Abstract C-Phycocyanin (C-PC), a water-soluble protein pigment, is one of the major constituents of Spirulina platenisi,. Which is a blue green algae and used in many countries as dietary supplements.The C-PC used in present study is a phycobiliprotein, and consist of two distinguishable protein subunits designated as £\ and £] subunits. The aim of this study was to screen pharmacological effects including analgesic, anti-inflammatory and hepatoprotective effects of C-PC in an animal model. Concomitantly, the antioxidant effect of C-PC was measured to confirm its free radical scavenger activity. The analgesic effect of C-PC measured by tail-flick test shown the latency (sec) of control mice is approximately 4-5 sec. After administration different doses of C-PC (0.1, 1.0, 10 mg/kg), the latency resulted in a dose-dependent phenomenon and prolong from 4 sec to 4.5, 5.6, and 6.9 sec, respectively. The anti-inflammatory effect of C-PC was analyzed in the £f ¡Vcarrageenan injected rat. The hind paw edema percentage change by injection of different doses (0.1~1.0 mg/kg) of C-PC, our results have indicated that C-PC possessed a dose-dependent inhibitory effect on carrageenan-induced edema formation. Intraperitoneal (i.p.) injection of different dose (0.01,0.1 1, 1.0 mg/kg) of C-PC on CCl4 (0.3 ml/kg) challenged rats have indicated that C-PC possessed a significant hepatotoprotective effect and significant decrease the serum levels of glutamate pyruvate transaminase (SGPT) and serum glutamate oxyloacetic transaminase (SGOT). Antioxidant effect of C-PC was measured by both xanthine oxidase method, and cytochrome c method. The IC50 of C-PC measured by xanthine oxidase method is 7.23 ¡Ó0.21 mg/ml. The IC50 value of C-PC estimated by cytochrome c method is approximately 6.1¡Ó0.74 mg/ml. Moreover, the direct active oxygen (¡PO2-) and hydrogen oxide (¡POH) radical scavenging (SOD-like) activity was also confirmed by using advance electron spin resonance (ESR) method. We used the spin-trapping technique to evaluate free radical scavenging ability of C-PC. The IC50 (¡PO2- ) and (¡POH) scavenging activities are 1.08 ¡Ñ 103 unit/g and 7.11 unit/g. Our results have shown the superoxide radical (¡POOH) scavenging activity of C-PC is approximately 3.28 mg/ml. By contrast, IC50 of the hydroxyl radical (¡POH) scavenging activity of C-PC is 4.75 mg/ml. Human hepatoma cells (HepG2 and 2.2.15 cells) and rat glioma C6 cells were cocultured with different concentration of C-PC (1.0, 10 and 20 £gg/ml)for 72 hours, the cell cycle regulation of C-PC was measured by flow cytometer. We found the C-PC possessed a G0/G1 phase-arrest effect on all of the three kind cancer cell line. Whereas, the cell cycle modulations of C-PC are more significant in human hepatoma cells. In conclusion, both our pharmacological screening tests and antioxidant bioactivity assay have indicated that C-PC is a potential antitumor, anti-inflammatory and analgesic agent.

