent and 2 avirulent isolates) were examined following culture of the bacterium in vitro, in tryptic soy broth, and in vivo, in dialysis tubing placed within the peritoneal cavity of carp. Two types of molecular weight cut off tubes (25 and 100 kDa) were used for the implants. Whole cell (WC), outer membrane protein (OMP) and ECPs of the bacteria grown in vitro and in vivo were analysed by 1 dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (1D SDS-PAGE). Additionally, 2D SDS-PAGE was used to analyse WC preparations of A. hydrophila grown in vitro and in vivo. The production of unique proteins and up and down-regulation of protein expression were observed in all the preparations of bacteria grown in vitro and in vivo. Unique bands were seen in the 1D SDS-PAGE at 58 and 55 kDa for WC and OMP preparations, respectively, for all the isolates cultured in vivo. Bands of increased intensity were observed at 70, 55, 50 and 25 kDa with WC preparations for the virulent isolates cultured in vivo. Analysis of WC preparations by 2D SDS-PAGE indicated differences in the expression of spots between bacteria cultured in vitro and in vivo. A number of unique spots, mostly between 30 and 80 kDa with pI values ranging from 5.0-6.0 were observed in the bacteria grown in vivo. The protein profiles of different preparations (WC, OMP, ECP) of bacteria cultured in vitro and in vivo were screened by 1D Western blot using antibodies from carp artificially infected with different isolates of A. hydrophila to identify potential vaccine candidates. The WC preparations of A. hydrophila (T4 isolate) grown in vitro were also analysed by 2D Western blot. A 50 kDa protein of A. hydrophila was found to be the most immunogenic molecule in both WC and OMP of bacteria grown both in vitro and in vivo. The protection efficacy of this protein was determined in goldfish by vaccinating fish with electro-eluted 50 kDa protein then challenging the fish with A. hydrophila. Fish were also passively immunised with fish sera raised to the 50 kDa protein and then challenged. The relative percentage survival (RPS) was 67 % in the vaccination trial, while the results were inconclusive for the passive immunisation trial. The 50 kDa protein was confirmed to be the S-layer protein of A. hydrophila following identification using matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS). Recombinant S-layer protein was then produced and the cross-protection efficacy of this protein against six virulent isolates of A. hydrophila was confirmed in a large scale vaccination trial using carp. The RPS value for the 6 isolates of A. hydrophila ranged from between 56 and 87 %. The results of this project suggest that the immunogenic S-layer protein of A. hydrophila could be used as a common antigen to protect fish against infection by different isolates of this pathogenic bacterium.

Proteomic techniques were used to evaluate the protein profile of the earthworm, (Lumbricus terrestris), following a bacterial challenge. One control group received no injection; a second control group received injections of phosphate buffer solution (PBS). The experimental group received injections of PBS containing (Aeromonas hydrophila). After incubation for 12 hours at 20°C, coelomic fluid was collected from each group for analysis by 2-D electrophoresis. There were significant differences in spot appearance and density between control and experimental groups. Sixteen spots showed a two-fold increase in density and 63 showed at least a two-fold decrease in density between samples from control and bacteria-challenged earthworms, respectively, suggesting up- and down-modulation of proteins potentially involved in the earthworm's response to bacterial challenge.

This study reports on a proteomic analysis of sorghum proteomes in response to salt and hyperosmotic stresses. Two-dimensional gel electrophoresis (2DE) in combination with mass spectrometry (MS) was used to separate, visualise and identify sorghum proteins using both sorghum cell suspension cultures and whole plants. The sorghum cell suspension culture system was used as a source of culture filtrate (CF) proteins. Of the 25 visualised CBB stained CF spots, 15 abundant and well-resolved spots were selected for identification using a combination of MALDI-TOF and MALDI-TOFTOF MS, and database searching. Of these spots, 14 were positively identified as peroxidases, germin proteins, oxalate oxidases and alpha-galactosidases with known functions in signalling processes, defense mechanisms and cell wall metabolism.

