Regenerative medicine is a rapidly developing field, merging engineering and biological life sciences to create biological replacements for damaged tissue and organ function. Development of cellular based therapies has the potential of curing present untreatable diseases and conditions, such as diabetes. The identification of protein expression patterns, that guide undifferentiated cells to different lineages, can provide important information about the progression of cellular differentiation at various stages. This research project utilizes proteomics and in vitro live-cell microscopy to investigate two distinct cellular systems: (1) the signaling pathways of calmodulin (CaM) in the differentiation of a human glioblastoma cell line; and (2) the effect of islet neogenesis associated protein (INGAP) on human islet-derived progenitor cells (hIPCs). Using a proteomic readout with a long term live-cell imagining approach, it was hypothesized that highly specific binding proteins of a CaM-mutant, and proteins in hIPCs perturbed by INGAP, could be identified and studied in vitro, characterizing specific signaling pathways which control the function of CaM in brain tumour cells and the mechanism(s) of INGAP in islet-derived progenitor cells. This thesis presents the utility of a proteomics and an in vitro cell microscopy approach to investigate therapeutic proteins, such as INGAP, on cell culture systems. The results have established the limitations and the utility of DIGE, differential binding of a CaM-mutant versus calcium-CaM, and the cell specific uptake feasibility of using the TAT-binding domain. In the hIPC system, proteomic, phenotypic, motility, proliferation and nuclear effects of INGAP were determined. Specifically, hIPCs exposed to INGAP had 50% decrease in average nuclear speed, the translocation of two identified proteins caldesmon and tropomyosin and INGAP was found to bind specifically to hIPCs. However, hIPCs had no changes in insulin specific hormone expression.

Sugarcane bacilliform virus (SCBV) is an economically important pathogen of sugarcane in Australia which limits access to foreign sugarcane germplasm. Although SCBV is present in the major cane growing regions worldwide, very little is known about its variability, virulence and the yield losses resulting from infection. The limited information on SCBV has resulted in quarantine measures being introduced to protect the Australian sugarcane industry, with a major consequence being restricted access to imported sugarcane germplasm for breeding programs. Foreign sugarcane germplasm plays an important role in breeding of new commercial varieties for the Australian sugar industry and is essential for the long term productivity, profitability and sustainability of the sugar industry. This study was aimed at characterising Australian isolates of SCBV to enable the development of reliable and robust molecular and/or antibody-based diagnostic tests which could be used to not only assess the impact of SCBV on the Australian sugarcane industry, but could also be used to screen imported sugarcane germplasm for the virus. SCBV virions (SCBV-IM) were purified from the sugarcane accession "Ireng Maleng" and the dsDNA genome was cloned and sequenced. The genome of SCBV-IM comprised 7687 bp with an organisation typical of other badnaviruses. When the entire nucleotide sequence of SCBV-IM was compared to that of the Moroccan SCBV isolate (SCBV-Mo), less than 75% similarity was present. Within the coding regions, ORF I, ORF II and ORF III had 83%, 71% and 73% nucleotide similarity to SCBV-Mo, respectively. At the amino acid level, ORFs I, II and III from SCBV-IM showed 91%, 84% and 85% similarity to the equivalent regions in SCBV-Mo, respectively. To further investigate the level of sequence variability within Australian SCBV isolates, virions were purified from three further sugarcane accessions and a 220 bp fragment of the reverse transcriptase-coding region was amplified. Five clones from each sub-population were selected and sequenced. Analysis of these sequences revealed considerable variability in the virus population with variability within one plant as great as it was between isolates. However, since the use of specific primers could also be selecting for a sub-population of SCBV sequences, it was possible that the variability may actually be greater than that reported. These results indicated that SCBV isolates are complex and variable and may represent a continuum of genetic variability. High molecular weight DNA species larger than the SCBV 7.6 kbp unit-length genome were found in DNA extracted from purified SCBV-IM virions. We confirmed that these high molecular weight nucleic acids were virus-specific and open circular in conformation. Using field inversion gel electrophoresis (FIGE), the SCBV-IM DNA was separated into four discrete bands with sizes ranging from between 1 to 4 genome copies. The DNA was shown to comprise overlapped individual genome-length molecules and not covalently-bonded continuous DNA strands. We presume that these DNA molecules are concatamers formed during replication as a result of a terminal overlap on the sense strand. The presence of these concatamers within virions may explain the observation of particles with lengths corresponding to one, two or three times the modal length of 130 nm. Four SCBV-infected Saccharum officinarum plants were examined for the presence of integrated viral DNA. Southern blot analysis of viral DNA and total DNA extracted from the same plant source were compared with, or without, restriction digestion. The resulting restriction patterns from viral and total DNA were almost identical suggesting that there were no integrated SCBV sequences in the sugarcane cultivars tested. Although larger-than-single-genome copy bands were detected in both the viral and the total DNA samples, this was probably due to the presence of genomic concatamers. SCBV integration studies using Southern analyses were further complicated by high sequence variability which precluded the restriction digestion of all viral DNA species. As such, some of the SCBV DNA species remain as concatamers which appear as larger-than-unit-length SCBV products. An antiserum derived from a mixture of purified SCBV isolates has been used routinely in the past to screen for SCBV infection, but the heterogeneity reported for badnaviruses has cast doubt on the ability of this antiserum to detect all SCBV isolates. We attempted to determine whether antiserum generated against proteins other than the viral capsid could be used to detect SCBV infections, thus improving the reliability and robustness of SCBV diagnosis. The complete coding regions of SCBV ORF I and ORF II were bacterially expressed and used as antigens for antiserum production. Both ORF I and II proteins were found to be highly immunogenic and generated high-titre antisera, designated AS-I and AS-II, respectively. The diagnostic utility of both antisera to detect SCBV in six different infected sugarcane plants was tested using both immunosorbent electron microscopy (ISEM) and western blots. The currently used SCBV antiserum (AS-V), generated against a mixture of purified SCBV isolates, was included for comparison. In western analyses, neither AS-I nor AS-V was able to conclusively detect SCBV in any of the six infected plants due to reactivity with numerous non-specific proteins. In contrast, AS-II reacted specifically with a protein of the expected size (~13.5 kDa) in 2/6 infected plants. When compared using ISEM, AS-V, AS-I and AS-II trapped virions from 6/6, 6/6 and 2/6 SCBV-infected plants, respectively. However, the number of virions trapped using AS-V was approximately 30-fold more than that trapped using either AS-I or AS-II. These results highlight the variability between SCBV isolates and suggest that ISEM with antisera raised against mixtures of viral proteins may be a useful tool for the detection of viral isolates.

