This thesis documents the subcellular localization and protein-protein interactions of two methyl recycling enzymes. These two enzymes, adenosine kinase (ADK) and S-adenosyl-L-homocysteine hydrolase (SAHH), are essential to sustain the hundreds of S-adenosyl-L-methionine (SAM)-dependent transmethylation reactions in plants. Both ADK and SAHH are involved in the removal of a competitive inhibitor of methyltransferases (MTs), S-adenosyl-L-homocysteine (SAH), that is generated as a by-product of the each transfer of a methyl group from SAM to a substrate. This research focused on understanding how SAH is metabolized in distinct cellular compartments to maintain MT activities required for plant growth and development. Localization studies using green fluorescent protein (GFP) fusions revealed that both ADK and SAHH localize to the cytoplasm and the nucleus, and possibly to the chloroplast, despite the fact that the primary amino acid sequence of neither protein contains detectable targeting signals. This suggested the possibility that these methyl-recycling enzymes may be targeted by specific protein-protein interactions. Moreover, deletion analysis of SAHH1 indicated that the insertion region (IR) of 41 amino acids (Gly150-Lys190), which is present only in plants and parasitic protozoan SAHHs among eukaryotes, is essential for nuclear targeting. This result suggested that the surface-exposed IR loop may serve as a binding domain for interactions with other proteins that may direct SAHH to the nucleus. To investigate protein-protein interactions, several methods were performed including co-immunoprecipitation, bimolecular fluorescence complementation, and pull-down assays. These results not only revealed that ADK and SAHH possibly interact through the IR loop of SAHH in planta, but also suggested that this interaction is either dynamic or indirect, requiring a cofactor/another protein(s) or post-translational modifications. Moreover, possible interactions of both ADK and SAHH with a putative Arabidopsis mRNA cap methyltransferase (CMT), which is localized predominantly in the nucleus, were also confirmed. These results support the hypothesis that the nuclear targeting of both SAHH and ADK can be mediated by the interaction with CMT. In addition, purification of Strep-tagged SAHH1 expressed in Arabidopsis identified a novel interaction between SAHH and aspartate-semialdehyde dehydrogenase (ASDH), an enzyme that catalyzes the second step of the aspartate-derived amino acid biosynthesis pathway. Analysis of ASDH-GFP fusions revealed that ASDH localizes to the chloroplast and the stromule-like structure that emanates from chloroplasts. Moreover the mutation in three amino acids (Pro164-Asp165-Pro166) located within the IR loop of SAHH disrupted its binding to ASDH which affected the plastid localization of SAHH, suggesting that the interaction between SAHH and ASDH is required for plastid-targeting of SAHH. Taken together, this thesis demonstrated that the localization of ADK and SAHH in or between compartments is possibly mediated by specific protein interactions, and that the surface-exposed IR loop of SAHH is crucial for these interactions.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Submitted by BRUNA MARQUES DOS SANTOS null (brunamarques.bio@gmail.com) on 2016-05-20T14:43:34Z No. of bitstreams: 1 Dissertação_Bruna_Marques_Santos.pdf: 2509707 bytes, checksum: d5b8e8138728f1d899f6f957bedf5425 (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2016-05-24T14:23:17Z (GMT) No. of bitstreams: 1 santos_bm_me_jabo.pdf: 2509707 bytes, checksum: d5b8e8138728f1d899f6f957bedf5425 (MD5) / Made available in DSpace on 2016-05-24T14:23:17Z (GMT). No. of bitstreams: 1 santos_bm_me_jabo.pdf: 2509707 bytes, checksum: d5b8e8138728f1d899f6f957bedf5425 (MD5) Previous issue date: 2016-03-30 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A emissão de dióxido de carbono (CO2) pelas atividades humanas vem aumentando desde a revolução industrial. Previsões indicam que ocorrerá um aumento expressivo da concentração atmosférica deste gás nos próximos anos. Este fato deve resultar em alterações metabólicas nas plantas e, por consequência, impactar o setor