Mestrado em Bioquímica
Staphylococcus pseudintermedius is an opportunistic pathogenic bacterium responsible for most skin and post-surgical infections in dogs. The number of bacterial strains resistant to β-lactam antibiotics is increasing and are the major challenges now faced by veterinary medicine. Bacteria that produce biofilm are more resistant to treatment and thus, the production of this structure is already considered a virulence factor. In a biofilm, bacteria are embedded in a matrix of extracellular polymeric substances (EPS) some of which are proteins. With the objective to know more of this array element, the characterization of the biofilm matrix proteome (BMP) from a highly virulent S. pseudintermedius strain isolated from a dog with severe pyoderma was performed. Biofilm was developed by culturing the S. pseudintermedius strain 5819/10 in specific media. The biofilm matrix was then be separated from bacterial cells and evaluated for their protein content and complexity. Finally, the proteome was separated by 1D electrophoresis and characterized by nanoLC-ESI-Q-TOF and analysed using bioinformatics tools. The BMP of strain S. pseudintermedius 5819/10 consisted in a diverse group of proteins, where 63% of the proteins could be related to either the extracellular region or the plasma membrane, as protein complexes, and most of them had functions essential to cell survival. However, it was not possible to establish a clear relation between them and biofilm formation. Proteins known to be involved in biofilm formation consisted mostly of regulator factors of biofilm formation as well as virulence factors of-mainly-bacterial cell adhesion and host colonization. The prevalence of adhesins and the almost total absence of proteins involved in EPS synthesis pointed to a biofilm matrix where cells are directly or indirectly closely glued together to each other.
Staphylococcus pseudintermedius (S.pseudintermedius) é uma bactéria patogénica oportunista, responsável pela maioria das infeções cutâneas e pós-cirúrgicas em cães. O número de estirpes resistentes a antibióticos β-lactâmicos está a aumentar constituindo actualmente um dos grandes desafios enfrentados pela medicina veterinária. As bactérias mais resistentes ao tratamento são aquelas que produzem biofilme sendo esta capacidade considerada um fator de virulência. Num biofilme, as bactérias estão envoltas numa matriz de substâncias poliméricas extracelulares (SPE), algumas das quais são proteínas. Tendo por objectivo obter mais informação acerca do biofilme, foi caracterizado o proteoma da matriz do biofilme de uma estirpe bastante virulenta de S. pseudintermedius isolada de um cão com piodermite profunda. Para tal cultivaram-se biofilmes da estirpe de S. pseudintermedius 5819/10 em meio apropriado, separou-se a matriz das suas células bacterianas e avaliou-se as proteínas presentes quanto ao seu conteúdo e complexidade. Posteriormente o proteoma foi separado por electroforese 1D, caracterizado por nanoLC-ESI-Q-TOF e analisado usando ferramentas bioinformáticas Constatou-se que o proteoma da matriz do biofilme da estirpe 5819/10 de S. pseudintermedius é muito diverso e que 63% das proteinas podem estar relacionadas com a região extracelular do biofilme ou da membrana plasmática na forma de complexos proteicos. Verificou-se também que a maioria das proteínas identificadas possui funções essenciais para a sobrevivência da bactéria mas não foi possível estabelecer uma relação clara entre elas e a formação de biofilmes. Algumas proteínas que se sabe estarem envolvidas na formação de biofilmes foram identificadas, tratam-se principalmente de factores reguladores da formação de biofilme e outros factores de virulência relacionados com a colonização de um hospedeiro a adesão bacteriana a uma superfície. A prevalência de adesinas e a ausência quase total de proteínas envolvidas na síntese de SPEs, forneceu dados que apoiam a hipótese que a matriz do biofilme do S. pseudintermedius 5819/10 seja constituída por células directamente ou indirectamente unidas entre si.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
