Localisation of key proteases involved in the assembly and repair of Photosystem II in cyanobacterium Synechocystis sp. PCC 6803.

PhDAll photosynthetic organisms use light as a source of energy, however prolonged excessively high light causes irreversible damage to the main photosynthetic complexes. In particular the D1 polypeptide of Photosystem II is susceptible to damage and must be degraded and replaced. While the concept of PSII repair has attracted intensive research, important details remain to be determined. The sub-cellular localisation of proteases involved in PSII repair and assembly is investigated here in the model cyanobacterium Synechocystis sp. PCC 6803, by employing fluorescent protein tagging and fluorescence imaging in vivo. Results show that all FtsH protease homologues in Synechocystis are localised to distinct regions of the plasma membrane (FtsH1) and thylakoids (FtsH2, FtsH3, FtsH4). Importantly, FtsH2, involved in PSII repair, remains within distinct thylakoid membrane zones when activated by high light, leading to the hypothesis of localised PSII repair centres in the thylakoid membranes. In order to assess composition of the FtsH2-defined membrane zones, a novel technique for isolating membrane sub-fractions by anti-GFP pulldowns was employed. Mass spectrometry identified potentially interacting and neighbouring proteins within the repair centres, whose content changes under different light exposure. Furthermore, observed changes in FtsH2 and FtsH4 distributions under iron and copper deprivation suggest functions in responses to other stress conditions. To find the locations of D1 synthesis during PSII repair and de novo assembly, the D1 C-terminal processing peptidase CtpA was similarly GFP-tagged and observed in vivo. Results suggest that D1 synthesis for PSII repair takes place in the thylakoid membranes, while D1 synthesis for de novo PSII biogenesis takes place in specialised regions at both edges of the thylakoid system, adjacent to the plasma membrane and protruding into the central cytoplasm. By localising crucial cellular enzymes in vivo, this study demonstrates functional compartmentalisation and membrane heterogeneity in a prokaryote.HARVEST Marie Curie ACTION

Empirical Study on the Effects of Acquisition Parameters for FTIR Hyperspectral Imaging of Brain Tissue

Fourier transform infrared (FTIR) spectroscopic imaging is a powerful technique for molecular imaging of pathologies associated with the nervous systems including multiple sclerosis research. However, there is no standard methodology or standardized protocol for FTIR imaging of tissue sections that maximize the ability to discriminate between the molecular, white and granular layers, which is essential in the investigation of the mechanism of demyelination process. Tissue sections are heterogeneous, complex and delicate, hence the parameters to generate high quality images in minimal time becomes essential in the modern clinical laboratory. This article presents an FTIR spectroscopic imaging study of post-mortem human brain tissue testing the effects of various measurement parameters and data analysis methods on image quality and acquisition time. Hyperspectral images acquired from the same region of a tissue using a range of the most common optical and collection parameters in different combinations were compared. These included magnification (4× and 15×), number of co-added scans (1, 4, 8, 16, 32, 64 and 128 scans) and spectral resolution (4, 8 and 16 cm−1). Images were compared in terms of acquisition time, signal-to-noise (S/N) ratio, and accuracy of the discrimination between three major tissue types in a section from the cerebellum (white matter, granular and molecular layers). In the latter case, unsupervised k-means cluster (KMC) analysis was employed to generate images from the hyperspectral images, which were compared to a reference image. The classification accuracy for tissue class discrimination was highest for the 4× magnifying objective, with 4 cm−1 spectral resolution and 128 co-added scans

2D-Raman correlation spectroscopy as a method to recognize of the interaction at the interface of carbon layer and albumin

