13 research outputs found
Secondary zoospores in the algal endoparasite Maullinia ectocarpii (Plasmodiophorea).
No full textThe present paper deals with the ultrastructure of zoospores produced by the plasmodiophorid Maullinia ectocarpii , living in the marine algal host Ectocarpus siliculosus. The zoospores described here are very similar to secondary zoospores of Polymyxa graminis and Phagomyxa sp. (the latter an algal endopara- site, also). Our results indicate that M. ectocarpii produces two types of plasmodia, and suggest that is a species with a complete life cycle, as it is known for all the Plasmodiophormycota that have been studied. Sporogenic and sporangial plasmodia produce, respectively, primary zoospores with parallel flagella within thick walled resting sporangia, and secondary zoospores with opposite flagella within thin walled sporangia.Fil: Parodi, Elisa Rosalia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Sur; ArgentinaFil: Caceres, Eduardo Jorge. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Laboratorio de Ficología y Micología; ArgentinaFil: Westermeier, Renato. Universidad Austral de Chile; ChileFil: Muller, Dieter G.. Universität Konstanz; Alemani
A HIGH RESOLUTION TEMPERATURE CLIMATOLOGY FOR THE GREATER ALPINE REGION (GAR)
No full textThe Greater Alpine Region (the GAR) covering the area between 4-19°E and 43-50°N and an altitude range between 0 and more than 4000 m asl. offers a challenging climate worth to be studied in any detail. However, it is surprising that up to now no comprehensive Alpine Temperature Climatology covering the whole region is existing. To overcome this deficiency as a first step we want to produce monthly temperature maps for this region in spatial resolution as high as possible. The period under investigation will be 1961-1990. In this paper we will describe the first steps of our initiative as well as the further plans
Avaliação da produção de alginato por Pseudomonas mendocina
No full textDissertação (mestrado) - Universidade Federal de Santa Catarina, Centro Tecnológico, Programa de Pós-Graduação em Engenharia de Alimentos, Florianópolis, 2011O alginato é um exopolissacarídeo constituído de quantidades variáveis de ácidos ?-D-manurônico e seu epímero C-5 ácido ?-L-gulorônico, unidos por ligações ?-1,4 glicosídicas. É extensamente utilizado na indústria de alimentos e biotecnológica. Atualmente a demanda do alginato para estas aplicações é suprida a partir da sua extração de algas marinhas, entretanto vários estudos reportam a produção de alginato por micro-organismos do gênero Pseudomonas e Azotobacter. A produção bacteriana de alginato apresenta-se como uma alternativa interessante e sua produção por micro-organismos, além de possibilitar a produção de biopolímeros de alta qualidade com características específicas e pré-determinadas, irá diminuir o impacto ambiental nas regiões em que as algas marinhas das quais é extraído são coletadas. Nos últimos anos, vários estudos relacionados à produção de alginato por micro-organismos foram realizados com o objetivo de avaliar sua produção e rota metabólica de biossíntese, para caracterizar o material produzido e para determinar as potencialidades de aplicação deste novo material. O conhecimento sobre a via metabólica de um organismo permite a compreensão da fisiologia celular e regulação de seu metabolismo, sendo a quantificação de fluxos metabólicos um importante objetivo, principalmente no que diz respeito à obtenção de produtos úteis comercialmente ou cientificamente. Neste estudo foi avaliado o efeito da relação C:N na produção de alginato. Foram realizados experimentos em frascos agitados a 240 rpm e 30°C, em meios contendo glicerol como fonte de carbono nas concentrações iniciais de 20 a 40 g.L-1 e (NH4)2SO4 como fonte de nitrogênio em concentrações de 0,5 a 2,0g.L-1. Fluxos extracelulares, medidos experimentalmente, foram utilizados para estimar fluxos intracelulares no metabolismo do glicerol em Pseudomonas mendocina na biossíntese de alginato. Foram utilizados modelos estequiométricos e técnicas de balanço de massa, conhecidas como Análise de Fluxo Metabólico (MFA). Para avaliação foi considerado o Estado Pseudo-estacionário (PSS). O objetivo foi avaliar o efeito das concentrações da fonte de carbono e da fonte de nitrogênio no fluxo de carbono. Os resultados indicaram um maior rendimento de bioconversão de substrato em alginato em concentrações menores de nitrogênio, sugerindo que nessas condições P. mendocina utiliza a fonte de carbono principalmente para produção de alginato. Assim, o aumento da relação C:N favorece a produção de alginato, diminuindo o fluxo de carbono na via Entner-Dourdoroff e o crescimento celular.Alginate is an exopolysaccharide consisting of variable amounts of ?-D-mannuronic acid and its C5-epimer ?-L -guluronic acid linked via ?