O presente trabalho teve como objetivo avaliar a associação da expressão gênica e proteômica com a maciez da carne de bovinos da raça Nelore. A partir de uma população de 90 animais foram selecionados três grupos experimentais por meio da análise de força de cisalhamento (FC) e índice de fragmentação miofibrilar (MFI), sendo: carne moderadamente macia, carne moderadamente dura e carne muito dura. A expressão dos genes foi avaliada por meio da análise de PCR em tempo real e a análise proteômica foi realizada com base na separação de proteínas por meio da eletroforese bidimensional (2D-PAGE) e caracterizção por espectrometria de massas com ionização eletrospray (ESI-MS/MS). A expressão da isoforma da calpastatina (CAST2) mostrou-se up regulated (P<0,05) nos grupos de carne moderadamente dura e muito dura. Os genes HSP90AA1, DNAJA1 e HSPB1, os quais representam as proteínas de choque térmico Hsp90, Hsp40 e Hsp27, respectivamente, mostraram expressão down regulated (P<0,05) no grupo de carne moderadamente macia em relação ao grupo de carne muito dura. Na análise proteômica, a expressão do spot protéico das enzimas metabólicas TPI e PGM1, proteína estrutural PFN1 e aminiopeptidase LAP3 se mostraram up regulated (P<0,05) no grupo de carne moderadamente macia, enquanto que a expressão das proteínas estruturais (ACTA1, ACTB, ACTG1 e MLC1), estresse oxidativo (PRDX6, PRDX2, PRDX1 and PARK7), proteínas de choque térmico (HSP90AA1, HSP90AB1, HSPA1A, HSPA1B, HSPA1L, HSPD1 e HSPB1), e co-chaperonas e regulação celular (CD37, STIP1 e ARHGDIA) se mostraram down regulated (P>0,05) no mesmo grupo experimental. Estes resultados fornecem uma visão importante de novos possíveis marcadores biológicos atuantes no processo de amaciamento da carne, o que pode colaborar para melhor entender e gerar novas estratégias de seleção nos programas de melhoramento genético de bovinos Nelore.
The objective of this study was to evaluate the association of gene expression and proteomics with meat tenderness in Nellore cattle. From population of 90 animals three experimental groups were selected by shear force (SF) and/or myofibrillar fragmentation index (MFI): moderately tender meat, moderately tough meat and very tough meat. Gene expression was evaluated by real-time PCR and proteomics analysis was performed based on protein separation by two-dimensional gel electrophoresis (2D-PAGE) and characterisation by eletrospray ionisation mass spectrometry (ESI-MS/MS). Expression of the calpastatin isoform (CAST2) was up-regulated (P<0.05) in the moderately tough and very tough meat groups. Expression of the HSP90AA1, DNAJA1 and HSPB1 genes, wich represent the heat shock proteins Hsp90, Hsp40 and Hsp27, respectively, were down-regulated (P<0.05) in the moderately tender meat in relation to the very tough group. In the proteomics analysis, the expression of the protein spots of metabolism TPI1 and PGM1, structural protein PFN1, and aminopeptidase LAP3 were up regulated (P<0.05) in the moderately tender meat, while the expression of structural proteins (ACTA1, ACTB, ACTG1 and MLC1), oxidative stress (PRDX6, PRDX2, PRDX1 and PARK7), heat shock protein (HSP90AA1, HSP90AB1, HSPA1A, HSPA1B, HSPA1L, HSPD1 and HSPB1) and co-chaperones and cellular regulatory (CD37, STIP1 and ARHGDIA) were down regulated (P>0.05) in the same experimental group. The present results suggest an important view of possible new biological markers in the meat tenderization process, wich permit to unsderstand and generate new strategies for selection in Nellore cattle breeding programs.