Les systèmes d'éclairage artificiels sont couramment utilisés pour la croissance des plantes en serre ou environnement contrôlé (culture hydroponique, hors sol, etc...). Leur principale fonction est d'améliorer la qualité et la quantité de la production agricole indépendamment des saisons et de l'ensoleillement. L'utilisation d'une source de rayonnement artificielle pour plantes (PARS pour "Plant Artificial Radiation Sources") signifie que la lumière du soleil n'a pas été l'unique source de lumière pour la production agricole, mais qu'elle a été remplacé ou complété par une source artificielle (PARS). Les serres ou les complexes de culture hydroponique avec éclairage artificiel (Plant Factory with Artificial Lighting, "PFAL"), notamment à LED, sont une technologie innovante pour l'agriculture moderne susceptible de changer fondamentalement certains concepts. Cependant, certaines difficultés persistent avec ces nouvelles techniques. Tout d'abord, par manque de formation ou d'information, certaines personnes ne comprennent pas les caractéristiques techniques des sources de lumière artificielle moderne. Deuxièmement, les mécanismes de photobiologie sous différents spectres sont encore mal définis en fonction des espèces de plantes. Troisièmement, le secteur agricole est une grande entité qui présente également une grande complexité de part la variabilité des cultures. En conséquence, les "PARS" ont généralement une faible efficacité et présentent une consommation énergétique élevée, ce qui devient le principal obstacle pour leur application. Les PARS et leurs systèmes sont la technique de base pour développer l'horticulture contrôlée, en particulier dans la culture hydroponique qui n'utilisent que les sources d'éclairage artificielle. Toutefois, la consommation d'énergie et les défauts de conception deviennent des difficultés rédhibitoires à leur mise en oeuvre. Par conséquent, la sélection d'une source de lumière efficace et l'optimisation des systèmes d'éclairage sont d'une grande importance. Connaître le type de spectre optimal pour une variété de plante ou d'algue nécessite donc des études plus approfondies. Les diodes électroluminescentes (LED) pour l'éclairage constituent une source de lumière de dernière génération compatible avec les puissances lumineuses nécessaires à la croissance de plantes. Par rapport aux sources traditionnelles, elles présentent des avantages incomparables tels qu'un rendement élevé, une longue durée de vie, un rayonnement relativement simple à contrôler par rapport aux sources de lumières classiques, une lumière dite "froide" (pas d'émission infra-rouge), de petite taille, robuste, etc. En outre, les systèmes d'éclairage à LED ("LED Lighting Systems", LLS) utilisent une alimentation en courant continu, ce qui est plus fiable et plus facile à contrôler. Par conséquent, les "LLS" deviennent de plus en plus populaire pour les chercheurs, ingénieurs, fabricants, biologistes et industriels du secteur agronomique. En particulier, les applications des LEDs pour la production agricole suscitent une vive attention dans le monde ces dernières années. Les sources de lumière à LED sont connues comme étant le choix idéal en horticulture sous conditions contrôlées (notamment vis-à-vis de leur faible consommation énergétique). / Artificial lighting systems can be used for plant growth in protected horticulture. Their main function is to improve the quality and quantity of agricultural products. Plant factory and greenhouse with supplemental lighting are the concrete manifestation of protected horticulture. The applications of Plant Artificial Radiation Sources (PARS) mean that the sunlight has not been the unique light source for agricultural production but can be replaced by PARS. Especially, Plant Factory with Artificial Lighting (PFAL) is an innovative technology for modern agriculture that fundamentally change the concept of farming. However, there are some problems for this new technique. First, some people do not understand well the characteristics of artificial light source. Second, photobiology mechanism under different spectra is not clear enough for all the species. Third, agricultural field is a large system of great complexity. As a result, the PARS usually have low efficiency and high energy consumption, which become the main obstacles for plant applications. PARS and their systems are the core technique to develop protected horticulture, especially for plant factory that can only use PARS for photosynthesis and agricultural production. However, the high energy consumption and design flaws become the bottleneck problems. Therefore, how to select the light sources and optimize the lighting systems are of great importance. Which kind of spectrum is optimal for plants or algae needs further study. Light emitting diode (LED) is the fourth (the latest) generation light source. Compared with traditional light sources, it has unparalleled advantages such as high efficiency, long lifetime, flexible spectrum, cool light, small size, robust, etc. Besides, LED lighting systems (LLS) use DC power supply, which is more reliable and easier to control. Therefore, LLS become more and more popular to the researchers, engineers, manufacturers and biologists. Particularly, LED applications for agricultural production also attract broad attention in the world in recent years. LED is known as the ideal choice to spread in the protected horticulture.

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Cyanobacterial algal bloom is a major water quality issue in inland lakes, reservoirs, and estuarine environments because of its scum and bad odor forming and toxin producing abilities. Health risks from cyanobacterial toxin can vary from skin irritations to fever, intestinal problems, and neurological disorders. Terminations of blooms also cause oxygen depletion leading to hypoxia and widespread fish kills. Adding to the problem, many species of cyanobacteria produce odorous compounds such as geosmin and 2-methylisoborneol (MIB) that cause “earthy-muddy” and “musty” odor in drinking water, which is also a serious issue in aquaculture and drinking water industry. Therefore continuous monitoring of cyanobacterial presence in recreational water bodies, surface drinking water sources, and water bodies dedicated for aquaculture is highly required for their early detection and subsequent issuance of a health warning and reducing the economic loss. Remote sensing techniques offers the capability of identifying and monitoring cyanobacterial blooms in a synoptic scale. Over the years, the scientific community has focused on developing methods to quantify cyanobacterial biomass using phycocyanin,an accessory photosynthetic pigment, as a marker pigment. However, because of the confounding influence of chlorophyll-a and other photo pigments, remote retrieval of phycocyanin signal from turbid productive water has been a difficult task. This dissertation analyzes the potential of remote sensing techniques and develops empirical and quasi-analytical algorithms to isolate the phycocyanin signal from the remote sensing reflectance data using a set of radiative transfer equations and retrieves phycocyanin concentration in the water bodies. An extensive dataset, consisting of in situ radiometric measurements, absorption measurements of phytoplankton, colored dissolved organic matter, detritus, and pigment concentration, was used to optimize the algorithms. Validations of all algorithms were also performed using an independent dataset and errors and uncertainties from the algorithms were discussed. Despite the simplicity, an empirical model produced highest accuracy of phycocyanin retrieval, whereas, the newly developed quasi-analytical phycocyanin algorithm performed better than the existing semi-analytical algorithm. Results show that remote sensing techniques can be used to quantify cyanobacterial phycocyanin abundance in turbid and hypereutrophic waters.

