Electron transfer by singlet excited state of porphyn and electron acceptor affinity in rigid medium: photoacoustic phase angle analysis

Photoacoustic spectroscopy provides information about both amplitude and phase of the response of a system to an optical excitation process. This paper presents the studies of the phase in the electron transfer process between octaethylporphyn (OEP) and quinone molecules dispersed in a polymeric matrix. It was observed a tendency in the phase behavior to small values only in the spectral region near to 620 nm, while for shorter wavelength did not show any tendency. These measurements suggested that the electron transfer to acceptor occurred with the participation of octaethylporphyn singlet excited state.

Simplified sample handing in mass spectrometry based protein research - focus on protein phosphorylation

The human genome comprises roughly 20 000 protein coding genes. Proteins are the building material for cells and tissues, and proteins are functional compounds having an important role in many cellular responses, such as cell signalling. In multicellular organisms such as humans, cells need to communicate with each other in order to maintain a normal function of the tissues within the body. This complex signalling between and within cells is transferred by proteins and their post-translational modifications, one of the most important being phosphorylation. The work presented here concerns the development and use of tools for phosphorylation analysis. Mass spectrometers have become essential tools to study proteins and proteomes. In mass spectrometry oriented proteomics, proteins can be identified and their post-translational modifications can be studied. In this Ph.D. thesis the objectives were to improve the robustness of sample handling methods prior to mass spectrometry analysis for peptides and their phosphorylation status. The focus was to develop strategies that enable acquisition of more MS measurements per sample, higher quality MS spectra and simplified and rapid enrichment procedures for phosphopeptides. Furthermore, an objective was to apply these methods to characterize phosphorylation sites of phosphopeptides. In these studies a new MALDI matrix was developed which allowed more homogenous, intense and durable signals to be acquired when compared to traditional CHCA matrix. This new matrix along with other matrices was subsequently used to develop a new method that combines multiple spectra from different matrises from identical peptides. With this approach it was possible to identify more phosphopeptides than with conventional LC/ESI-MS/MS methods, and to use 5 times less sample. Also, phosphopeptide affinity MALDI target was prepared to capture and immobilise phosphopeptides from a standard peptide mixture while maintaining their spatial orientation. In addition a new protocol utilizing commercially available conductive glass slides was developed that enabled fast and sensitive phosphopeptide purification. This protocol was applied to characterize the in vivo phosphorylation of a signalling protein, NFATc1. Evidence for 12 phosphorylation sites were found, and many of those were found in multiply phosphorylated peptides

Selection of microorganisms producer of lipase for fat removal from biodiesel purification water

The objective this study has been the selection of lipase productor microorganism, for removal of oils and grease, in the pre-treatment of biodiesel wastewater washing. For this, analyses of the physicist-chemistries characteristics had been made with the wastewater of the biodiesel washing, and then it had been isolated and chosen, by means of determinations of the lipase activity. Following, it was made a test of fat biodegradation, in the conditions: pH (5.95), temperature (35 ºC), rotation (180 rpm) and ammonium sulfate as nitrogen source (3 g L-1) and establishing as variable the two microorganism preselected and the time (24; 48; 72; 96 and 120 h). The biodiesel purification wastewater had presented high potential of environmental impact, presenting a concentration of O of 6.76 g L-1. From the six isolated microbiological cultures, two microorganisms (A and B) had been selected, with enzymatic index of 0.56 and 0.57, respectively. The treatment of the wastewater using the isolated microorganism (Klebsiella oxytoca) had 80% of the fatty removal in 48 h.

TANDEM-trauma

Kirjallisuusarvostelu

TANDEM demokratiasta

Kirjallisuusarvostelu

Jauhekaari- ja tandem-MIG/MAG-hitsauksen vertailu

Tässä kanditaatintyössä vertaillaan jauhekaari- ja tandem-MIG/MAG-hitsausta kirjallisuustutkimuksena. Vertailun kohteena ovat hitsausprosessien tuottavuus ja kustannukset sekä hitsausprosesseilla saavutettava laatu. Lisäksi kandidaatintyössä esitetään hitsausprosesseille tyypilliset käyttökohteet CASE -esimerkkeinä.