FAPESP: 15/13021-1

La microsporidie Encephalitozoon cuniculi, parasite intracellulaire obligatoire, pathogène de l'homme, est responsable d'infections opportunistes chez des sujets immunodéprimés. Sa spore est protégée par une épaisse paroi protéo-chitineuse pour laquelle peu de données sur les constituants protéiques sont disponibles. Dans ces travaux, nous avons décrit le protéome exprimé dans les stades tardifs de développement d'E. Cuniculi. Grâce à des extractions protéiques séquentielles et une double stratégie d'analyse protéomique, après électrophorèse et "Shotgun", 177 protéines différentes ont pû être identifiées, permettant d'obtenir une vision globale de la physiologie de la spore. L'exploitation de ces données et le développement d'un crible bioinformatique a permis l'identification d⇋ 4 protéines de paroi. Le premier modèle dynamique de morphogenèse de la paroi microsporidienne a été proposé grâce au suivi de la localisation de ces protéines durant le cycle de développement

This study is focused on changes in protein expression in a glioblastoma cell line during infection with tick-borne encephalitis virus. Newly synthesized proteins were distinguished from previously synthesized proteins using bioorthogonal chemistry (BONCAT method) to observe changes in protein synthesis. Labelled proteins were visualized using two-dimensional PAGE and western blotting followed by Click reaction on membrane. Differences in protein pattern between control and infected cells were observed.

La linea generale su cui si sviluppa il mio progetto di dottorato è la ricerca e misurazione quantitativa di biomarcatori, ed è inserito nel più ampio campo della diagnostica medica. Un biomarcatore è definito in modo chiaro dalla Food and Drug Administration statunitense come “una caratteristica che sia oggettivamente misurabile e valutabile come indicatore di processi fisiologici o patologici o di risposta a trattamento con farmaci”. Il primo progetto di ricerca di questo periodo di dottorato consiste in un’analisi proteomica differenziale su siero di cordone ombelicale e fluido amniotico alla ricerca di biomarcatori di ritardo di crescita intrauterino, una patologia altrimenti detta IUGR (Intra Uterine Growth Restriction). Nel progetto in questione sono stati analizzati campioni di siero di cordone ombelicale e fluido amniotico provenienti da madri al termine della gravidanza, per le quali sia stato diagnosticato (si parla di “casi”) o meno (“controlli”) un ritardo di crescita intrauterino. Con la tecnica della elettroforesi bidimensionale su gel di poliacrilammide (2D-PAGE) la moltitudine di proteine presenti nei suddetti campioni è stata dipanata, in modo da identificare separatamente le singole proteine sovra- o sottoespresse in caso di patologia. Il risultato è stato l’identificazione di 14 proteine uniche nel siero e 11 nel cordone ombelicale, concorrenti complessivamente a determinare un quadro d’insieme della patologia. Queste proteine sono coinvolte in processi alterati nel caso di ritardo di crescita intrauterino, in particolare: coagulazione del sangue, pressione sanguigna, difese immunitarie, omeostasi di ferro e rame, stress ossidativo. I risultati forniti dall’elettroforesi bidimensionale sono stati validati in modo incrociato con Western Blot, applicato su 5 proteine scelte per importanza fisiologica o livello di sovra- o sottoespressione. Di particolare spicco nel determinare il quadro complessivo di modulazione proteica sono state: transferrina (coinvolta nel trasporto di ossigeno nel sangue), kininogeno (coinvolta nei processi di coagulazione e di modulazione della pressione sanguigna), fibrinogeno (coinvolta nel processo di coagulazione del sangue), angiotensinogeno (uno dei soggetti implicati nella regolazione della pressione sanguigna) e componente C3 del complemento (complesso proteico implicato nella cosiddetta “immunità innata”). Diverse condizioni patologiche sono innescate da un’ alterazione di un meccanismo fisiologico in particolare: la comunicazione