florestal brasileiro. Os cloroplastos são as organelas-chave na fixação do CO2 e início do particionamento do carbono nas plantas. Alterações na disponibilidade de CO2 podem afetar o metabolismo dessas organelas. O objetivo do presente trabalho foi avaliar se o cultivo de plantas jovens de Eucalyptus urograndis em ambiente enriquecido com CO2 resulta em alterações no proteoma cloroplastidial. Para tanto, primeiramente foram avaliados diferentes métodos de isolamento de cloroplastos quanto aos seguintes parâmetros: morfologia dos cloroplastos observada em microscopia de luz (1); rendimento protéico após isolamento plastidial (2); grau de contaminação por proteínas não cloropastidiais (3); e abundância em número de proteínas identificadas e já descritas como plastidiais (4). Após a definição da melhor metodologia para obtenção do proteoma cloroplastidial, mudas de Eucalyptus urograndis de aproximadamente três meses de idade foram cultivadas sob concentrações atmosféricas controladas de CO2 (400 e 1000 ppm) durante dez semanas. A avaliação do proteoma plastidial, por buscas restringentes contra um banco de dados de sequências protéicas de Eucalyptus grandis, resultou na identificação de 816 proteínas em E. urograndis, das quais 80% já haviam sido descritas como plastidiais. O mapeamento in silico de vias metabólicas resultou na identificação de todas as proteínas envolvidas no ciclo de Calvin-Benson, além da detecção de um aumento discreto, porém significativo na abundância de enzimas-chave: PGK, GAPDH, FBA, FBPase, SBPase e RPI. Embora a avaliação da eficiência quântica do fotossitema II tenha indicado ausência de alteração fotossintética, as plantas tratadas com 1000 ppm de CO2 apresentaram fechamento estomático em resposta à condição ambiental imposta, além da diminuição na área do tecido vascular foliar. Esta é a primeira caracterização do proteoma cloroplastidial do gênero Eucalyptus, cujos resultados indicam que a atmosfera enriquecida com CO2 causou respostas na espécie, incluindo um aumento na abundância de proteínas envolvidas na fixação de carbono. Os resultados apresentados aqui podem auxiliar na compreensão das respostas bioquímicas estimuladas por um aumento na concentração atmosférica de CO2 em plantas do tipo C3, além de contribuir para programas de melhoramento que visem obter plantas adaptadas às condições climáticas futuras. / Carbon dioxide (CO2) emissions from human activities have increased since the industrial revolution. Global projections indicate that there will be a significant increase in the atmospheric concentration of this gas in the coming years. This fact can result in metabolic changes in plants and, consequently, affect the Brazilian forest sector. Chloroplasts are key organelles in carbon fixation and early carbon partitioning in plants. Changes in the availability of CO2 may affect the metabolism of these organelles. The goal of the present study was to assess whether the cultivation of seedlings of Eucalyptus urograndis under a CO2 enriched environment could result in changes in the chloroplast proteome. For this purpose, different chloroplast isolation methods were evaluated to the following parameters: chloroplast morphology observed in bright-field microscopy (1); protein yield after plastid isolation (2); degree of contamination by non-plastidic proteins (3); and abundance in the number of identified proteins described as plastidic (4). After determining the best methodology for the isolation of the chloroplast proteome, E. urograndis seedlings about three months old were grown under CO2 controlled atmospheric concentrations (400 and 1000 ppm) for ten weeks. Evaluation of the plastid proteome, using stringent search against a protein sequence database from Eucalyptus grandis, resulted in the identification of 816 proteins in E. urograndis, from which 80% were already described as plastidic. In silico metabolic pathway mapping resulted in the identification of all proteins involved in the Calvin-Benson cycle and detection of a slight but significant increase in the abundance of key enzymes: PGK, GAPDH, FBA, FBPase, SBPase, and RPI. Although the assessment of the quantum efficiency of photosystem II suggested