-1,4-glycosidic bonds. It is widely used in applications in the food and biotechnology industries. Currently the demand for these applications of alginate is supplied from the extraction of seaweed, however several studies report the production of alginate by micro-organisms of the genus Pseudomonas and Azotobacter. The bacterial production of alginate appears to be an interesting alternative and its production by micro-organisms, besides enabled the production of high quality polymers with specific characteristics and pre-determined, will reduce the environmental impact in areas where the seaweed is collected. In recent years, several studies relating the production of alginate by micro-organisms were carried out to evaluate their production and metabolic pathway of biosynthesis, to characterize the material produced and to determine the potential application of this new material. The knowledge about the metabolic pathway of an organism allows the understanding of cell physiology and regulation of their metabolism. The quantification of metabolic fluxes is an important goal, specially with regard to obtaining commercially or scientifically useful products. This study assessed the effect of C: N ratio in the production of alginate. Experiments were performed in shaken flasks at 240rpm and 30°C in medium containing glycerol as carbon source at initial concentrations of 20, 30 and 40 g.L-1 and (NH4)2SO4 as a nitrogen source at initial concentrations of 0,5; 1,0; 1,5 and 2,0 g.L-1. Extracellular fluxes, measured experimentally, were used to estimate intracellular fluxes of glycerol metabolism in Pseudomonas mendocina in the biosynthesis of alginate. It was used stoichiometric models and mass balance techniques, known as metabolic flux analysis (MFA). For evaluation was considered the pseudo-steady state (PSS). The objective was to evaluate the effect of concentrations of carbon and nitrogen sources in the flow of carbon. The results indicated a higher yield of bioconversion of substrate in alginate in lower concentrations of nitrogen, suggesting that in these conditions P. mendocina uses the carbon source mainly for alginate production. Thus, increased C:N ratio favors the production of alginate, decreasing the flow of carbon into the Entner-Dourdoroff pathway and cell growth
Proteomic analysis of inflammatory protein expression patterns in cell culture and transgenic animal models for Alzheimer's disease
No full textDementia is a syndrome characterized by failure of recent memory and other cognitive functions that is usually insidious in onset but steadily progresses with age. Alzheimer’s disease (AD) is the most common form of senile dementia. It is neuropathologically characterized by extracellular and perivascular aggregation of amyloid β (Aβ) peptide, by the generation of intracellular neurofibrillary tangles due to a hyperphosphorylation of tau protein and by an increased rate of neuronal degeneration. The degenerative process starts 20-30 years before the clinical onset of the disease. Clinical diagnosis of AD is difficult but possible, but can only be confirmed by biopsy or autopsy. At present, no biological marker exists for early diagnosis of AD during life. Therefore, identification of biomarkers for AD would be of great value for clinical diagnosis of incipient AD. Recent studies have proven the involvement of inflammatory processes in the neurodegenerative events in AD. Inflammation may not be the first event in the progression of the disease, but it involves activation of glia cells including microglia and astrocytes and subsequent release of proinflammatory mediators. Cytokines released such as IL-1, TNF-α and IL-6 are the main proinflammatory cytokines that can modulate inflammatory responses as well as glial proliferation and activation. Oxidative stress triggered by inflammatory processes causes changes in proteins such as tyrosine nitration or lipid peroxidation. Aβ deposits, tau hyperphosphorylation, inflammation and oxidative stress may finally lead to changes in synaptic connectivity and efficacy including perturbation of long-term potentiation (LTP), important in the formation of memory. Proteomic technology used in these studies is a recent technology which is a two step process: separation of proteins and their subsequent analysis by mass spectrometry. Moreover, this technology can provide new information concerning the expression level, post-translational modification of specific proteins as well as their conformational changes during disease progression. In our study, this technology was modified and improved, e.g by the miniaturization of the complete process. Proteomic technology was also used in parallel with other methods such as chromatography in order to increase the sensitivity of detection by mass spectrometry.
This study aimed:
1) To establish that cytokine treatment of human microglia cells is an efficient method to
study certain aspects of AD pathogenesis. For this analysis, a map of protein expression
in normal and in treated microglia cells was made.
2) To map protein expression in APP/PS2 transgenic mice, a model for human AD, in
order to compare human AD brain with murine models.