Allergic diseases, for example asthma and eczema, are nowadays considered belonging to the most common chronic diseases amongst children in the West, but the cause for this increase in allergy prevalence is unknown. Since studies have indicated a connection between children's exposure of microorganisms during infancy and risk of developing allergic disease, it is suggested that this exposure is a crucial factor in question of allergy development or not. Other studies have established differences in microflora composition between healthy children and children with allergic disease, and several studies have shown that probiotic therapy can give positive results in both prevention and treatment of allergic diseases. The aim of this master's thesis was to develop a method, using PCR and denaturing gradient gel electrophoresis, to study the diversity of Lactobacillus spp. in fecal samples retrieved from a study of the probiotic strain L. reuteri ATCC 55730. The developed method was successful in detecting lactobacilli in fecal samples, but three other bacterial genera commonly found in humans were also amplified. Comparison of average numbers of detected bacterial strains and lactobacilli strains between samples belonging to the probiotics and placebo groups, respectively, showed higher numbers for the probiotics group. Also, the only fecal samples that contained L. reuteri belonged to the probiotics group. Although the results are far from statistically significant, they support the theories that probiotics may influence the intestinal microbiota.

A esquizofrenia é uma doença crônica, grave e incapacitante que afeta cerca 24 milhões de pessoas em âmbito mundial. É caracterizada por uma desorganização no pensamento que prejudica a funcionalidade do indivíduo. Existem intervenções que são efetivas e contribuem para a diminuição da prevalência do transtorno, pois ajudam o portador a levar uma vida produtiva e integrada à sociedade, porém devem ser ministradas nos estágios iniciais da doença. No entanto, existe uma grande dificuldade em se diagnosticar a esquizofrenia precocemente devido a sua complexidade e às sutilezas dos seus sintomas apresentados antes do surgimento da psicose. O cérebro não é acessível a exames invasivos in vivo e por esse motivo a exploração de fluidos periféricos é de grande importância. As plaquetas e neurônios serotonérgicos possuem características bioquímicas e morfológicas em comum que possibilitam a comparação entre a estrutura e a função de ambos e, por causa dessa similaridade, muitos trabalhos utilizam plaquetas como modelo para o estudo de doenças neuropsiquiátricas, inclusive a esquizofrenia. A detecção precoce da esquizofrenia é um objeto de investigação atual e relevante não somente para revolucionar os meios atuais de diagnóstico, mas também para desenvolver novos tratamentos aplicados aos estágios iniciais da doença, diferenciar os subgrupos de doentes e monitorar as intervenções preventivas. A proposta do presente trabalho é fazer o estudo da expressão de proteínas em plaquetas de pacientes esquizofrênicos e controles com o objetivo de identificar proteínas candidatas a biomarcadores utilizando técnicas proteômicas quantitativas e confiáveis, como 2D-DIGE e a espectrometria de massas. / Schizophrenia is a disabling, serious, and chronic illness, which affects about 24 million people worldwide. It is characterized by a severe disorganization of the thoughts that harms the social life of patients becoming them dependent of the family and/or government. There are effective treatments that contribute to decrease the prevalence of the disorder because they improve the life and social conditions of the patients, but they are only advantageous if the intervention is made in the early stages of the disease. It is difficult to obtain early diagnosis due to the complexity of the disease and its insidious symptoms before the beginning of the psychosis. The brain is not easily accessed in vivo and, because of this, it is very important to study the peripheral tissues like blood, which makes the use of the platelets very interesting. Furthermore, platelets and serotonergic neurons share biochemical and morphological characteristics that allows the comparison between structure and function of both. From these similarities many authors has used platelets as a neuron model to study many neurodegenerative diseases including schizophrenia. The early detection of schizophrenia is a current and suitable goal, not only to improve the early diagnosis but also to develop new treatments, differentiate the subtypes, and monitor the preventive interventions. The purpose of this project is to do a comparative screening of expressed proteins in platelets from schizophrenics and controls with the objective of finding differently expressed proteins that could be candidates to biomarkers using 2D-DIGE and mass spectrometry.