the absence of changes in the photosynthesis rate, plants treated with 1000 ppm of CO2 presented stomatal closure in response to the imposed environmental condition. A decreased area of the leaf vascular tissue was also detected in young leaves. This is the first characterization of chloroplast proteome of the genus Eucalyptus. Our results indicate that the CO2 enriched atmosphere stimulated metabolic responses, including an increase in the abundance of proteins involved in carbon fixation. Results showed here will assist on the understanding of the biochemical responses stimulated by an increase in the atmospheric CO2 concentration in C3-type plants, and contribute to breeding programs that aim to obtain plants adapted to future climate conditions. / FAPESP: 2014/07454-0

Orientador: Marcelo Menossi Teixeira / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-11T15:42:58Z (GMT). No. of bitstreams: 1 Vicentini_Renato_D.pdf: 5901500 bytes, checksum: 7c8fe505e4c1675900f96c6643895d32 (MD5) Previous issue date: 2008 / Resumo: As células de plantas são altamente organizadas e muitos processos biológicos estão associados com estruturas subcelulares específicas. A localização subcelular é uma característica chave das proteínas, visto que está relacionada com a função biológica. A determinação da localização subcelular usando a predição é uma estratégia altamente desejável, principalmente porque as abordagens experimentais demandam um tempo considerável. Com o objetivo de desenvolver um método para melhorar a predição de localização subcelular, diversos algoritmos foram integrados visando à exploração ótima do potencial de cada um. O desempenho com 90% de exatidão deste novo método foi claramente superior a todos os métodos utilizados em sua criação. Usando esta estratégia foi realizada a primeira análise em larga escala da localização subcelular do proteoma de cana-de-açúcar (com 11.882 proteínas preditas), sendo encontrado que a maioria das proteínas estão localizadas em quatro compartimentos: núcleo (44%), citoplasma (19%), mitocôndria (12%), e extracelular (11%). Adicionalmente foi observado que cerca de 19% das proteínas são localizadas em múltiplos compartimentos. Outros resultados foram capazes de identificar um conjunto de proteínas de cana-de-açúcar que podem apresentar duplo direcionamento pelo uso de variações na extremidade amino. Utilizando expressão transiente em células da epiderme de cebola, foi investigada a localização subcelular de 96 proteínas de cana com fusão a proteína GFP. As construções contendo fusão amino- e carboxi-terminal dos genes foram expressas, e a localização das proteínas de fusão foi detectada por microscopia de fluorescência. É relatado também a caracterização do gene ScBAK1, um receptor do tipo quinase com repetições ricas em leucina, que apresenta similaridade de seqüência com o gene brassinosteroid insensitive1-associated receptor kinase1. Foi mostrado que transcritos desse gene se acumulam em níveis muito mais altos nas células da bainha do feixe vascular do que nas células do mesófilo, e que a fusão ScBAK1-GFP é localizada na membrana plasmática. Essa distribuição espacial e esse padrão de expressão indicam que a ScBAK1 pode estar potencialmente envolvida em cascatas de sinalização celular intermediadas por altos níveis de açúcar na folha. Ainda considerando estudos de localização subcelular, é conhecido que seqüências de nucleotídeos que flanqueiam o códon de início da tradução afetam a eficiência traducional dos mRNA, e podem indicar a presença de sítios de inicio de tradução (TIS) alternativos. O multi-direcionamento pode ser um reflexo da variabilidade traducional destas outras formas da proteína. Neste estudo foi desenvolvido um método computacional para investigar o uso de TISs alternativos na síntese de novas variantes protéicas que podem apresentar localização subcelular diferente. Visando contribuir para o nosso entendimento da complexidade do genoma da cana-de-açúcar, foi empregada uma análise em larga escala dos TIS nesta espécie. Também é demonstrado que os transcritos com expressão induzida apresentam um forte TIS quando comparados com os reprimidos, e que os transcritos constitutivos