3) To identify highly nitrated proteins in brains of transgenic animals. Several proteins
were found to be modified after injury.
4) To provide evidence for instability of synapses in AD brains. To start with this study,
the technologies used to map mouse brain cytosolic proteins were improved.
5) To isolate synaptosomal membranes from the whole brain and to analyse it by massspectrometry.
For mapping synaptic membrane protein expression in controls or
transgenic mouse models, the technology was miniaturizated and optimized. This study is
still in progress
Respostas bioquímicas e moleculares em mexilhões Perna perna (Linné, 1758) expostos ao óleo diesel
No full textDissertação (mestrado) - Univesidade Federal de Santa Catarina, Centro Tecnológico. Programa de Pós-Graduação em Biotecnologia
Nucleases sintéticas: caracterização bioquímica e mecanismo de ação sobre DNA
No full textTese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Físicas e Matemáticas. Programa de Pós-Graduação em QuímicaFoi estudada a interação de cinco complexos metálicos de Cu2+ com moléculas de DNA. Foram estudados o complexo macrocíclico Cu2BMXD, o complexo mononuclear CuMFF e os complexos binucleares Cu2L-dtb, Cu2L-H e Cu2L-NO2 (estes diferenciam-se por substituições no anel fenólico terminal do ligante). Os estudos foram feitos pela incubação com o plamídio pBSK-II em diferentes condições e presença de inibidores e ligantes específicos do DNA. A constante de ligação intrínseca dos complexos com o DNA foi determinada por titulação UV-Vis. Todos os complexos foram capazes de causar quebras no DNA. O complexo Cu2BMXD tem uma grande afinidade por DNA, sendo ativo na degradação oxidativa destas moléculas através de um ciclo catalítico que envolve a formação de Cu+ e radicais HO×. O complexo CuMFF possui menor afinidade pelo DNA, mas é capaz de clivar estas moléculas hidroliticamente. Já os complexos Cu2L-X apresentam um mecanismo misto de degradação de DNA, sendo o Cu2L-dtb o mais ativo, seguido do Cu2L-NO2 e Cu2L-H. O complexo Cu2L-dtb é o que apresenta a maior tendência de degradação oxidativa do DNA. Entretanto, para todos os complexos a via hidrolítica é aumentada com o aumento do pH. A constante de ligação intrínseca destes complexos com o DNA é menor do que para os anteriores, porém, parece ser ela a responsável pela diferença de atividade encontrada entre os complexos Cu2L-H e Cu2L-NO2
Unravelling the mechanisms of resistance to imatinib mesylate in chronic myeloid leukemia: a proteomic approach
No full textImatinib mesylate is a potent inhibitor of the Bcr-Abl tyrosine kinase, an oncoprotein that plays a key role in the development of chronic myeloid leukemia. Consequently, imatinib is used as front-line therapy for this disease. A major concern in imatinib treatment is the emergence of resistance to the drug. The aim of this study was to obtain further insights into the Bcr-Abl activity-independent mechanisms underlying imatinib resistance, in chronic myeloid leukemia. The imatinib-resistant KCL22R and sensitive KCL22S cells were used as experimental model. None of the already described resistance mechanisms has been detected so far in KCL22R cells; therefore additional mechanisms independent of Bcr-Abl kinase activity could be envisaged. Moreover, KCL22S cells exhibited typical features of the quiescent hematopoietic Ph+ stem cells, thereby representing a good experimental model to investigate imatinib resistance. To this aim differentially expressed proteins between KCL22S and KCL22R cells were characterized using a proteomic approach: two-dimensional differential gel electrophoresis (2D-DIGE) coupled with Tandem Mass Spectrometry. 51 proteins were identified: 27 over-expressed and 24 under-expressed in KCL22R cells versus KCL22S cells. Bioinformatic analysis with GeneSpring and Ingenuity Pathway Analysis (IPA) softwares showed that several of these proteins were involved in the modulation of redox balance and activation of anti-apoptotic pathways mediated by NF-kB and Ras-MAPK signaling. Since the Erk pathway has been shown to influence chemotherapeutic drug resistance of hematopoietic cells, the level of activation of Erk in KCL22R and KCL22S cells was investigated. This analysis demonstrated that continuous activation of Erk occurred in KCL22R cells as compared to sensitive cells. Interestingly, examination of the most statistically significant protein network showed that several differentially expressed proteins, between KCL22R and KCL22S cells, were directly or indirectly connected with Erk. In particular, among them, this study focused on two SH2-containing, non-receptor protein tyrosine phosphatases: Shp1 (PTPN6) and Shp2 (PTPN11). It has been shown that Shp2 positively regulates the Ras-Erk pathway and is activated by phosphorylation. This study demonstrated that the level of phosphorylation and hence of activation of Shp2 in KCL22R cells was higher than in KCL22S cells. In addition the knock-down of Shp2 expression, in combination