Multiple forms of cotyledonary b-galactosidases from Vigna unguiculata quiescent seeds

Cotyledonary b-galactosidases were isolated and partially purified from Pitiúba cowpea (Vigna unguiculata (L.) Walp.) quiescent seeds. The purification steps consisted of precipitation of the crude extract with ammonium sulphate in the range of 20-60% saturation, acid precipitation, DEAE-Sephadex ion-exchange chromatography and Lactosyl-Sepharose affinity chromatography. This purification process gave rise to three b-galactosidases-rich fractions: b-gal I, b-gal II and b-gal III, which were purified about 5, 509, and 62 fold, respectively. They reached maximal enzyme activity at different pH ranges: 3.5-4.5 for b-gal I, 3.0-3.5 for b-gal II, and 3.0-4.0 for b-gal III. Their maximal activities were reached when the temperature of the assay medium was 60° C, and preincubation of the enzymes at different temperatures has shown that they were heat-stable up to 50° C. There were no significant differences among the partially purified enzymes as far as their response to the different effectors tested, except for Mn2+ and EDTA, which affected differently b-gal I, b-gal II, and b-gal III. They were slightly affected by Mg2+, Ca2+, Zn2+, Co2+, tartarate, molybdate, glucose, and lactose, strongly inhibited by Cu2+ and galactose, and inactivated by Hg2+. These chemical and physical properties are similar to the ones found for other plant b-galactosidases. Although through this process of purification three isoforms of this enzyme were obtained, isoelectric focusing in polyacrylamide slab gel of these enzyme-proteins suggest that cotyledons of Pitiúba cowpea quiescent seeds possess four isoforms of b-galactosidases.

Hitsauslankojen kallistuskulmien vaikutus hitsausliitoksen ominaisuuksiin S355-rakenneteräksen tandem-MAG-pienahitsauksessa

Tämä kandidaatintyö on tutkimus hitsauslankojen kallistuskulmien vaikutuksesta hitsausliitoksen ominaisuuksiin tandem-MAG-pienahitsauksessa S355-rakenneteräkselle. Kokeellinen osuus suoritettiin Lappeenrannan teknillisen yliopiston hitsaustekniikan laboratoriossa jatkotutkimuksena Binbin Yanin diplomityölle ”Influence of wire configurations of welding S355 structural steel with tandem GMAW”. Tutkimuksen tavoitteena on määrittää tandem-MAG-hitsauksessa käytettyjen hitsauslankojen kallistuskulmien vaikutus hitsausliitoksen ominaisuuksiin. Teoriaosuuden tavoitteena on selvittää myös muiden hitsausparametrien vaikutus hitsausliitoksen ominaisuuksiin sekä perehtyä hitsisulan käyttäytymiseen terästen tandem-MAG-hitsauksessa.

Purification of Hydrocarbon Waste Streams

Purification of hydrocarbon waste streams is needed to recycle valuable hydrocarbon products, reduce hazardous impacts on environment, and save energy. To obtain these goals, research must be focused on the search of effective and feasible purification and re-refining technologies. Hydrocarbon waste streams can contain both deliberately added additives to original product and during operation cycle accumulated undesired contaminants. Compounds may have degenerated or cross-reacted. Thus, the presence of unknown species cause additional challenges for the purification process. Adsorption process is most suitable to reduce impurities to very low concentrations. Main advantages are availability of selective commercial adsorbents and the regeneration option to recycle used separation material. Used hydrocarbon fraction was purified with various separation materials in the experimental part. First screening of suitable materials was done. In the second stage, temperature dependence and adsorption kinetics were studied. Finally, one fixed bed experiment was done with the most suitable material. Additionally, FTIR-measurements of hydrocarbon samples were carried out to develop a model to monitor the concentrations of three target impurities based on spectral data. Adsorption capacities of the tested separation materials were observed to be low to achieve high enough removal efficiencies for target impurities. Based on the obtained data, batch process would be more suitable than a fixed bed process and operation at high temperatures is favorable. Additional pretreatment step is recommended to improve removal efficiency. The FTIR-measurement was proven to be a reliable and fast analysis method for challenging hydrocarbon samples.