possuem uma alta freqüência de TIS alternativos. O mesmo ocorre para os genes com altas taxas evolutivas, e transcritos específicos de folhas e entrenós, levantando a hipótese de que esses genes possam codificar diferentes polipeptídeos / Abstract: Plant cells are highly organized and many biological processes are associated with specialized subcellular structures. Subcellular localization is a key feature of proteins, since it is related to biological function. The determination of subcellular localization using computational prediction is a highly desirable strategy because experimental approaches are time-consuming. In order to develop a method for the enhanced prediction of subcellular localization, the outputs of some prediction tools were integrated so as to optimally exploit the potential of each one. The prediction performance (with 90%of accuracy) of this new method was clearly superior to all the methods used to create the predictor. Using this method, the first in silico genome-wide subcellular localization analysis was performed for sugarcane (with 11,882 predicted proteins). It was found that most of the proteins are localized to four compartments: nucleus (44%), cytosol (19%), mitochondria (12%), and secretory destination (11%). It is also shown that about 19%of the proteins are localized to multiple compartments, and that a potential set of sugarcane proteins can show dual targeting by use of N-truncated form of proteins. The subcellular localization of 96 sugarcane proteins fused with GFP were evaluated using transient expression in onion epidermal cells. Constructs containing the Nand C-terminal fusion of genes encoding both endogen and GFP proteins were transiently expressed, and the localization of the fusion proteins were detected by fluorescent microscopy. It was reported the characterization of ScBAK1, a sugarcane leucine-rich repeat receptor-like kinase, with sequence similarity to brassinosteroid insensitive1-associated receptor kinase1. We have found that ScBAK1 transcripts accumulated at higher levels in bundle-sheath than in mesophyll cells. ScBAK1-GFP fusions were localized to the plasma membrane. This spatial distribution and expression pattern indicates that ScBAK1 might be potentially involved in cellular signaling cascades mediated by high levels of sugar in this organ. The nucleotide sequence flanking the translation initiation codon affects the translational efficiency of eukaryotic mRNAs, and may indicate the presence of an alternative translation initiation site (TIS) to produce proteins with different properties. Multi-targeting may reflect the translational variability of these other protein forms. In this study it was also developed a computational method to investigate the usage of alternative TISs for the synthesis of new protein variants that might have different subcellular localization. To contribute to our understanding of the genome complexity of sugarcane, we undertook a genome wide TIS analysis in sugarcane data. It is demonstrated that up-regulated transcripts show a stronger TIS when compared with the down-regulated, and that ubiquitous transcripts have a high frequency of alternative TIS in the next downstream AUG codon. The same occurs for fast-evolving genes, and leaf and internodes specific transcripts, that may encode different polypeptides by N-terminal polymorphism / Doutorado / Genetica Vegetal e Melhoramento / Doutor em Genetica e Biologia Molecular

Maspina (mammary serpin) foi identificada em 1994 como uma serpina (serine protease inhibitor) que apresenta atividade de supressão tumoral. Foi classificada como uma serpina devido à homologia na sequência de aminoácidos, porém, maspina não apresenta atividade de inibição de serina proteases. Entre os efeitos biológicos de maspina estão a modulação da adesão, a inibição do crescimento e a invasão tumoral, a inibição da angiogênese, o efeito pró-apoptótico e o controle da resposta ao stress oxidativo, propriedades que contribuem para supressão tumoral. Esta diversidade de funções se reflete nos inúmeros ligantes de maspina e na sua localização subcelular, já que é encontrada na membrana plasmática, no citoplasma, núcleo e mitocôndrias. A