No description available.

A dissertation submitted to the Faculty of Science, University of Witwatersrand, in fulfilment of the requirements for the degree of Master of Science. Johannesburg, 2014.

No description available.

The PI3K/AKT/mTOR signaling pathway is commonly dysregulated in cancer. The goal of this thesis project was to assess the hypothesis of a strong correlation between PI3K/AKT/mTOR pathway activity and the response to targeted therapies, by using a protein quantitation technique called immuno-matrix assisted laser desorption/ionization (iMALDI). The use of iMALDI as a clinical tool was demonstrated by automating an established iMALDI assay for quantifying plasma renin activity. The results from the automated method gave high correlation coefficients of ≥0.98 with a clinical LC-MS/MS method and could be performed significantly faster than with manual sample preparation. The 7.5-fold faster analysis compared to LC-MS/MS, reduction in human error, and higher throughput, demonstrated the suitability of this assay for clinical use. The automated iMALDI platform was then adapted for use with cancer cell lines and tissue analysis, targeting the kinases AKT1 and AKT2 as surrogate proteins for signaling pathway activity. Using minute amounts (10 µg/capture), AKT1 and AKT2 expression and phosphorylation stoichiometry (PS) were successfully quantified via their C-terminal tryptic peptides, which encompassed key phosphorylation sites. After assay optimization, the assays were analytically validated for linear range, accuracy, and interferences. In addition, PS cut-off values based on measurement errors were established for confident PS quantitation. The functionality of the assay was demonstrated with cell lines, and flash-frozen and FFPE tissue lysates, with, on average, lower AKT1/AKT2 measurements obtained from FFPE samples. The developed assays were sensitive and precise enough to detect differences between matched normal and adjacent tumor tissues. To answer the hypothesis, patient-derived xenograft (PDX) mouse-model tumors treated with Herceptin, Everolimus, a combination of both (E+H), or with no treatment, were assessed for molecular patterns linked to tumor response. One mouse from the E+H group showed a partial response, with elevated total and phosphorylated AKT1/AKT2. Unfortunately, overlapping values between treatment groups were obtained in this study, and the large within-group spread and the low number of biological replicates made it difficult to confirm a definite correlation between PI3K/AKT/mTOR pathway activity and response to treatment. A follow-up study with additional protein targets, a larger number of samples, and serial biopsies will be required to determine if there is, in fact, a correlation between PI3K/AKT/mTOR pathway activity and response to treatment. / Graduate / 2019-10-05

The classification of the Azotobacteraceae to the level of genus and species has been uncertain since the studies of Beijerinck in 1901. This dissertation represents an effort to establish a system of classification more complete than the one now in use. In this study, both qualitative and quantitative taxonomy were used in order to establish a well founded classification scheme. Qualitative methods included certain important morphological and physiological characteristics, isoenzyme patterns, and immunological reactions. Quantitative methods included numerical taxonomy (based on total morphological and physiological characteristics) and numerical analysis of protein profiles. All the data from these experiments were subject to comparison with other genotypic and phenotypic data.

