The impact of a structurally conserved second sphere residue in lytic polysaccharide monooxygenase catalysis

Lytic polysaccharide monooxygenases (LPMOs) are redox enzymes that enhance the degradation of recalcitrant polysaccharides available in biomass. Understanding the catalytic mechanism of LPMOs is of supreme importance in achieving the full potential of LPMOs in biomass conversion into valuable products. However, limited research covers the roles of individual amino acids involved in LPMO catalysis. This master’s thesis work provides insight of the impact of the gatekeeper residue on LPMO catalysis, as the gatekeeper residue is structurally conserved in all LPMOs families. The gatekeeper residue in AfAA11B (E160) and NcAA9C (Q180) was mutated to Glu, Gln, Asp, or Asn to observe the catalytic effect of changing the length and charge of the gatekeeper residue functional group. A gatekeeper residue of carboxylic acid nature introduced a negative charge near the copper center, whereas an amide gatekeeper residue was neutrally charged. The impact of the distance between the copper center and the gatekeeper functional group was assessed when the gatekeeper residue was either Glu or Asp, or Gln or Asn. Altering the length and charge of the gatekeeper side-chain were demonstrated to have a clear impact on several essential features for LPMO catalytic activity. AfAA11B and NcAA9C with a gatekeeper residue with relatively long side-chain of negative charge were identified with low redox potential of the copper site with low peroxidase activity and high oxidase activity. This high oxidase activity was associated with high catalytic activity of substrate oxidation in the presence of O2 and an external reductant during turnover. Substrate turnover with H2O2 supply was most efficient for the AfAA11B with a gatekeeper residue containing a long carboxylic acid functional group in the side chain. Furthermore, altering the electronic properties of the NcAA9C copper center also affected the transportation of radicals generated in the active site from the reaction between NcAA9C-Cu(I) and H2O2 in the absence of a substrate. As such, our results suggest two different routes through the LPMO to transport the oxidative holes. This study demonstrates the importance of the gatekeeper residue in two different LPMO families (AA9 and AA11) with different substrate specificity (cellulose and chitin). The gatekeeper residue appears essential in critical LPMO features such as electronic properties of the copper center, oxidase activity, peroxidase activity, and substrate turnover. Conclusively, this study provides a basis for future studies on the LPMO catalytic mechanisms of recalcitrant polysaccharide degradation.Lytisk polysakkarid monooksygenaser (LPMOer) er redoksenzymer som bidrar til nedbrytning av vanskelig nedbrytbare karbohydrater tilgjengelig i biomasse. Forståelse av den katalytiske mekanismen til LPMOer er essensiell for å oppnå LPMOers fulle potensial i nedbrytningen av biomasse til verdifulle produkter. Likevel er det begrenset forskning som dekker rollene til individuelle aminosyrer involvert i LPMO katalyse. Denne masteroppgaven gir innsikt i «gatekeeper» residuens påvirkning av LPMO katalyse, fordi «gatekeeperen» er konservert i alle LPMO familier. «Gatekeeperen» i AfAA11B (E160) og NcAA9C (Q180) ble mutert til Glu, Gln, Asp eller Asn for å observere den katalytiske effekten av å endre lengden og ladningen til den funksjonelle gruppen i «gatekeeperen». En «gatekeeper» med en karboksylsyre som funksjonell gruppe i sidekjeden introduserte en negativ ladning nær koppersenteret, imens et amid «gatekeeper» residue var nøytralt ladet. Effekten av avstanden mellom koppersenteret og «gatekeeperens» funksjonelle gruppe ble analysert ved å mutere «gatekeeperen» til enten Glu eller Asp, eller Gln eller Asn. Variasjon av lengde og ladning på «gatekeeperens» sidekjede ble vist å påvirke flere essensielle egenskaper avgjørende for LPMO katalytisk aktivitet. AfAA11B og NcAA9C med en «gatekeeper» med en relativt lang, negativt ladet sidekjede ble identifisert med lave redokspotensialer for koppersetet med påfølgende lav peroksidaseaktivitet og høy oksidaseaktivitet. Denne høye oksidaseaktiviteten ble assosiert med høy katalytisk aktivitet ved substratoksidasjon hvor O2 og en ekstern reduktant var til stede. Substratomsetning med tilførsel av H2O2 var mest effektiv for LPMOen med en «gatekeeper» med en lang, karboksylsyre funksjonell gruppe i sidekjeden. Videre påvirket også variasjonen i koppersenterets elektroniske egenskaper for NcAA9C transporten av radikaler generert ved reaksjonen mellom LPMO-Cu(I) og H2O2 i fravær av substrat. Våre resultater antyder at det finnes to ulike transportruter for de frie radikalene gjennom LPMOer. Denne studien demonstrerer viktigheten til «gatekeeperen» i to ulike LPMO familier (AA9 og AA11) med ulik substratspesifisitet (cellulose og kitin). «Gatekeeperen» virker essensiell for viktige egenskaper til LPMOen, slik som elektroniske egenskaper til koppersenteret, oksidaseaktivitet, peroksidaseaktivitet, og substratomsetning. Denne studien gir et grunnlag for videre forskning av LPMOers katalytiske mekanisme for nedbrytning av vanskelige nedbrytbare karbohydrater.M-KJEM