attraverso la fosforilazione delle proteine, facente parte del gruppo delle cosiddette modificazioni post-traslazionali (PTM, Post Translational Modifications). Queste sono modificazioni chimiche delle proteine nella fase successiva alla loro sintesi ribosomiale, che a valle di una serie di passaggi trasforma in una molecola effettiva l’informazione potenziale contenuta nel DNA. Alterazioni in questo processo biologico sono causa di gravi malattie come cancro, neurodegenerazione e diabete e gli studi degli scienziati hanno fatto emergere la necessità di nuovi materiali per la cattura delle fosfoproteine, soggetti rari in un mare di proteine non fosforilate. Allo scopo una tecnologia promettente è lo stampo molecolare di polimeri (Molecular Imprinting of Polymers, MIP), che sfrutta la molecola oggetto di analisi per formare attorno a sé una tasca complementare ad essa, in grado di riconoscerla e legarla successivamente quando rimossa dalla matrice stessa. Data poi la dimensione nanometrica delle proteine, lavorare sulla stessa scala dimensionale favorisce l’efficacia del riconoscimento delle molecole, come avviene per l’interazione antigene-anticorpo. L’idea che nasce da questa premessa è quindi quella della sintesi e caratterizzazione di nanoparticelle stampate molecolarmente per il riconoscimento di fosfoproteine e fosfopeptidi: il secondo progetto seguito durante questo periodo di dottorato di ricerca. La fase sperimentale ha portato a sintetizzare e caratterizzare fisicamente nanoparticelle (cioè particelle aventi almeno una dimensione al di sotto dei 100 nanometri) di copolimeri con 4 ricette diverse, in presenza o meno del templato: fosfotirosina protetta all’azoto terminale con fluorenilmetossicarbonile (Fmoc). La fase di verifica delle proprietà di legame è stata effettuata su una ricetta delle quattro, preparata stavolta con metodo standardizzato, ovvero non sotto forma di nanoparticelle. Si è valutata la capacità di legare fosfotirosina in sue soluzioni a concentrazione crescente. I risultati vedono una tendenza di legame a saturazione nel polimero stampato, mentre un accumulo crescente di substrato nel caso del polimero non stampato. La capacità di legame del MIP è attorno ai 3μg fosfotirosina/mg polimero. Ma l’individuazione dei marcatori di patologie è solo una fase del processo della diagnosi medica: una volta individuato un marcatore di patologia è necessario mettere a punto un metodo di misura dello stesso. Questo metodo dovrà essere il più possibile sensibile alle concentrazioni dell’analita nel campione in esame e specifico per esso, in modo da non confondere un segnale derivante da un fondo di molecole con la sua presenza. Un grosso beneficio alla condizione del paziente può anche derivare dalla possibilità di misurare l’analita nel luogo dove si trova la persona, attuando così quella che si chiama “point of care diagnostics”. A tale scopo esistono delle tecniche analitiche che più si prestano alla realizzazione di dispositivi portatili semplici: una di queste è la risonanza plasmonica superficiale (Surface Plasmon Resonance, SPR). La terza parte del mio periodo di dottorato è stata quindi dedicata alla messa a punto di un metodo di rilevazione di un ormone peptidico, l’epcidina, sfruttando la suddetta tecnica. L’analita in questione è il principale regolatore del metabolismo del ferro, e la realizzazione di una metodica analitica sensibile ed efficace per la sua misurazione è considerata essere effettivamente necessaria dalla comunità scientifica internazionale. I risultati ottenuti in questa parte sperimentale sono stati la creazione di una curva di calibrazione per l’epcidina in concentrazioni nel range pato-fisiologico 1-100ng/mL, una verifica della scarsa interazione del nostro sistema di cattura dell’epcidina nei confronti di un suo competitore, una sua forma troncata, più la valutazione della costante di affinità dell’analita per il legante utilizzato nella cattura, risultata essere 50nM, quindi in linea con precedenti misurazioni in soluzione.