Submitted by Ana Damasceno (ana.damasceno@unipampa.edu.br) on 2017-06-07T21:02:40Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) PA³P uma nova ferramenta para determinação in silico da alergenicidade e antigenicidade de proteínas.pdf: 4093415 bytes, checksum: 0bc76850b4fffd6e3c97f8f6a339dc0b (MD5) / Made available in DSpace on 2017-06-07T21:02:40Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) PA³P uma nova ferramenta para determinação in silico da alergenicidade e antigenicidade de proteínas.pdf: 4093415 bytes, checksum: 0bc76850b4fffd6e3c97f8f6a339dc0b (MD5) Previous issue date: 2016-05-05 / Uma das preocupações envolvendo a expressão de diferentes proteínas heterólogas em alimentos é a possibilidade do desenvolvimento de reações alérgicas ou antigênicas nos consumidores finais. A Food and Agriculture Organization of the United Nations juntamente com a World Health Organization definiram que uma sequência de aminoácidos seria considerada alergênica/antigênica quando apresentasse ao menos 35 % de identidade em uma janela de 80 aminoácidos ou 6 – 8 aminoácidos contíguos e idênticos quando comparada com sequências de alergénos conhecidos. Para categorizar proteínas alergênicas ou antigênicas foi construída uma plataforma (chamada Plataforma de Análise da Alergenicidade e Antigenicidade de Proteínas – PA³P – e disponível em http://lpa.saogabriel.unipampa.edu.br:8080/pa3p/index.jsp ou http://pluriserver.com.br/pa3p/) que congrega um conjunto de ferramentas específicas para tais análises. A plataforma fora construída através da linguagem de programação Java e HTML. Desta forma, foram separados grupos de proteínas alergênicas/antigênicas de não alergênicas/não antigênicas com redução dos casos de falsos positivos ou falsos negativos. As análises complementares utilizadas neste trabalho são necessárias, pois a metodologia proposta pela Food and Agriculture Organization of the United Nations juntamente com a World Health Organization é insuficiente, gerando resultados duvidosos. A plataforma construída neste trabalho obteve valores de 98 % de sensibilidade e 96 % de especificidade, comparada com 89 % e 85 %, respectivamente, dos testes utilizados isoladamente. Esta plataforma pode ser utilizada para embasar a decisão de utilizar determinada proteína na construção de um Organismo Geneticamente Modificado com finalidade nutricional. / A major drawback involving heterologous protein expression in organisms used for human consumption is the possibility of allergenic or antigenic reactions. Both Food and Agriculture Organization of the United Nations and World Health Organization determined that a potentially allergenic protein world present at least 35 % identity in a 80 amino acid window or 6 – 8 contiguous and identical amino acids when compared with known allergens. In order to assess the allergenic or antigenic potential of a given protein, the Platform for Analysis of Allergenicity and Antigenicity of Proteins (PA³P – available at http://lpa.saogabriel.unipampa.edu.br:8080/pa3p/index.jsp or http://pluriserver.com.br/pa3p/) was developed. The platform was constructed using Java and HTML programming languages. It was p ssible to discriminate allergenic/antigenic from non-allergenic/non-antigenic proteins, with reduction of false positive and false negative samples. The additional analyses used in the platform are necessary, since the insufficient FAO and WHO proposed methods, rendering doubtable results. In our tests, PA³P presented 98% sensibility and 96% specificity when compared with the web tools used independently (89 and 85%, respectively). This tool has a potential to contribute to decision making regarding the of a given protein in GMO construction with nutritional intent.

Mosquitos transgênicos incapazes de transmitir malária podem ser um controle alternativo, mas atualmente não estão disponíveis. O estudo da interação mosquitopatógeno é importante para melhorar o desenho de genes. A proteína circumsporozoita (CSP) tem dois domínios conservados que podem estar envolvidos na penetração dos esporozoítos na glândula salivar. Nosso objetivo foi expressar peptídeos contendo essas regiões na hemolinfa do mosquito usando o sistema de expressão transiente vírus Sindbis e a tecnologia de transgênese. Se a CSP está envolvida neste processo, os peptídeos competirão com spz impedindo a penetração. Cinco vírus sindbis e quatro linhagens transgênicas foram construídos e desafiados por P. gallinaceum. Nossos resultados mostram que os peptídeos não impediram a penetração de spz na glândula salivar, principalmente porque os peptídeos recombinantes não foram produzidos ou detectados. Aprimorar o desenho de genes, usando a otimização de códons e outras tecnologias, será essencial para a expressão de proteínas exógenas em mosquitos transgênicos. / Transgenic mosquitoes that impair malaria transmission can be an alternative control but currently an effective line is not available. A better understanding of mosquito interaction with pathogens is very important to improve refractory transgene design. Circumsporozoite protein (CSP) has two conserved domains that could be involved in spz penetration into mosquito salivary glands. Our aim was to express peptides encompassing these conserved regions in the mosquito hemolymph using Sindbis virus transient expression system and transgenesis technology. If CSP is involved in this process these peptides will compete with sporozoites impairing its penetration. Five Sindbis virus and four transgenic lines were constructed and challenged with P. gallinaceum. Our results showed these peptides could not impair sporozoites penetration in salivary glands, mainly because the recombinant proteins could not be produced or detected. Improving transgene design using codon usage and other technologies will be essential for expressing foreign proteins in transgenic mosquitoes.