Online Pattern Recognition for the ALICE High Level Trigger

The ALICE High Level Trigger has to process data online, in order to select interesting (sub)events, or to compress data efficiently by modeling techniques.Focusing on the main data source, the Time Projection Chamber (TPC), we present two pattern recognition methods under investigation: a sequential approach "cluster finder" and "track follower") and an iterative approach ("track candidate finder" and "cluster deconvoluter"). We show, that the former is suited for pp and low multiplicity PbPb collisions, whereas the latter might be applicable for high multiplicity PbPb collisions, if it turns out, that more than 8000 charged particles would have to be reconstructed inside the TPC. Based on the developed tracking schemes we show, that using modeling techniques a compression factor of around 10 might be achievableComment: Realtime Conference 2003, Montreal, Canada to be published in IEEE Transactions on Nuclear Science (TNS), 6 pages, 8 figure

Perturbed angular correlations for Gd in gadolinium: in-beam comparisons of relative magnetizations

Perturbed angular correlations were measured for Gd ions implanted into gadolinium foils following Coulomb excitation with 40 MeV O-16 beams. A technique for measuring the relative magnetizations of ferromagnetic gadolinium hosts under in-beam conditions is described and discussed. The combined electric-quadrupole and magnetic-dipole interaction is evaluated. The effect of nuclei implanted onto damaged or non-substitutional sites is assessed, as is the effect of misalignment between the internal hyperfine field and the external polarizing field. Thermal effects due to beam heating are discussed.Comment: 37 pages, 15 figures, accepted for publication in NIM

Configurable Electronics with Low Noise and 14-bit Dynamic Range for Photodiode-based Photon Detectors

We describe the principles and measured performance characteristics of custom configurable 32-channel shaper/digitizer Front End Electronics (FEE) cards with 14-bit dynamic range for use with gain-adjustable photon detectors. The electronics has been designed for the PHOS calorimeter of ALICE with avalanche photodiode (APD) readout operated at -25 C ambient temperature and a signal shaping time of $1 {\mu}s$. The electronics has also been adopted by the EMCal detector of ALICE with the same APD readout, but operated at an ambient temperature of +20 C and with a shaping time of 100ns. The CR-RC2 signal shapers on the FEE cards are implemented in discrete logic on a 10-layer board with two shaper sections for each input channel. The two shaper sections with gain ratio of 16:1 are digitized by 10-bit ADCs and provide an effective dynamic range of 14 bits. Gain adjustment for each individual APD is available through 32 bias voltage control registers of 10-bit range. The fixed gains and shaping times of the pole-zero compensated shapers are defined prior to FEE production by the values of a few R and C components. For trigger purposes, "fast OR" outputs with 12-bit dynamic range are available. FPGA based slave logic, combined with a USB processor supports a variety of remote control and monitoring features, including APD gain calibration. The measurements presented here for APDs at -25 C ambient temperature and $1 {\mu}s$ shaping time achieve an average RMS noise level of 0.25 ADC counts or 290 electrons.The linearity over the dynamic range is better than 1%, as is the uniformity of shaping time and gain over 32 channels. Due to the excellent correspondence of the output pulse shape with offline fit, a differential timing resolution of less than 1.5 ns between channels has been achieved at ca. 2 GeV, i.e. at 1.5% of the dynamic range of PHOS

