Diss. (sammanfattning) Linköping : Linköpings universitet, 2005. / Härtill 7 uppsatser.

<p>This thesis covers studies on endogenous proteolytic enzymes and their impact on fish muscle proteins and texture. The studies have been performed using Atlantic salmon (<i>Salmo salar</i>) and cod (<i>Gadus morhua</i>) subjected to different treatments and storage conditions.</p><p>The textural properties were very different in the two species. Salmon fillets were significantly softer and less resilient than cod fillets, and the properties changed somewhat differently during storage experiments. Different proteolytic enzymes have been reported to participate in muscle softening. Some of these enzymes were investigated, and specific proteolytic activities were detected throughout the storage periods. Collagenase-like enzymes seem to be the most important for cod muscle texture. Microorganisms and/or microbial enzymes seem not to be important for changes in salmon muscle texture. Results suggest that the cathepsin B-like enzymes are important for salmon texture. The activities of the proteolytic enzymes may be greatly affected by the muscle pH, and by the treatment(s) the fish are subjected to. In any case, changes caused by differences in proteolytic activities may need some time to be detectable or have significant impact on fish quality.</p><p>When cod fillets are stored in ice, it is highly recommended to keep the temperature low. Even a relatively mild temperature abuse was sufficient to result in less favorable textural characteristics, and make the fillets seem older than their days of storage.</p><p>Salmon fillets are often subjected to cold-smoking. The smoking temperature was important for the solubility properties of the muscle proteins, and for their composition, but did not affect the proteolytic activity. The effects of the processing parameters were most important early in the product’s shelf life, as the differences caused by the different smoking temperatures were reduced by further storage of the smoked samples.</p> / Paper II and III are reprinted with kind permission of Elsevier, sciencedirect.com

This thesis covers studies on endogenous proteolytic enzymes and their impact on fish muscle proteins and texture. The studies have been performed using Atlantic salmon (Salmo salar) and cod (Gadus morhua) subjected to different treatments and storage conditions. The textural properties were very different in the two species. Salmon fillets were significantly softer and less resilient than cod fillets, and the properties changed somewhat differently during storage experiments. Different proteolytic enzymes have been reported to participate in muscle softening. Some of these enzymes were investigated, and specific proteolytic activities were detected throughout the storage periods. Collagenase-like enzymes seem to be the most important for cod muscle texture. Microorganisms and/or microbial enzymes seem not to be important for changes in salmon muscle texture. Results suggest that the cathepsin B-like enzymes are important for salmon texture. The activities of the proteolytic enzymes may be greatly affected by the muscle pH, and by the treatment(s) the fish are subjected to. In any case, changes caused by differences in proteolytic activities may need some time to be detectable or have significant impact on fish quality. When cod fillets are stored in ice, it is highly recommended to keep the temperature low. Even a relatively mild temperature abuse was sufficient to result in less favorable textural characteristics, and make the fillets seem older than their days of storage. Salmon fillets are often subjected to cold-smoking. The smoking temperature was important for the solubility properties of the muscle proteins, and for their composition, but did not affect the proteolytic activity. The effects of the processing parameters were most important early in the product’s shelf life, as the differences caused by the different smoking temperatures were reduced by further storage of the smoked samples. / Paper II and III are reprinted with kind permission of Elsevier, sciencedirect.com

A generalized quantum chemical approach for electron transport in molecular devices is developed. It allows to treat the devices where the metal electrodes and the molecule are either chemically or physically bonded on equal footing. Effects of molecular length and hydrogen bonding on the current-voltage (I-V) characteristics of molecular devices are discussed. An extension to include the vibration motions of the molecule has been derived and implemented. It provides the inelastic electron tunneling spectroscopy (IETS) of molecular devices with unprecedented accuracy, and reveals important information about the molecular structures that are not accessible in the experiment. The IETS is shown to be a powerful characterization tool for molecular devices. An effective elongation method has been developed to study the electron transport in nanoand bio-electronic devices at hybrid density functional theory level. It enables to study electronic structures and transportation properties of a 40 nm long self-assembled conjugated polymer junction, a 21 nm long single-walled carbon nanotubes (SWCNT), and a 60 basepairs DNA molecule. It is the first time that systems consisting of more than 10,000 electrons have been described at such a sophisticated level. The calculations have shown that the electron transport in sub-22 nm long SWCNT and short DNA molecules is dominated by the coherent scattering through the delocalized unoccupied states. The derived length dependence of coherent electron transport in these nanostructured systems will be useful for the future experiments. Moreover, some unexpected behaviors of these devices have been discovered. / QC 20101203