localização subcelular de maspina guarda importante relação com sua função, já que foi demonstrado que sua localização nuclear está correlacionada com bom prognóstico em diversos tumores e seu efeito supressor de tumor foi observado somente quando maspina está localizada no núcleo. Entre os ligantes de maspina estão a HDAC1, IRF6, GST, HSP90 e HSP70, β1 integrina, uPAR e colágeno tipo I e III. O mecanismo molecular envolvido na regulação dessas atividades não foi elucidado, e até o momento, somente um gene e uma proteína de maspina foram descritos, desta forma alterações pós-traducionais devem estar envolvidas na regulação dessas atividades. Com objetivo de verificar se há modificações pós-traducionais em maspina, utilizamos células MCF10A, que expressam grande quantidade dessa proteína, e submetemos seu extrato proteico à separação por gel bidimensional seguido de western blot. Identificamos quatro formas de maspina com a mesma massa molecular (42kDa), mas pontos isoelétricos distintos. Três destas formas são sensíveis ao tratamento com fosfatase ácida, o que sugere que estas sejam fosforiladas. Utilizamos ainda peroxidovanadato de sódio, um potente inibidor de tirosina fosfatase para investigar o papel da fosforilação de maspina em resíduos de tirosina. Através de western blot e imunofluorescência, observamos que o tratamento das células com o inibidor resultou no aumento dos níveis celulares de maspina assim como no seu acúmulo no citoplasma. Deste modo, concluímos que existem três diferentes fosfoformas de maspina em células MCF10A e ainda a inibição de tirosinas fosfatases aumentam os níveis de maspina e resultam no acúmulo da proteína no citoplasma. Esses resultados sugerem que a fosforilação pode estar envolvida na localização subcelular de maspina e na regulação dos seus níveis proteicos na célula. / Maspin (mammary serpin) was identified in 1994 as a serpin (serine protease inhibitor) which presents tumor suppressor activity. It was classified as a serpin due to its homology in amino acids sequence; however, maspin doesn\'t exhibit serine protease inhibition activity. Among maspin biological effects are modulation of cell adhesion, inhibition of tumor growth, invasion and angiogenesis, a pro-apoptotic effect and control of oxidative stress response, properties which contribute to tumor suppression. This functional diversity reflects maspin numerous ligands and its subcellular localization, since it is found on the plasma membrane, in the cytoplasm, nucleus and in mitochondria. Maspin subcellular localization is closely related to its function, as its nuclear localization correlates with good prognostic in several tumors and maspin tumor suppressor activity is only observed when it is located in the nucleus. Among maspin ligands are histone H1 deacetylase, IRF6, GST, HSP90 e HSP70, β1 integrin, uPAR and type I and III collagen. The molecular mechanisms involved in the regulation of maspin biological activities are poorly understood. So far, only one gene and one protein have been assigned to maspin, so posttranslational modification should be involved. In order to verify posttranslational modification in maspin, we utilized MCF10A cells, which express great amount of this protein, and we submitted its proteic extract to 2D-SDS-PAGE followed by western blot. We identified four maspin forms with the same molecular mass (42kDa), but different isoelectric point. Three of these forms are sensitive to acidic phosphatase treatment, suggesting that they are phosphorylated maspin forms. We also utilized sodium peroxovanadate, a potent tyrosine phosphatase inhibitor to investigate the role of maspin tyrosine phosphorylation. Through western blot and immunofluorescence analyses, we observed that cell treatment resulted in increase in maspin cellular levels as well as its cytoplasmic accumulation. Thus, we concluded that there are three diferente maspin phosphoforms in MCF10A cells and yet tyrosine phosphatase inhibition increases maspin levels and results in accumulation of the protein in the cytoplasm. These data suggest that phosphorylation may be involved in maspin subcellular localization and regulation of its protein levels in the cell.