Orientador: Marco Aurélio Zezzi Arruda / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-22T00:56:12Z (GMT). No. of bitstreams: 1 Silva_MarceloAnselmoOseasda_D.pdf: 21762318 bytes, checksum: 0f395a27c9b28a3d7414579ff560bd4a (MD5) Previous issue date: 2012 / Resumo: Inúmeros trabalhos envolvendo uma nova área de estudos denominada metalômica destacam a necessidade de melhor compreender a interação entre íons metálicos e proteínas. Esta Tese empregou o girassol (Helianthus annuus L.), uma planta da família das oleaginosas que apresenta uma relevante importância econômica na produção de óleo vegetal, bem como em processos de fitorremediação, em um estudo metalômico para avaliação de possíveis alterações na expressão de proteínas mediante o desenvolvimento das plantas irrigadas com íons selenito ou zinco. Inicialmente, o estudo avaliou quatro diferentes métodos para a adequada para extração e separação de proteínas das folhas de girassol empregando a técnica de eletroforese bidimensional em gel de poliacrilamida (2DPAGE), sendo que o melhor procedimento consistiu na extração empregando tampão na presença de fenol. A posterior caracterização das proteínas, nos spots proteicos, empregando espectrometria de massas também foi conduzida evidenciando, pela primeira vez para o organismo estudado, a expressão de 12 novas proteínas. Um estudo ionômico foi desenvolvido para avaliar a distribuição destes elementos nas diferentes estruturas das plantas, buscando correlação com os processos biológicos que estariam ocorrendo nos girassóis. O estudo revelou uma correlação entre o nível de selênio e enxofre nas plantas sugerindo a incorporação do selênio em aminoácidos utilizados para síntese de proteínas. O emprego da técnica de ablação a laser acoplada ao espectrômetro de massas com plasma acoplado indutivamente (LA-ICP-MS) também apresenta destaque na pesquisa, complementando os dados obtidos no estudo ionômico, por meio da identificação de selênio nos spots proteicos extraídos das folhas dos girassóis e separadas por 2D-PAGE. Desenvolveu-se também desenvolver um método para análise quantitativa direta e simultânea de selênio e enxofre nas folhas das plantas empregando a hifenação LA-ICP-MS, buscando correlação entre os dois elementos / Abstract: Several papers highlight a new research area called metallomic, and points out the necessity to better understand the relationship between metallic ions and proteins. This Thesis has employed the sunflower (Helianthus annuus L.), which is considered an oilseed plant with significant economical importance for the production of vegetal oil, and also used in fitoremediation processes, to carry out a metallomic study focused on the evaluation of possible changes in proteins expression due to the irrigation of the plants with selenite or zinc ions. Initially, the study evaluated four different methods for the adequate extraction and separation of sunflower leaf proteins using the two-dimensional gel electrophoresis (2DPAGE) technique and the best result was obtained using a phenol based extraction procedure. Further protein characterization in protein spots were carried out using mass spectrometry and revealed for the first time for the studied organism, the expression of 12 new proteins. An ionomic study was also developed to investigate the distribution of these elements in the different structures of the plants, looking for the correlation of obtained data with biological processes that could be occurring in the sunflowers. The study showed a correlation between selenium and sulfur levels, suggesting the incorporation of selenium into amino acids used for the synthesis of proteins. The application of laser ablation technique coupled to the inductively coupled plasma mass spectrometer (LA-ICP-MS) has also been highlighted in this research, complementing the results obtained in the ionomic study through the identification of selenium in sunflower leaves protein spots. In addition, it was evaluated an analytical procedure for the direct and simultaneous quantitative determination of selenium and sulfur in the leaves of the plants using LA-ICP-MS, searching for a correlation between both elements / Doutorado / Quimica Analitica / Doutor em Ciências