Indiana University-Purdue University Indianapolis (IUPUI) / This project examines the impact of confounding variables that have limited the accuracy of remotely predicting phycocyanin in three Indiana drinking and recreational water reservoirs. In-situ field reflectance spectra were collected from June to November 2006 over a wide range of algal bloom conditions using an ASD Fieldspec (UV/VNIR) spectroradiometer. Groundtruth samples were analyzed for chlorophyll a, phycocyanin, total suspended matter, and other water quality constituents. Previously published spectral algorithms for the detection of phycocyanin were evaluated against lab measured pigment concentrations using linear least squares regression. Algorithm performance varied across study sites (best performing models by reservoir resulted in r2 values of 0.32 to 0.84). Residuals of predicted versus measured pigment concentrations were analyzed against concentration of potential confounding water constituents. Residual analysis revealed optically active constituents contributed between 25% and 95% of original phycocyanin model errors. Inclusion of spectral variables into models to account for significant confounders resulted in improved spectral estimates of phycocyanin (r2 = 0.56 to 0.93).

The goal of this research is to identify and characterize enzymes involved in bilin attachment to the phycobiliprotein allophycocyanin in the cyanobacterium Synechococcus sp. PCC 7002. Candidates for lyases responsible for attachment of phycocyanobilin to allophycocyanin are two cpeS-like genes termed cpcS and cpcU, and one cpeT-like gene termed cpcT. In vitro bilin attachment reactions were conducted in the presence of the recombinant substrate apo-allophycocyanin (HT-ApcAB). Size exclusion HPLC showed that CpcS and HT-CpcU form a 1:1 heterodimeric complex and that HT-ApcAB is present as a monomer (áâ). Absorbance and fluorescence spectroscopy illustrated that both CpcS and HT-CpcU were required to get holo-allophycocyanin with phycocyanobilin attached to the cysteine-81 residue. Absorbance of the product at 615 nm was consistent with holo-monomeric allophycocyanin. Experiments were performed with HT-ApcD ApcB and HT-ApcF ApcA, but size exclusion HPLC showed they were in aggregated form.

The goal of this research is to investigate the role of the slr1098 gene in the cyanobacterium Synechocystis sp. PCC 6803, a gene with similarity to cpcE which encodes a subunit of an enzyme involved in bilin attachment to phycocyanin. This protein is hypothesized to be involved in oligomerization of phycocyanin due to previous results showing the mutant made shorter phycocyanin rods. The recombinant Slr1098 protein was produced and purified from E. coli cells. Binding assays showed interaction between Slr1098 and both apo- and holo-phycocyanin, but not to apo-allophycocyanin. Slr1098 blocked bilin addition at Cys-82 on CpcB by the CpcS/CpcU bilin lyase. Size exclusion chromatography and sucrose density gradient analysis of complexes formed suggest that Slr1098 strongly interacts with all intermediate forms of phycocyanin and may be an important checkpoint in the biosynthesis and oligomerization of this protein, but that by itself, Slr1098 does not increase oligomerization of phycocyanin.