Orientador: Edi Lúcia Sartorato / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T15:12:43Z (GMT). No. of bitstreams: 1 DeMoraes_VanessaCristineSousa_D.pdf: 4935241 bytes, checksum: ce8c385da6e3872d30f08426c629d34d (MD5) Previous issue date: 2013 / Resumo: O alargamento do aqueduto vestibular (EVA) é uma malformação da orelha interna que pode ser identificado por tomografia computadorizada ou ressonância magnética. O EVA é um dos principais sinais clínicos da Síndrome de Pendred (PDS), uma doença genética com padrão de herança autossômico recessivo causada na maioria dos casos por mutações no gene SLC26A4. Além de EVA, o bócio e defeito na organificação do iodeto na tireóide são achados clínicos típicos da PDS. Por sua vez, mutações no gene SLC26A4 têm também sido observadas em indivíduos com surdez não sindrômica associada ao EVA. Recentemente os genes FOXI1 e KCNJ10 também foram implicados na PDS. O gene FOXI1 é um fator de transcrição do gene SLC26A4. Medições electrofisiológicas mostraram que a alteração da pendrina, proteína codificada pelo gene SLC26A4, em modelos animais levava indivíduos à surdez pela falta do potencial endococlear devido à perda de expressão de canais potássio. Sendo atribuído ao gene KCNJ10 a função de manutenção do potencial endococlear. Desta maneira, o presente estudo teve como objetivo avaliar a ocorrência de mutações nos genes SLC26A4, FOXI1 e KCNJ10 em 60 indivíduos brasileiros portadores de perda auditiva sensorioneural, associada ou não a alterações no aqueduto vestibular. Foram encontradas 14 diferentes alterações no gene SLC26A4, das quais 3 ainda não haviam sido descritas na literatura (P142L, G149R e C282Y) e 4 já haviam sido descritas, porém ainda não haviam sido caracterizadas funcionalmente (T193I, Q413R, L445W e R776C). Dessa forma, foi realizada a análise funcional e a co-localização celular da proteína Pendrina com estas 7 variações alélicas. Não foi encontrada nenhuma evidência de contribuição digênica relacionada ao gene FOXI1 e/ou KCNJ10, uma vez que nenhum paciente desta casuística com alteração no gene SLC26A4 apresentou mutações nesses genes. Além disso, no grupo composto por 30 indivíduos surdos que não apresentam EVA, ficou evidente que o rastreamento do gene SLC26A4 não foi suficiente para explicar a perda auditiva nesses pacientes, uma vez que foram encontradas apenas alterações em um alelo do gene. Por outro lado, no grupo formado por 30 indivíduos surdos que apresentam EVA, o rastreamento do gene SLC26A4 possibilitou o esclarecimento do diagnóstico etiológico da perda auditiva em 5 pacientes que apresentaram mutações nos dois alelos do gene SLC26A4 / Abstract: Enlargement of the vestibular aqueduct (EVA) is a malformation of the inner ear that can be identified by computed tomography or magnetic resonance imaging. EVA is the main feature of Pendred syndrome (PDS), a genetic disease with autosomal recessive inheritance pattern, in most cases caused by mutations in the SLC26A4 gene. Besides EVA, goiter and defective organification of iodide in the thyroid are other typical clinical signs of PDS. In turn, SLC26A4 gene mutations have been also observed in patients with non-syndromic deafness associated with EVA. Recently the genes FOXI1 and KCNJ10 were also implicated in the PDS. The FOXI1 gene is a transcription factor of SLC26A4 gene. Electrophysiological measurements in animal models showed that the mutated pendrin, the protein encoded by the SLC26A4 gene, led individuals to deafness by the lack of endocochlear potential due to loss of expression of potassium channels. Being assigned to the KCNJ10 gene the maintenance of endocochlear potential. Thus, the present study aimed to evaluate the occurrence of mutations in SLC26A4, and KCNJ10 FOXI1 genes in 60 Brazilian patients with sensorineural hearing loss, with or without changes in the vestibular aqueduct. We found 14 different mutations in SLC26A4 gene, of which 3 had not yet been described in the literature (P142L, G149R and C282Y) and 4 had already been described, but had not been characterized functionally yet (T193I, Q413R, L445W and R776C). Thus, we performed the functional analysis and cellular co-localization of Pendrin protein with these 7 allelic variants. We found no evidence of digenic contribution related to FOXI1 and/or KCNJ10 genes, since no patient in with mutations in SLC26A4 gene showed mutations in these genes. In addition, the screening of SLC26A4 gene in 30 deaf individuals with no EVA was not sufficient to explain the hearing loss in these patients, since mutations were found only in one allele of the gene. On the other hand, the screening of SLC26A4 gene in 30 deaf individuals with EVA allowed the elucidation of the etiology of hearing loss in 5 patients with mutations in both alleles of this gene / Doutorado / Genetica Animal e Evolução / Doutora em Genética e Biologia Molecular