ABSTRACT The aim of this study is develop and test an inexpensive molecular tool kit to be used for diagnostic PCR for diseases such as Leber hereditary optic neuropathy (LHON) and Cystic fibrosis(CF). By developing and optimizing recombinant Taq polymerase and making a DNA size ladder from plasmids pPSU1 and pPSU2 the financial cost for the tool kit would be reduced significantly compared to the commercial components. With an inhouse method both the recombinant Taq polymerase and the pPSU1 and pPSU2 plasmids were purified from the E.coil strain DH5-α. Thereafter to analyse the components of the tool kit both conventional PCR and Real-time PCR to make sure that the tool kit would work for both types of PCRs.     The homemade Taq polymerase proved to be able to sustain in room temperature for at least 24 h and the polymerase also showed that it works with different primers such as LHON, CF and Beta-globin in both endpoint and probe base real-time PCR. The homemade size marker produced a reliable in agarose gel electrophoresis but requires optimization for continued usage for smaller PCR products.     In conclusion the homemade Taq polymerase will be used in future PCR analysis in the laboratory and the recombinant production process as well. Meanwhile the homemade size marker did not work sufficiency enough to be continuously used with gel electrophoresis in the laboratory without being further modified.

Technique of pulsed field gel electrophoresis (PFGE) has found widespread use in the analysis of the genome of all life organisms. It is applied to the separation of the large DNA molecules above thousands base pairs up to millions of base pairs in size, where using conventional gel electrophoresis techniques are not possible (for instance large bacterial, yeast, fungal or mammalian chromosome). Presented work was realized as a comparative analysis of genome of several carotenogenic yeasts. The conditions of isolation and analysis of chromosomal yeast DNA were optimized. A lysis of yeast cells and deproteination of DNA within agarose chops was shown as the most appropriate method for DNA isolation. Cultivation to late exponential phase (50 hours) is the most suitable to obtaining intact DNA in sufficient amount and quality. Carotenogenic yeasts undergo the random mutagenesis using alkylation reagent ethyl methanesulfonate (EMS). Genome of pigment overproducing mutants was analyzed by pulsed field gel electrophoresis and amount of carotenoids by high pressure liquid chromatography (HPLC). However, overproduction of beta-carotene was analyzed in mutant strains Rhodotorula glutinis (10.6 g/l of biomass enriched 0,34 mg/g of beta-carotene) and Cystofilobasidium capitatum (8.5 g/l of biomass enriched 0,23 mg/g of beta-carotene). Selection of mutant strains overproducing carotenoid pigments was in presented experiment series successful in almost all analyzed strains except in the case of the strain Rhodotorula aurantiaca.

This term project deals with the classification of 1D gel electrophoresis samples. It describes the theoretical information about gel electrophoresis, various types of errors, processing of the image and its classification using the cluster analysis. One of the main goals is creation of images with the highest quality as possible. A realization of pre-processing and detection of the sample borders is made in the MATLAB environment. And finally, classification of samples is done with subsequent statistical analysis.

Recently much attention is given to lanthanides and their complexes as excellent catalysts for cleavage of nucleic acids. The thesis has been focused on the cleavage of plasmid and bacterial DNA by ions Nd3+ and Y3+ and by different carriers containing the lanthanide compounds. The creation of single-stranded nicks and double-stranded ones in the plasmid DNA molecules was studied by agarose gel electrophoresis. Verification of the cleavage of bacterial DNA was made by polymerase chain reaction using primers specific for the domain Bacteria and genus and species-specific primers. The results will be used in the development of the method that will allow perfect carriers`s coverage verification with the magnetic perovskit nucleus and other carriers with the lanthanide compounds.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.