Phycoerythrin (PE) present on the outer phycobilisome (PBS) rods in Fremyella diplosiphon contains covalently attached phycoerythrobilin (PEB) chromophores for efficient photosynthetic light capture. Chromophore ligation on phycobiliprotein subunits occurs through bilin lyase catalyzed reactions. The cpeY and cpeZ genes in F. diplosiphon were shown to attach PEB on alph-82 of PE. To better understand the individual functions of cpeY and cpeZ in native cyanobacteria, we characterized PBS and PE purified from cpeY and cpeZ deletion mutants and compared them with wild type (WT). Both cpeY and cpeZ mutants generated much less PE than WT as well as assembling much less PE into the PBS. PE purified from cpeY mutant had phycocyanobilin on alpha-PE in place of PEB. The mutation of cpeZ affected the biosynthesis and accumulation of beta-PE with a red-shifted absorbance compared to WT PE. CpeY was shown to function as a bilin lyase, and CpeZ possibly functions as a chaperone.

Indiana University-Purdue University Indianapolis (IUPUI) / Water is an essential resource for life on Earth, and monitoring its quality is an important task for mankind. However, the amount of water quality data collected by the traditional method is insufficient for the conservation and sustainable management of this important resource. This challenge will be exacerbated by increasing harmful algal blooms at the global scale. To fill this gap, Earth Observations (EO) have been proposed to help stakeholders make their decisions, but the use of EO for monitoring inland water quality is still in development. In this context, the main objective of this study was to improve the estimation of cyanobacteria via remote sensing data. To achieve this goal, the water type classification was first used to identify the dominant optically active constituents within aquatic environments. This information is crucial for understanding the optical properties of inland waters and selecting the best remote sensing algorithm for specific optical water types. The next research question was to develop a universal structure for retrieval of the inherent optical properties of several important aquatic systems around the world, which can be used as a corner stone for developing a globally applicable remote sensing algorithm. The third research topic of this dissertation is about removing the interference of chlorophyll-a with the absorption strength at 620 nm where phycocyanin exhibits its diagnostic absorption so that the estimation of phycocyanin concentration can be improved. Despite the novelty of the proposed remote sensing algorithms which are able to accommodate distinct water optical properties, there are abundant opportunities for improving the parameterization of the proposed models to retrieve inland water quality and optical properties when a global database of optical and water quality measurements is available. Considering the current advancement in spaceborne technology and the existence of a coordinate effort for global calibration and validation of remote sensing algorithms for monitoring inland waters, there is a high potential for operational assessment of harmful cyanobacterial blooms using the remote sensing algorithms proposed in this dissertation.

A multi-plasmid, co-expression system was used to recreate the biosynthetic pathway for phycobiliproteins from the cyanobacterium Synechococcus sp. PCC 7002 in E. coli. This system efficiently produced chromophorylated allophycocyanin (ApcA/ApcB), -phycocyanin, and -phycocyanin. This system was used to demonstrate that CpcS-I and CpcU proteins are both required attaching PCB to allophycocyanin subunits ApcD (AP-B) and ApcF (18). The N-terminal, AP-like domain of ApcE (LCM99) was produced in soluble form and shown to have intrinsic bilin lyase activity. In addition, this system was used to chromophorylated CpcA from Synechococystis sp. PCC 6803 with a non-cognate bilin; PEB with the aid of CpcEF type bilin lyase. However, the CpcSU type lyase displays much higher specificity for PCB (the native bilin in these species) than PEB. Next, using a heterologous, co-expression system in E. coli, the PEB ligation activity of putative lyase subunits CpeY, CpeZ, and CpeS was tested on the CpeA and CpeB subunits from F. diplosiphon. CpeY/CpeZ was found to ligate PEB on CpeA, although CpeY alone had only 60% chromophorylation activity compared to CpeYZ together. Studies with site-directed variants of CpeA (C82S and C139S), revealed that CpeY/CpeZ attached PEB at Cys-82 on HT-CpeA. The CpeS bilin lyase ligated PEB at both Cys-82 and Cys-139 of CpeA, but the yield of attached PEB at Cys 82 was much lower than observed with CpeY or CpeY/CpeZ. However, CpeS efficiently attached PEB to Cys-82 of CpeB. Purified PE from cpeY deletion mutants in F. diplosiphon was found to have PCB added on α-PE instead of PEB, which was likely performed by CpcEF in vivo. However, a cpeZ knock-out mutant is affected in chromophorylation of both  and  subunits of PE with a red-shifted absorbance compared to wild type PE probably due to missing PEB on PE subunits. Next a new type of bilin lyase isomerase for PEII ( subunit) named MpeZ from Synechococcus sp. RS 9916, was analyzed using the E. coli heterologous coexpression system. MpeZ acted as bilin lyase/isomerase chromophorylating α-PEII (MpeA) with PUB on Cys 83.