A cell is considered to be the smallest unit of life. It carries out a variety of biochemical reactions through the activities of proteins and protein enzymes. In order to perform functions, proteins must be in their native folded state together with the correct environmental conditions. A slight change in pH or temperature could cause disruption to the electrostatic interactions within the protein, thus leading to conformational change and the loss of activity. Studies have shown that solubility could be enhanced by increasing the number of charges on the protein surface. And from the studies of extremophiles, we learned that the presence of non-polar aromatic residues could be a key for thermostable proteins. Thus, charges are important to determine the function and adaptation of proteins.Over the decades, large amount of protein sequence and structure information relating to molecular biology has been produced. By employing algorithms, computational and statistical techniques, it is possible to analyse these data to solve biological problems. Often these investigations are based mainly on sequences since their numbers outstrip the number of available structures. However, adding structures would allow us to investigate problems such as the relationship between charges, sequence, structure and functions, which is the aim of this study.In this thesis, the relationships between proteins and function were examined by various electrostatic features derived from charges and also geometric properties from structures. One interesting finding is that the averaged value of pH of maximum stability of proteins within a subcellular location was highly correlated to the pH of that subcellular compartment, which was due to pKas (of histidines), and their locations on the proteins. We also found that the size of the largest non-charged patch on the protein surface correlates with solubility and provides a predictor with a maximum accuracy of 76%. The use of novel charge-based methods shows little improvement in distinguishing between enzymes and non-enzymes. However, the method of using real charges with grid size of 1 angstrom has paved a way into the idea of using charges and dipoles pattern from enzyme active site to distinguish different enzymes. Finally, a web-tool for displaying conserved residues on 3D protein structure is made available to the public for identifying residues that may be of functional importance.

Cancer remains one of the world’s most devastating diseases, with more than 10 million new cases every year. However, traditional treatments have proven insufficient for successful medical management of cancer due to the chemotherapeutics’ difficulty in achieving therapeutic concentrations at the target site, non-specific cytotoxicity to normal tissues, and limited systemic circulation lifetime. Although, a concerted effort has been placed in developing and successfully employing nanoparticle(NP)-based drug delivery vehicles successfully mitigate the physiochemical and pharmacological limitations of chemotherapeutics, work towards controlling the subcellular fate of the carrier, and ultimately its payload, has been limited. Because efficient therapeutic action requires drug delivery to specific organelles, the subcellular barrier remains critical obstacle to maximize the full potential of NP-based delivery vehicles. The aim of my dissertation work is to better understand how NP-delivery vehicles’ structural, chemical, and physical properties affect the internalization method and subcellular localization of the nanocarrier. In this work we explored how side-chain and backbone modifications affect the conjugated polymer nanoparticle (CPN) toxicity and subcellular localization. We discovered how subtle chemical modifications had profound consequences on the polymer’s accumulation inside the cell and cellular retention. We also examined how complexation of CPN with polysaccharides affects uptake efficiency and subcellular localization. This work also presents how changes to CPN backbone biodegradability can significantly affect the subcellular localization of the material. A series of triphenyl phosphonium-containing CPNs were synthesized and the effect of backbone modifications have on the cellular toxicity and intracellular fate of the material. A mitochondrial-specific polymer exhibiting time-dependent release is reported. Finally, we present a novel polymerization technique which allows for the controlled incorporation of electron-accepting benzothiadiazole units onto the polymer chain. This facilitates tuning CPN emission towards red emission. The work presented here, specifically, the effect that side-chain and structure, polysaccharide formulation and CPN degradability have on material’s uptake behavior, can help maximize the full potential of NP-based delivery vehicles for improved chemotherapeutic drug delivery.