Medical diagnosis is the process of attempting to determine and/or identify a possible disease or disorder. This process is revealed by biomarkers, defined by The Food and Drug Administration (FDA) as “characteristics that are objectively measured and evaluated as indicators of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention”. The process of biomarker discovery has been boosted in the last years by proteomics, a research discipline that takes a snapshot of the entire wealth of proteins in an organism/ tissue/ cell/ body fluid. An implementation of the analysis methods can help in isolate proteins present in the low range of concentrations, like biomarkers very often are. An established biomarker can further be measured with the help of biosensors, devices that can be employed in the point-of care diagnostics. This PhD thesis shows and discusses the results of three projects in the field of protein biomarkers discovery and quantification. The first project exploited proteomics techniques to find relevant protein markers for Intrauterine Growth Restriction (IUGR) in cordonal blood serum (UCS) and amniotic fluid (AF). A 14 proteins in UCS and 11 in AF were successfully identified and found to be differentially expressed. Molecularly Imprinted Polymers (MIPs) directed towards proteins and peptides containing phosphotyrosine were then produced, with the final goal of selectively extract phosphopeptides from a peptide mixture. An alteration of the phosphorylation pattern is often associated to important diseases like cancer. The polymers were produced as nanoparticles, that were characterized with Dynamic Light Scattering (DLS) and Atomic Force Microscopy (AFM). A recipe was also tested for binding capacity towards phosphotyrosine. A Surface Plasmon Resonance (SPR) biosensor to quantify hepcidin hormone was finally produced. This is the major subject in iron homeostasis in vertebrates and marker of iron unbalance diseases. A calibration curve was made and affinity/kinetic parameters for the ligand employed were measured.

Winter rye produces ice-modifying antifreeze proteins upon cold treatment. Two of these antifreeze proteins are members of the large, highly conserved, ??-1,3-endoglucanase family. This project was designed to identify glucanase genes that are expressed during cold acclimation, wounding, pathogen infection, drought or treatment with the phytohormones ethylene and MeJa. Additionally, a more detailed proteomic analysis was to be carried out to evaluate the glucanase content of the apoplast of cold-acclimated (CA) winter rye. Results of 2D SDS-PAGE analysis revealed that non-acclimated whole leaf protein extracts contain at least two ??-1,3-endoglucanses while CA whole leaf protein extracts contain at least three ??-1,3-endoglucanses. Subsequent 2D SDS-PAGE analysis was conducted on the apoplast extracts of NA and CA winter rye plants revealed the limitations of standard 1D SDS-PAGE. The 2-dimensional gel analysis revealed that there is a minimum of 25 proteins within the apoplast of CA winter rye, including at least 5 ??-1,3-endoglucanases. Genome walking was used to isolate cold-responsive glucanase genes. The five genes isolated were designated scGlu6, scGlu9, scGlu10, scGlu11 and scGlu12. The cis-element pattern within the promoter of each gene was evaluated using online databases of documented plant cis elements. As expected, all of the promoters contained elements associated with cold, biotic and abiotic stresses, light regulation, and development. The expression patterns predicted by the cis elements in each promoter were compared to the mRNA abundance produced by each gene as detected by semi-quantitative reverse transcriptase PCR. In most cases, the abundance of transcripts arising from each gene loosely corresponded to the expression pattern predicted by the cis elements the corresponding promoter. Transcripts of scGlu9, 10 and 11 were present in cold-treated tissues and are candidates for ??-1,3-endoglucanases with antifreeze activity. The results presented in this thesis provide additional insight into the apoplast proteome of CA winter rye plants as well as the complexity of the signals controlling the proteins that reside there. Although there are still a number of unresolved questions, this research opens new directions for future studies in the cold acclimation process in winter rye and specifically for the contribution of ?? -1,3-endoglucanses.

