Proceedings of the 1986 EPA/APCA Symposium on Measurement of Toxic Air Pollutants /

"EPA Report No. 600/9-86-013"--P. ii.

Biosynthesis of fluorescent allophycocyanin alpha-subunits by autocatalysis in Escherichia coli

APC (allophycocyanin) is widely used for fluorescence tagging and may be a promising antioxidant agent for use within the food and pharmaceutical industries. Chromophore attachment to apo-ApcA (apo-APC alpha-subunit without chromophore) can be auto-catalysed both in vitro and in vivo. In the present study, a plasmid containing genes of apo-ApcA and chromophore synthetases (HOI (ferredoxin-dependent haem oxygenase) and PcyA (phycocyanobilin:ferredoxin oxidoreductase)] was constructed and expressed in Escherichia coli. The results show that holo-ApcA (APC alpha-subunit with chromophore) can be synthesized by autocatalysis in E. coli. Recombinant holo-ApcA showed the same spectral and fluorescent properties as PC (phycocyanin) and could serve as a good substitute for native PC for fluorescent tagging. Moreover, recombinant ApcA can inhibit hydroxyl and peroxyl radicals more strongly than holo-ApcA and native APC. The EC50 values were 296.4 +/- 22.4 mu g/ml against hydroxyl radicals and 38.5 +/- 2.6 mu g/ml against peroxyl radicals.

Recombination and heterologous expression of allophycocyanin gene in the chloroplast of Chlamydomonas reinhardtii

Heterogeneous expression of multiple genes in the nucleus of transgenic plants requires the introduction of an individual gene and the subsequent backcross to reconstitute multi-subunit proteins or metabolic pathways. In order to accomplish the expression of multiple genes in a single transformation event, we inserted both large and small subunits of allophycocyanin gene (apcA and apcB) into Chlamydomonas reinhardtii chloroplast expression vector, resulting in papc-S. The constructed vector was then introduced into the chloroplast of C. reinhardtii by micro-particle bombardment. Polymerase chain reaction and Southern blot analysis revealed that the two genes had integrated into the chloroplast genome. Western blot and enzyme-linked immunosorbent assay showed that the two genes from the prokaryotic cyanobacteria could be correctly expressed in the chloroplasts of C. reinhardtii. The expressed foreign protein in transformants accounted for about 2%-3% of total soluble proteins. These findings pave the way to the reconstitution of multi-subunit proteins or metabolic pathways in transgenic C. reinhardtii chloroplasts in a single transformation event.

Combinational biosynthesis of a fluorescent cyanobacterial holo-alpha-allophycocyanin in Escherichia coli

Allophycocyanin ( APC) is a phycobiliprotein with various biological and pharmacological properties. An expression vector containing five essential genes in charge of biosynthesis of cyanobacterial APC holo-alpha subunit ( holo- ApcA) was constructed, resulting in over- expression of a fluorescent holo- ApcA in E. coli. After being cultured for 16 h, the dry cell density reached 22.5 gl(-1), and the expression of holo- HT- ApcA was up to 1 gl(-1) broth. The recombinant protein showed similar spectral features to native APC.

Biosynthesis of fluorescent cyanobacterial allophycocyanin trimer in Escherichia coli

Allophycocyanin (APC), a cyanobacterial photosynthetic phycobiliprotein, functions in energy transfer as a light-harvesting protein. One of the prominent spectroscopic characteristics of APC is a strong red-shift in the absorption and emission maxima when monomers are assembled into a trimer. Previously, holo-APC alpha and beta subunits (holo-ApcA and ApcB) were successfully synthesized in Escherichia coli. In this study, both holo-subunits from Synechocystis sp. PCC 6803 were co-expressed in E. coli, and found to self-assemble into trimers. The recombinant APC trimer was purified by metal affinity and size-exclusion chromatography, and had a native structure identical to native APC, as determined by characteristic spectroscopic measurements, fluorescence quantum yield, tryptic digestion analysis, and molecular weight measurements. Combined with results from a study in which only the monomer was formed, our results indicate that bilin synthesis and the subsequent attachment to apo-subunits are important for the successful assembly of APC trimers. This is the first study to report on the assembly of recombinant ApcA and ApcB into a trimer with native structure. Our study provides a promising method for producing better fluorescent tags, as well as a method to facilitate the genetic analysis of APC trimer assembly and biological function.