Maspina é uma proteína supressora de tumor e metástase e sua localização subcelular está relacionada ao prognóstico do câncer de mama. Nosso grupo mostrou em MCF-10A que quando fosforilada maspina se acumula no citoplasma. Porém, esta correlação ainda não foi relatada in vivo. Aqui investigamos a expressão, fosforilação e localização subcelular de maspina ao longo do desenvolvimento da glândula mamária murina. Maspina foi detectada no estágio mais tardio da gestação, na lactação e na involução. Os níveis de fosforilação de maspina são maiores no período de lactação do que na involução. Interessantemente, a porcentagem de células que apresenta maspina no núcleo é maior na fase de involução do que na fase de lactação Estes dados mostram que a correlação entre níveis de fosforilação de maspina e localização subcelular também é observada in vivo e que esses processos são reguladas ao longo do desenvolvimento na glândula mamária murina. / Maspin is a protein with tumor and metastasis suppressing activity and its subcellular localization is related to breast cancer prognosis. Using MCF-10A cells as a model system, our group demonstrated a correlation between maspin phosphorylation and cytoplasmic accumulation. Here we investigated maspin expression, phosphorylation levels and subcellular localization in vivo during the murine mammary gland development. Maspin was detected in late pregnancy, during lactation and involution. Maspin phosphorylation levels is higher during lactation than during involution. Interestingly, the percentage of cells which present nuclear maspin is higher in the involution than in lactation. These data show that the correlation between maspin phosphorylation and subcellular localization is also observed in vivo and these processes are regulated during murine mammary gland development.

Sucrose is the major transported photoassimilate in plants and is degraded concurrently by two enzymes: invertases and sucrose synthase. Sucrose synthase catalyzes the reversible conversion of UDP and sucrose to form fructose and UDP-glucose, the latter being the activated substrate for many metabolic processes including cellulose biosynthesis. There is evidence that sucrose synthase is phosphorylated as a regulatory mechanism of carbon allocation at a conserved N-terminal serine residue. The phosphorylation or dephosphorylation at this specific site has also been found to shift the protein localization in a tissue and species specific manner. A literature study of the functional regulation of sucrose synthase in plants has highlighted several scientific questions: Is sucrose synthase cellular localization regulated by phosphorylation of an N-terminal conserved serine residue? What are the regulatory mechanisms underlying within and between species variation in sucrose synthase localization? Does sucrose synthase associate with the cellulose synthase enzyme complex? Can cellulose biosynthesis be increased by over-expression of the membrane-associated form of sucrose synthase? The aim of this M.Sc study was to determine the subcellular localization of Eucalyptus grandis sucrose synthase 1 (EgSUSY1) fusion proteins expressed in Arabidopsis thaliana plants. This was investigated through modifying the 11th serine residue of EgSUSY1 into either a non-polar alanine residue that cannot be phosphorylated (S11A), or into a negatively charged glutamic acid residue which may mimic phosphorylation at this site (S11E). The modified proteins were translationally fused to green fluorescent protein (GFP) and expressed in transgenic Arabidopsis thaliana. The proteins’ subcellular localization were analysed in planta using laser scanning confocal microscopy (LSCM). Findings in this study point to the peripheral localization of modified and unmodified GFPEgSUSY1 proteins with a prominent cytoplasmic component. No evidence was found for the localization of modified or unmodified GFP-EgSUSY1 proteins within the extracellular matrix. The current study did not establish nor negate plasma membrane association of any of the GFP-EgSUSY1 fusion proteins. It was concluded that alternative methodologies need to be explored to further address issues surrounding subcellular localization of sucrose synthase. These studies will not only aid in defining the role of this enzyme in carbon allocation, but also add to our expanding knowledge of cellulose biosynthesis and cell wall formation. Copyright 2011, University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. Please cite as follows: Sauer, J 2011, The subcellular localization of eucalyptus grandis sucrose synthase 1 (EgSUSY1) fusion proteins expressed in Arabidopsis thaliana, MSc dissertation, University of Pretoria, Pretoria, viewed yymmdd < http://upetd.up.ac.za/thesis/available/etd-02102012-102209 / > C12/4/111/gm / Dissertation (MSc)--University of Pretoria, 2011. / Genetics / unrestricted