碩士
國立中正大學
化學所
95
1. The non-enzymatic conjugated addition product of glucose or aldehyde derivatives and glycation reaction of protein are the main cause of vascular complications of diabetes. If the concentration of glucose remains at high level in the body, the amount of ?dicarbonyl compounds, such as glyoxal and methylglyoxal, also increases. Glyoxal is obtained from glucose and amino acid oxidation, lipid peroxidation; methylglyoxal is mainly from glucose degradation intermediate product: G-3-P self-decompose. These reactive ?dicarbonyl compounds react with protein, lipids and amino acids to form saccharides as the final product. This study makes use of LC/NSI/MS to investigate the reaction of glyoxal and methylglyoxal with the amino acids on protein. First, we use LC/NSI/MS to confirm that glyoxal and methylglyoxal react with N?acetyl-cysteine and N?acetyl-L-lysine to form cross-linked products. Next, we use somatostain, which contains 14 amino acids and disulfide bond to react with glyoxal and methylglyoxal respectively and we have detected their addition and cross-linked products. Finally, we investigated the selectivity of glyoxal and methylglyoxal with human hemoglobin. We found that there was a single glyoxal addition on at Lys-11 of ?globin, and Lys-16, Lys-144 and Cys-93 of β-globin:, whereas methylglyoxal were found to add ?globin at Arg-31and β-globin at Lys-144 and Arg-104. We also confirm that glyoxal forms hydroimidazolone with ?globin at Arg-92. Similar hydroimidazolone also occurs with methylglyoxal and ?globin at Arg-31and Arg-92. However, the cross-linked products of glyoxal or methylglyoxal with human hemoglobin have not been identified. 2. In cancer research, inflammation is a very crucial and dangerous factor. During infection and inflammation, the activated macrophages and neutrophils will produce excess superoxide anion and NO which will react rapidly to produce peroxynitrite. Peroxynitrite could lead to breakage and mutation of DNA, and it also reacts with protein to form 3-nitrotyrosine. Many inflammation and neural degradation diseases are related to 3-nitrotyrosine, including eye inflammation, retinal ischemia, and cancer. In 2000, a report showed that using anti-3-nitrotyrosine together with Western blotting and mass spectromety effectively identified proteins containing 3-nitrotyrosine in mouse’s retinal. This study tried to determine proteins that contain 3-nitrotyrosine in human urine using 2D-PAGE and mass spectrometry. First, we eliminated most salts in our urine sample using acetone precipitation and centrifugal device, then separated the proteins by 2D-PAGE. Next, we used anti-3-nitrotyrosine antibody-base Western blotting to locate the nitrated protein. Before identifying the proteins in 2D-PAGE, the gel was digested. Hence, different gel microwave digestion conditions were carried out to find the best conditions. The urine samples were then analyzed using this condition. Currently, we successfully identified 3 kinds proteins containing 3-nitrotyrosine, including, protein complex 4, epsilon 1 subunit; adaptor related; LIM domain only 6[Homo sapiens] and Hypothetical protein FLJ32940 isoform 1. We also found a protein, transmembrane protein 16 F[Homo sapiens], containing nitrotryptothan.

PsbO (manganese-stabilizing protein) is the largest extrinsic protein of photosystem II, located on the lumen side of photosystem. It is present in all known oxyphototrophic organisms. PsbO facilitates photosynthetic water splitting, which takes place in an oxygen evolving center (Mn4CaO5 cluster) of photosystem II. This work is focused on PsbO of higher plants and its isoforms, particularly their evolution and functions. Bioinformatic analyses revealed that majority of higher plants express exactly two psbO isoforms. A phylogenetic tree of PsbO sequences has an unusual topology. The two paralogous isoforms do not diverge at the base of the phylogenetic tree, as anticipated, but rather at the end of particular branches, at the level of family or lower taxonomic unit. In this work we propose and discuss several hypotheses concerning evolution of PsbO isoforms. The work further includes detailed analysis and identification of protein spots assigned to PsbO on 2D IEF-SDS PAGE gels of potato thylakoid proteins. We identified predominant version of PsbO isoform in most of the spots. We did not succeed to find any posttranslational modification. We optimized a method of psbO expression in E. coli and subsequent purification, which yielded relatively big amount of properly folded recombinant protein. Analysis of...