Photodiode read-out of the ALICE photon spectrometer PbWO4PbWO_{4} crystals

Proposal of abstract for LEB99, Snowmass, Colorado, 20-24 September 1999The PHOton Spectrometer of the ALICE experiment is an electromagnetic calorimeter of high granularity consisting of 17280 lead-tungstate (PWO) crystals of dimensions 22x22x180 mm3, read out by large-area PIN-diodes with very low-noise front-end electronics. The crystal assembly is operated at -25C to increase the PWO light yield. A 16.1x17.1 mm2 photodiode, optimized for the PWO emissio spectrum at 400-500 nm, has been developed. The 20x20 mm2 preamplifier PCB is attached to the back side of the diode ceramic frame. The charge sensitive preamplifier is built in discrete logic with two input JFETs for optimum matching with the ~150pF PIN-diode. A prototype shaper has been designed and built in discrete logic. For a detector matrix of 64 units the measured ENCs are between 450-550e at -25C. Beam tests demonstrate that the required energy resolution is reached.Summary:The PHOton Spectrometer of the ALICE experiment is an electromagnetic calorimeter of high granularity consisting of 17280 lead-tungstate (PWO) crystals of dimensions 22x22x180 mm3, coupled to large-area PIN-diodes with matching low-noise preamplifiers. PHOS is optimized for measuring photons, pi0s and eta mesons in the momentum ranges 0.5-10, 1-10 and 2-10 GeV/c, respectively, and is designed for the expected large number of particles that will be produced in central Pb-Pb collisions. Lead tungstate (PWO) is a fast scintillating crystal with a rather complex emission spectrum, consisting of two components: a blue component peaking at 420 nm and a green component peaking at 480-520 nm. The light yield of PWO at room temperature is low compared with other heavy scintillating crystals, for instance BGO. However, the yield depends strongly on the temperature with a coefficient of ~-2 degree. At the selected operating temperature of -25C the yield is about a factor of 3 higher compared to room temperature. Still, in order to reach the required energy resolution for a PHOS channel, an ENC noise of less than 600e for the PIN-diode-preamplifier-shaper stage is required. This is a very low value taking into account the high capacitance of 150-200 pF of the large area PIN-diodes. In collaboration with the PHOS project, the company AME (Horten, Norway) has designed and produced a PIN-photodiode optimized for the cross-section and spectral responsivity of the PHOS PWO crystal. The photodiode has an active area of 17.1x16.1 mm2 and is fabricated on n-type silicon material of thickness 280 um. The wafer specific resistivity is between 3000 and 6000 ohm-cm, which corresponds to a depletion voltage of 70V. The photodiode response is optimized for the spectral region 400-500 nm in order to match the PWO emission spectrum. The PIN-diode is mounted on a ceramic substrate 0.65 mm thick. On this substrate the diode is surrounded by a ceramic frame. The preamplifier PCB of dimension 20x20 mm2 is attached to the back side of the frame. The PIN-diode and bondings to ground and preamplifier input are protected by an optically transparent epoxy layer. The front side of the PIN-diode is glued onto the endface of the PWO crystal with optically transparent glue (Melt-Mount Quick-Stick, Cargille Laboratories, USA). Each crystal is wrapped in White Tyvek to ensure maximum light collection efficiency and optical insulation between the crystals. The PHOS detector consists of four independent modules, each with 4320 channels. The crystal assembly with the photo detectors are operated at -25 +/- 0.3C. The power dissipation per module is ~1 kW. The charge sensitive preamplifier is an operational amplifier built in discrete logic and with two input JFETs (BF861A). Using two JFETs in parallel gives the lowest noise for detector capacitance >100 pF. A prototype shaper, comprising three amplification stages, has been designed and built in discrete logic. For a PIN-diode with capacitance ~150 pF and a leakage current <1 nA under cooling, calculations give optimum time differentiation and integration constants around 3 microsec. For a detector matrix of 64 units the measured ENCs are between 450-550 e at -25C. Beam tests of this matrix show that the required energy resolution for the PHOS is reached