with imatinib treatment, significantly reduced the activation of Erk 1/2 in KCL22R cells and produced a reversion of the KCL22R phenotype, suggesting that Shp2 plays a role in the Bcr-Abl activity-independent mechanisms of imatinib resistance. Interestingly this study also demonstrated that Shp1, that was found down-regulated in KCL22R cells, interacted with Shp2 and that Shp1 played a negative role in the Shp2 activation in KCL22S cells. Moreover Annexin A1 and Hsp70, belonging to the same protein network, were found down-regulated in KCL22R cells. They could also play a role in imatinib resistance by the direct or indirect interaction with Shp2. Taken together these results suggest that a reduced Shp1 expression in KCL22R cells could contribute to a continuous Shp2 activation, sustaining a Bcr-Abl activity-independent pathway of proliferation and survival to imatinib treatment. These two proteins could be used as putative biomarkers to evaluate the efficacy of imatinib treatment and to develop new combinatorial therapeutic approaches
A proteomic analysis of lipid raft and GPI anchored proteins in Caenorhabditis elegans
No full textGlycosylphosphatidylinositol (GPI) anchored proteins are a unique group of membrane proteins found on the surface and certain intracellular compartments of eukaryotic cells. They are bound to the membrane by a GPI moiety and have a
number of important functions, including digestion, endocytosis and signal transduction. GPI anchored proteins also reside within lipid rafts, which are microdomains on the phospholipid bilayer composed of sphingolipids and cholesterol. Rafts are thought to be capable of forming semi-stable “islands”of lipids and proteins that act as a platform for a number of important cellular processes, such as T-cell activation, caveolin mediated endocytosis and protein compartmentalisation. The majority of research into rafts has been carried out in single cellular organisms or cell cultures, and their importance within development has been poorly understood.
In this project a proteomic analysis of lipid raft and GPI anchored proteins was made for the proteome of the model organism Caenorhabditis elegans. We found a total of
327 predicted GPI anchored proteins from the C. elegans genome via a novel four-program prediction method and validated three of those proteins with mass spectrometric (MS) identification. The GPI biosynthesis pathway genes of C. elegans were also elucidated via a bioinformatics search. 41 lipid raft proteins were identified
using MS, which accounts for the largest number of such proteins found in the worm. This project will hopefully become a starting point for the research of GPI anchored
proteins and lipid rafts within the nematode, and shine a light on the properties of these important classes of proteins within the context of a developmentally complex
organism
Identification and characterisation of the Arabidopsis thaliana cell wall proteome: unravelling novel cell wall proteins and new potential functions of the plant extracellular matrix
No full textThe application of the proteomic approach has facilitated efforts directed toward the mapping of the entire Arabidopsis cell wall proteome. Proteins were sequentially extracted from purified cell walls using 0.2 M CaC1(_2) followed by a urea buffer. The extracts were resolved via large format two dimensional polyacrylamide gel electrophoresis (2-D PAGE) and were visualised via Coomassie brilliant blue staining. The aim was to identify and characterise as many cell wall proteins as possible, with the hope of identifying novel cell wall proteins. Out of 325 spots visualised on the 2-D polyacrylamide gel, 144 gave a positive protein identification representing 104 different proteins. The identified proteins were divided into 3 categories. The first category included proteins that have been previously identified as plant cell wall proteins. The second category was designated to include novel cell wall proteins (hypothetical proteins) and the third category was made up of proteins, which had recognised functions, but had never hitherto been known to be secreted to the extracellular matrix. Among the identified novel cell wall proteins there were several that shared high homology with protein kinases. These proteins possessed all the characteristics of secreted polypeptides, such as the cleavable N-terminal signal peptide, and were found to lack both the transmembrane domain and the endoplasmic reticulum retention tetrapeptides (HDEL and KDEL). These observations suggested that, as in animal cells, plant cells had extracellular protein kinase activity (phosphorylation). This was supported by the recent discovery that plant cells secrete ATP to the extracellular matrix (Thomas et al., 2000). Verification of the occurrence of extracellular protein kinase activity was further strengthened by the identification of phosphorylated bona fide cell wall proteins and stress responses caused by the depletion extracellular ATP