In modern nanomaterial production, including those for medical purposes, carbon based materials are important, due to their inert nature and interesting properties. The essential attribute for biomaterials is their biocompatibility, which indicates way of interactions with host cells and body fluids. The aim of our work was to analyze two types of model carbon layers differing primarily in topography, and developing their interactions with blood plasma proteins. The first layer was formed of pyrolytic carbon C (CVD) and the second was constructed of multi-walled carbon nanotubes obtained by electrophoretic deposition (EPD), both set on a Ti support. The performed complex studies of carbon layers demonstrate significant dissimilarities regarding their interaction with chosen blood proteins, and points to the differences related to the origin of a protein: whether it is animal or human. However the basic examinations, such as: wettability test and nano sctatch tests were not sufficient to explain the material properties. In contrast, Raman microspectroscopy thoroughly decodes the phenomena occurring at the carbon structures in contact with the selected blood proteins. The 2D correlation method selects the most intense interaction and points out the different mechanism of interactions of proteins with the nanocarbon surfaces and differentiation due to the nature of the protein and its source: animal or human. The 2D correlation of the Raman spectra of the MWCNT layer+HSA interphase proves an increase in albumin β-conformation. The presented results explain the unique properties of the Clayers (CVD) in contact with human albumin

Heterogeneous localisation of membrane proteins in Staphylococcus aureus

The bacterial cytoplasmic membrane is the interface between the cell and its environment, with multiple membrane proteins serving its many functions. However, how these proteins are organised to permit optimal physiological processes is largely unknown. Based on our initial findings that 2 phospholipid biosynthetic enzymes (PlsY and CdsA) localise heterogeneously in the membrane of the bacterium Staphylococcus aureus, we have analysed the localisation of other key membrane proteins. A range of protein fusions were constructed and used in conjunction with quantitative image analysis. Enzymes involved in phospholipid biosynthesis as well as the lipid raft marker FloT exhibited a heterogeneous localisation pattern. However, the secretion associated SecY protein, was more homogeneously distributed in the membrane. A FRET-based system also identified novel colocalisation between phospholipid biosynthesis enzymes and the respiratory protein CydB revealing a likely larger network of partners. PlsY localisation was found to be dose dependent but not to be affected by membrane lipid composition. Disruption of the activity of the essential cell division organiser FtsZ, using the inhibitor PC190723 led to loss of PlsY localisation, revealing a link to cell division and a possible role for FtsZ in functions not strictly associated with septum formation

Photoactivation: The light-driven assembly of the water oxidation complex of Photosystem II

Photosynthetic water oxidation is catalyzed by the Mn4CaO5 cluster of photosystem II. The assembly of the Mn4O5Ca requires light and involves a sequential process called photoactivation. This process harnesses the charge-separation of the photochemical reaction center and the coordination environment provided by the amino acid side chains of the protein to oxidize and organize the incoming manganese ions to form the oxo-bridged metal cluster capable of H2O-oxidation. Although most aspects of this assembly process remain poorly understood, recent advances in the elucidation of the crystal structure of the fully assembled cyanobacterial PSII complex help in the interpretation of the rich history of experiments designed to understand this process. Moreover, recent insights on the structure and stability of the constituent ions of the Mn4CaO5 cluster may guide future experiments. Here we consider the literature and suggest possible models of assembly including one involving single Mn2+ oxidation site for all Mn but requiring ion relocation.Peer reviewedMicrobiology and Molecular Genetic

Study of the human eye lenses analysed by Raman spectroscopy after cataract surgery. Environmental aspects.