光学活性重组别藻蓝蛋白的初步研究

藻胆蛋白是某些原核和真核藻类中一类重要的捕光天线蛋白，由脱辅基蛋白和开环四吡咯结构的色基共价结合组成。天然藻胆蛋白具有抗肿瘤、抗病毒、抗炎抗氧化等多种生物活性，而借助基因工程技术可以高效表达构效稳定的藻胆蛋白，应用于医药、食品、生化试剂等领域。 本研究在本实验室以前研制的抗肿瘤新药雷普克（rAPC）以及光学活性重组C-藻蓝蛋白α亚基的基础上，将别藻蓝蛋白α亚基基因apcA连入表达载体pCDFDuet-1中，构建了酶催化连接与自主催化连接两种新型载体，使大肠杆菌同时合成脱辅基蛋白与色基，而且二者能够在菌体内结合，表达出具有光学活性的重组别藻蓝蛋白α亚基。以重组菌BL21(DE3)作为研究对象，对酶催化连接的重组蛋白进行了5L规模的发酵，菌体密度OD600值达到23.52，并初步优化了发酵工艺，摸索了如何减少发酵过程中包涵体的生成。 采用金属螯合亲和层析的方法，建立了重组蛋白快速高效的纯化工艺。对比两种重组蛋白的光谱活性，发现藻蓝蛋白裂合酶的存在影响了重组蛋白的荧光吸收特性，推测藻胆蛋白的荧光特性可能是主要由脱辅基蛋白和色基空间构象决定，而不是肽链的一级结构。这些研究结果为解释藻胆蛋白荧光活性的机制提供了线索。

Special issue on the 6th Portuguese Conference on Automatic Control (Controlo 2004)

The Portuguese Association of Automatic Control (APCA) organizes, every two years, the Portuguese Conference on Automatic Control. Its 6th edition (Controlo 2004) was held from 7 to 9 June, 2004 at the University of Algarve, Faro, Portugal, by its Centre for Intelligent Systems (CSI). CONTROLO 2004 International Program Committee (IPC) has decided, from the very start, to ask for submission of full draft papers, to encourage special sessions with well-defined themes, and for student papers. All papers have been reviewed by three separate reviewers. From the 122 contributions submitted, the IPC selected 89 oral papers, 20 special session papers, and 5 student posters. CONTROLO 2004 Technical Programme consists of 33 oral sessions (5 being special sessions) and 1 poster session, covering a broad range of control topics, both from theory and applications. The programme also includes three plenary lectures, given by leading experts in the field, Professors Ricardo Sanz, João Miranda Lemos and Rolf Isermann.

Special section on intelligent control systems and signal processing

The IFAC International Conference on Intelligent Control Systems and Signal Processing (ICONS 2003) was organized under the auspices of the recently founded IFAC Technical Committee on Cognition and Control, and it was the first IFAC event specifically devoted to this theme. Recognizing the importance of soft-computing techniques for fields covered by other IFAC Technical Committees, ICONS 2003 was a multi-track Conference, co-sponsored by four additional Technical Committees: Computers for Control, Optimal Control, Control in Agriculture, and Modelling, Identification and Signal Processing. The Portuguese Society for Automatic Control (APCA) hosted ICONS 2003, which was held at the University of Algarve, Faro, Portugal.

Face recognition robust to head pose from one sample image

Most face recognition systems only work well under quite constrained environments. In particular, the illumination conditions, facial expressions and head pose must be tightly controlled for good recognition performance. In 2004, we proposed a new face recognition algorithm, Adaptive Principal Component Analysis (APCA) [4], which performs well against both lighting variation and expression change. But like other eigenface-derived face recognition algorithms, APCA only performs well with frontal face images. The work presented in this paper is an extension of our previous work to also accommodate variations in head pose. Following the approach of Cootes et al, we develop a face model and a rotation model which can be used to interpret facial features and synthesize realistic frontal face images when given a single novel face image. We use a Viola-Jones based face detector to detect the face in real-time and thus solve the initialization problem for our Active Appearance Model search. Experiments show that our approach can achieve good recognition rates on face images across a wide range of head poses. Indeed recognition rates are improved by up to a factor of 5 compared to standard PCA.

Prise en compte du risque environnemental dans les systèmes irrigués sahéliens : étude de cas au Sénégal

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Prise en compte du risque environnemental dans les systèmes irrigués sahéliens : étude de cas au Sénégal

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Validación de dos herramientas para la evaluación de cambios en las actitudes de alumnos en situaciones frecuentes en cuidado paliativo

Objective: To determine the psychometric properties of two scales designed to examine attitudes regarding palliative care: Comfort Scale in Palliative Care (CSPC, Pereira et al.) and Tanatophobia Scale (TS, Merrill et al.)Method: Seventy-seven students who completed an online course on psychosocial aspects of palliative care offered by the Latin American Association of Palliative Care participated in the study. They also completed the scales before and after the course. Construct validity and reliability of the CSPC and the TS were assessed using a Principal Components Analysis, internal reliability coefficient and test-retest reliability. Further, comparative statistics between the pre-course and post-course results were obtained in order to determine changes in attitudes.Results: The Principal Components Analysis showed satisfactory fit to the data. 3 components were extracted: two for the CSPC and one for the TS, which explained 55.37% of the variance. Internal consistency coefficients were satisfactory in all cases and Cronbach´s Alphas were satisfactory for all the scales, particularly for the CSPC. Test-retest reliability in t1 and t2 was found to be non significant, indicating that measures were not related in time. Regarding pre-course/post-course comparisons, significant changes in comfort assisting patients (p = 0.004) and comfort assisting families (p = 0.001) following the course were identified, but changes in thanatophobia were non significant (p > 0.05).Conclusions: both scales are valid and reliable. Attitudes regarding the practice of palliative care and how they change, particularly regarding psychosocial issues, can be accurately measured using the examined scales.