Single-step purification of crotapotin and crotactine from Crotalus durissus terrificus venom using preparative isoelectric focusing

We describe the isolation of crotoxin, a presynaptic B-neurotoxin, as well as its subunits B (crotactine) and A (crotapotin) from lyophilized Crotalus durissus terrificus venom by a single-step preparative isoelectric focusing procedure. From 98 mg of dried venom protein 20.1 mg of crotactine and 13.1 mg of crotapotin were recovered in the first step of focalization and 4.2 mg in a second run. These values correspond to 35.7% of the total venom protein applied. Crotactine separated in the 9.3-7.0 pH range (tubes 1-6) and crotapotin in the 1.8-2.8 pH range (tubes 15-19) and both were homogeneous by SDS-PAGE and N-terminal amino acid analysis. Crotactine, a 12-kDa protein, presented hemolytic and phospholipase A2 activity. Thus, using isoelectric focusing we simultaneously purified both toxins in high yields. This method can be used as an alternative for the purification and characterization of proteins from other snake venoms under conditions in which biological activity is retained

Large-scale production and purification of recombinant protein from an insect cell/baculovirus system in Erlenmeyer flasks: application to the chicken poly(ADP-ribose) polymerase catalytic domain

A simple and inexpensive shaker/Erlenmeyer flask system for large-scale cultivation of insect cells is described and compared to a commercial spinner system. On the basis of maximum cell density, average population doubling time and overproduction of recombinant protein, a better result was obtained with a simpler and less expensive bioreactor consisting of Erlenmeyer flasks and an ordinary shaker waterbath. Routinely, about 90 mg of pure poly(ADP-ribose) polymerase catalytic domain was obtained for a total of 3 x 109 infected cells in three liters of culture

Purification of bovine pancreatic glucagon as a by-product of insulin production

A process for purifying bovine pancreatic glucagon as a by-product of insulin production is described. The glucagon-containing supernatant from the alkaline crystallization of insulin was precipitated using ammonium sulfate and isoelectric precipitation. The isoelectric precipitate containing glucagon was then purified by ion-exchange chromatography on Q-Sepharose FF, gel filtration on Sephadex G-25 and ion-exchange chromatography on S-Sepharose FF. A pilot scale test was performed with a recovery of 87.6% and a purification factor of 8.78 for the first chromatographic step, a recovery of 75.1% and a purification factor of 3.90 for the second, and a recovery of 76.2% and a purification factor of 2.36 for the last one. The overall yield was 50%, a purification factor of 80.8 was obtained and the fraction containing active glucagon (suitable for pharmaceutical preparations) was 84% pure as analyzed by HPLC

Cloning, expression and purification of recombinant streptokinase: partial characterization of the protein expressed in Escherichia coli

We cloned the streptokinase (STK) gene of Streptococcus equisimilis in an expression vector of Escherichia coli to overexpress the profibrinolytic protein under the control of a tac promoter. Almost all the recombinant STK was exported to the periplasmic space and recovered after gentle lysozyme digestion of induced cells. The periplasmic fraction was chromatographed on DEAE Sepharose followed by chromatography on phenyl-agarose. Active proteins eluted between 4.5 and 0% ammonium sulfate, when a linear gradient was applied. Three major STK derivatives of 47.5 kDa, 45 kDa and 32 kDa were detected by Western blot analysis with a polyclonal antibody. The 32-kDa protein formed a complex with human plasminogen but did not exhibit Glu-plasminogen activator activity, as revealed by a zymographic assay, whereas the 45-kDa protein showed a Km = 0.70 µM and kcat = 0.82 s-1, when assayed with a chromogen-coupled substrate. These results suggest that these proteins are putative fragments of STK, possibly derived from partial degradation during the export pathway or the purification steps. The 47.5-kDa band corresponded to the native STK, as revealed by peptide sequencing