Celem pracy było sprawdzenie, czy materiał soczewki oka ludzkiego, uzyskany w wyniku jej usunięcia metodą fakoemulsyfikacji jest dobrym materiałem diagnostycznym oraz uzyskanie informacji o składzie soczewki, strukturze i jej zmianach, które możemy uzyskać badając próbki za pomocą spektroskopii Ramana i EPR. Praca zawiera informacje o wpływie środowiska na oko człowieka oraz o zastosowaniach powyższych metod w badaniach środowiskowych. W pracy opisano budowę i funkcje oka ludzkiego, jej zaburzenia chorobowym procesem katarakty, zabieg fakoemulsyfikacji, aspekty środowiskowe związane z tematem pracy oraz teoretyczne podstawy spektroskopii Ramana i EPR. Przedstawiono wyniki badań dziewięciu próbek, a także wykonano i porównano widma soczewek usuniętych metodą fakoemulsyfikacji i usuniętych metodą tradycyjną. Mapowanie ramanowskie soczewki usuniętej tradycyjnie wskazuje, że zawiera ona fluorescencyjny chromofor. Badania wykazały różnice między próbkami oraz różnice w obrębie próbek. Wyszczególniono dwie frakcje: białą, odznaczającą się wyższym tłem i żółtą, z nową zagregowaną formą białka charakteryzującą stopień zaawansowania choroby. Frakcja żółta zawiera więcej tryptofanu i fenyloalaniny, konformacji α-helisy, a mniej β-kartki,. Widma EPR wskazują na obecność wolnych rodników.Aim of this study was to examine whether the human eye lenses material, obtained by the removal of phacoemulsification is a good diagnostic material and to obtain information on the composition of the lenses, their structure and the modifications that can be obtained using Raman and EPR spectroscopy. The study contains information about the impact of the environment on the human eye and the applications of these methods in environmental research. There is described the structure and functions of the human eye, the cataract disease, phaco surgery, environmental aspects related to the topic of work and the theoretical basis of Raman and EPR spectroscopy. There are presented results of the nine samples, the spectra for samples obtained by phaco surgery were compared with that disposed in the traditional method. Raman mapping of the whole cataractous lens shows the presence of fluorescent chromophore. Studies have shown differences between the samples and within the sample. There was specified two factions: the white characterized by higher background and the yellow with a new form of aggregated protein characterizing the extent and severity of the disease. Yellow fraction contains more phenylalanine, tryptophan, α-helix and less β-sheet conformation. Higher content of tryptophan shows color fraction and protein degradation. The EPR spectra shows the presence of free radicals

The impact of urban climate on human health and well being

The aim of the study was to show the impact of the climate on the health and wellbeing of people living in cities. The essence is to understand the influence of various factors on the human system, which may, in many cases, cause discomfort, health damage and in long term contribute to increased mortality. The first part of the study is focused on characterizing urban climate, its components and factors. To obtain the first goal it was important to take into account each city separately and its factors such as: radiation balance, average air temperature, air humidity, air circulation, precipitation and air pollution. The next part of the study contains the description of various stimuli, that may have either direct or indirect influence on human health. That group of factors includes: photochemical, thermal, chemical and acoustic stimuli. The study includes also a chapter concerning meteoropathy, its reasons, effects and occurrence of meteoropathy diseases of people living in cities. The report also includes the importance of urban greenery in urban climate.Praca ma na celu wykazanie jaki wpływ na zdrowie oraz samopoczucie ludzi żyjących w mieście ma klimat tam panujący. Istotą jest poznanie wpływu poszczególnych czynników na ustrój człowieka, które powodować mogą złe samopoczucie, uszczerbki na zdrowiu czy też przyczyniać się do zwiększenia umieralności. W pierwszej części pracy scharakteryzowano klimat obszarów miejskich, przedstawiono jego składowe oraz opisano, co go kształtuje. Uwzględniono więc bilans promieniowania, temperaturę powietrza, wilgotność powietrza, cyrkulację powietrza, opady atmosferyczne oraz zanieczyszczenia powietrza charakterystyczne dla miast. W następnej części pracy znajduje się opis różnego rodzaju bodźców, działających bezpośrednio i pośrednio na organizm człowieka. Uwzględniono bodźce fotochemiczne, termiczno-wilgotnościowe, chemiczne oraz akustyczne. Praca zawiera rozdział dotyczący meteoropatii, jej przyczyn, skutków oraz występowania chorób meteorotropowych u ludzi żyjących w mieście. Praca uwzględnia również znaczenie zieleni w klimacie miasta

Protective Coatings in the Power Boilers which are Used to Combust Waste – Surfacing Anticorrosive Layers

General requirements and technological solutions of incinerating plants were presented. Heat processing of waste in fluidized and stoker-fired boilers were described. A problem of corrosion and the methods of preventing it in boilers used to combust waste were shown. Materials of increased resistance to corrosion used in power industry were characterised

Protective Coatings in the Power Boilers Used to Incinerate Waste – Fuel Characteristics of Waste as the Source of Energy

A general characteristics of waste management was presented. Municipal waste was characterised and its fuel properties were provided. Numerous thermal processes of waste utilisation were described such as an incinerating process, free-oxygen technology - pyrolysis, technology with oxygen deficiency - gasification and the plasma technology