Mosquitos transgênicos incapazes de transmitir malária podem ser um controle alternativo, mas atualmente não estão disponíveis. O estudo da interação mosquitopatógeno é importante para melhorar o desenho de genes. A proteína circumsporozoita (CSP) tem dois domínios conservados que podem estar envolvidos na penetração dos esporozoítos na glândula salivar. Nosso objetivo foi expressar peptídeos contendo essas regiões na hemolinfa do mosquito usando o sistema de expressão transiente vírus Sindbis e a tecnologia de transgênese. Se a CSP está envolvida neste processo, os peptídeos competirão com spz impedindo a penetração. Cinco vírus sindbis e quatro linhagens transgênicas foram construídos e desafiados por P. gallinaceum. Nossos resultados mostram que os peptídeos não impediram a penetração de spz na glândula salivar, principalmente porque os peptídeos recombinantes não foram produzidos ou detectados. Aprimorar o desenho de genes, usando a otimização de códons e outras tecnologias, será essencial para a expressão de proteínas exógenas em mosquitos transgênicos. / Transgenic mosquitoes that impair malaria transmission can be an alternative control but currently an effective line is not available. A better understanding of mosquito interaction with pathogens is very important to improve refractory transgene design. Circumsporozoite protein (CSP) has two conserved domains that could be involved in spz penetration into mosquito salivary glands. Our aim was to express peptides encompassing these conserved regions in the mosquito hemolymph using Sindbis virus transient expression system and transgenesis technology. If CSP is involved in this process these peptides will compete with sporozoites impairing its penetration. Five Sindbis virus and four transgenic lines were constructed and challenged with P. gallinaceum. Our results showed these peptides could not impair sporozoites penetration in salivary glands, mainly because the recombinant proteins could not be produced or detected. Improving transgene design using codon usage and other technologies will be essential for expressing foreign proteins in transgenic mosquitoes.

No description available.

Electric field gradient focusing (EFGF) utilizes a hydrodynamic flow and an electric field gradient to focus and concentrate charged analytes and order them in a separation channel according to electrophoretic mobility. Elution can be achieved by decreasing the applied voltage or increasing the hydrodynamic flow. EFGF has the advantages of concentrating a large volume (100 micro-L) of target proteins without significant band broadening and simultaneously removing unwanted components from the sample. Two types of EFGF devices have been investigated to concentrate and separate proteins: a fiber-based EFGF device and a hydrogel-based EFGF device. Using fiber-based EFGF with UV detection, a concentration factor as high as 15,000 and a concentration limit of detection as low as 30 pM were achieved using bovine serum albumin as a model protein. I also demonstrated the potential of using fiber-based EFGF for quantitative protein analysis. Simultaneous desalting and protein concentration as well as online concentration of ferritin and simultaneous removal of albumin from a sample matrix were also performed using this fiber-based EFGF system. In the approach of utilizing hydrogel-based EFGF, online concentration of amyloglucosidase indicated a concentration limit of detection of approximately 20 ng/mL (200 pM) from a sample volume of 100 micro-L. Both voltage-controlled and flow-controlled elution methods were demonstrated using a 3-component protein mixture. Concentration of human α1-acid glycoprotein with simultaneous removal of human serum albumin was also described. A tandem EFGF system, which integrates fiber-based and hydrogel-based EFGF sections, was also investigated to selectively concentrate and separate proteins in a mixture. By carefully controlling the voltages applied to both sections, charged analytes with high mobilities were trapped in the fiber-based section, analytes with intermediate mobilities in the hydrogel-based section, and analytes with low mobilities not at all. A 3-way switching valve was incorporated in the system to purge the analytes with high mobilities periodically. Selective concentration and separation of myoglobin from a mixture were performed using the tandem EFGF system. Based on the experimental results described in this dissertation, EFGF shows potential for selective isolation, concentration, and quantitation of trace analytes such as proteins in biomedical samples.