Real-time TPC Analysis with the ALICE High-Level Trigger

The ALICE High-Level Trigger processes data online, to either select interesting (sub-) events, or to compress data efficiently by modeling techniques. Focusing on the main data source, the Time Projection Chamber, the architecure of the system and the current state of the tracking and compression methods are outlined.Comment: 6 pages, 5 figures, to be published in NIM

Fear of cancer recurrence and its predictive factors among Iranian cancer patients

Fear of cancer recurrence (FOCR) is one of the most important psychological problems among cancer patients. In extensive review of related literature there were no articles on FOCR among Iranian cancer patients. Aim: The aim of present study was to investigation FOCR and its predictive factors among Iranian cancer patients. Materials and Methods: In this descriptive-correlational study 129 cancer patients participated. For data collection, the demographic checklist and short form of fear of progression questionnaire was used. Logistic regression was used to determine predictive factors of FOCR. Result: Mean score of FOCR among participants was 44.8 and about 50% of them had high level of FOCR. The most important worries of participants were about their family and the future of their children and their lesser worries were about the physical symptoms and fear of physical damage because of cancer treatments. Also, women, breast cancer patient, and patients with lower level of education have more FOCR. Discussion: There is immediate need for supportive care program designed for Iranian cancer patients aimed at decreasing their FOCR. Especially, breast cancer patients and the patient with low educational level need more attention

‘I know I'm not invincible’: An interpretative phenomenological analysis of thyroid cancer in young people

Objective. Thyroid cancer is one of the most common cancers affecting young people and carries an excellent prognosis. Little is known about the psychosocial issues that face young people diagnosed with a treatable cancer. This study explored how young people experienced diagnosis, treatment, and how they made sense of an experience which challenged their views on what it means to have cancer. Method. Semi-structured interviews were conducted with eight young people diagnosed with either papillary or follicular thyroid cancer, and analysed with interpre- tative phenomenological analysis (IPA). Results. Two inter-related aspects of their experience are discussed: (1) the range of feelings and emotions experienced including feeling disregarded, vulnerability, shock and isolation; (2) how they made sense of and ascribed meaning to their experience in the light of the unique nature of their cancer. A thread running throughout the findings highlights that this was a disruptive biographical experience. Conclusions. Young people experienced a loss of youthful immunity which contrasted with a sense of growth and shift in life perspective. Having a highly treatable cancer was helpful in aiding them to reframe their situation positively but at the same time left them feeling dismissed over a lack of recognition that they had cancer. The young peoples’ experiences point to a need for increased understanding of this rare cancer, more effective communication from health care professionals and a greater understanding of the experiential impact of this disease on young people. Suggestions to improve the service provision to this patient group are provided

Search for composite and exotic fermions at LEP 2

A search for unstable heavy fermions with the DELPHI detector at LEP is reported. Sequential and non-canonical leptons, as well as excited leptons and quarks, are considered. The data analysed correspond to an integrated luminosity of about 48 pb^{-1} at an e^+e^- centre-of-mass energy of 183 GeV and about 20 pb^{-1} equally shared between the centre-of-mass energies of 172 GeV and 161 GeV. The search for pair-produced new leptons establishes 95% confidence level mass limits in the region between 70 GeV/c^2 and 90 GeV/c^2, depending on the channel. The search for singly produced excited leptons and quarks establishes upper limits on the ratio of the coupling of the excited fermio