Branschen för bioteknik har länge varit stark i Sverige men har under de senaste åren utsatts för större konkurrens både vad gäller fler utländska satsningar och konkurrens om kapital nödvändigt för utveckling. Branschen karakteriseras av många små företag, innovationshöjd, höga tröskelkostnader för produktgodkännande samt stark reglering av produkter på marknaden. Företagen konkurrerar med olika strategier baserat på produkt och dess marknad. Det är tydligt att små företag i branschen inte själva täcker en hel värdekedja utan fokuserar sin kompetens på en del av kedjan. Företagen är i hög grad beroende av externa intressenter så som partners, distributörer och myndigheter. Arbetet syftar till att förstå om och hur dessa företag använder ekonomistyrning som stöd för strategiutveckling. Två praktikfall med tydligt olika strategier men liknande historia och storlek har studerats empiriskt och analyserats utifrån litteraturen. I båda fallen ses ett tydligt externt fokus och en kontinuerlig strategiutveckling. Externa parametrar viktiga för företagens strategiutveckling visade sig framförallt vara de direkt vetenskapliga men också hur marknaden förändras med avseende på reglering av licenser, patent och attityder till produkten. En viss systematik sågs i insamling av externa parametrar men på grund av osäkerheten i dess betydelse för företagens produkter samt företagens storlek saknades en plattform för behandling och systematisk analys av dessa. Klassisk ekonomistyrning användes som styrverktyg endast för korta perspektiv och var fokuserat på operationella mått. Sammanfattningsvis kan små bioteknikbolag med fördel använda strategisk ekonomistyrning i högre utsträckning för att säkrare och mer systematiskt behandla, analysera och integrera extern information som stöd för strategiutveckling.

Litteraturstudier har genomförts med syftet att utveckla minst en ny detektionsmetod som skulle kunna ersätta den metod som företaget Cavidi använder sig av idag. Cavidi hade specificerat krav som metodförslaget skulle uppfylla. Dessa krav var att metoden skulle vara snabb, lättanvänd, billig, ha hög känslighet och kunna förvaras i rumstemperatur. Två lovande metoder som baseras på två olika principer valdes ut. Den ena metoden bygger på att enkelsträngat DNA med en specifik nukleinsyrakomposition syntetiseras. Denna sekvens har egenskapen att spontant bilda en sekundärstruktur som kan bilda komplex med en fluorofor. Detta ökar dess fluorescens. Ökningen detekteras med fluorescensspektroskopi. Den andra metoden baseras på ett optomagnetiskt fenomen vilket innebär att ett magnetiskt fält påverkas av polariserat ljus. Metoden går ut på att virioner först renas fram från blodplasma och att de fäster på jonbytarkulor under rådande buffertförhållanden. Magnetiska nanopartiklar tillsätts som binder till jonbytarkulornas lediga ytor. Om många virioner har bundit till jonbytarkulorna finns det en större mängd fria nanopartiklar i lösningen. Antalet fria nanopartiklar i lösningen är proportionellt mot mängden HIV i provet och kan då detekteras med en fotodetektor. Fördelarna med dessa metoder är att processen blir billigare, snabbare och har en hög känslighet. Metoderna är lättanvända och använder färre komponenter jämfört med Cavidis nuvarande metod. Därmed blir Cavidis produkter billigare och tillgängliga för fler människor.

Rapid and accurate species identification and antibiotic susceptibility testing are of great importance for patients with sepsis and to stop over- and misuse of antibiotics contributing to antibiotic resistance. QuickMIC™ is a rapid antibiotic susceptibility testig system based on a microfluidic technology solution developed by Gradientech that measure MICs on bacteria from positive blood culture bottles. By combining QuickMIC™ with a rapid system for detection and identification, the time to detection, identification and antibiotic susceptibiolity testing could be shortened with days compared to pipelines used today which could mean the difference of life and death for patients. The T2Bacteria® panel and T2Dx® instrument developed by T2 biosystems is an FDA-cleared test for rapid detection and identification of bacteria from whole blood based on magnetic molecular resonance technology. The time to result of the T2Dx® instrument is 3-4 hours and the time to result for QuickMIC™ is 2-4 hours. In this project, the possibilities and benefits of such a pipeline have been studied by comparison to a pipeline typically used today. Time, accuracy and practical aspects have been investigated during the project and the results are promising for future further studies.

Detta arbete är en beställning från Vidilab AB som går ut på att kartlägga resistensmekanismer hos nematoder samt ta fram en resistensassay. Beställningen grundar sig i problematiken kring dagens metoder för resistensbestämning, som går ut på att analysera förekomsten av ägg i avföringen från betande djur innan respektive efter avmaskning. Vidilab efterlyser en molekylär diagnostikmetod för resistensbestämning innan boskapsdjuren behandlas med anthelmintika. Som avgränsning har projektet riktat in sig på resistensmekanismer hos nematoden Haemonchus contortus som i första hand parasiterar får. I projektet har i huvudsak förslag på tre tekniker tagits fram som skulle kunna användas för resistensbestämning hos H. contortus. Dessa tre metoder är allelspecifik PCR, RT-PCR och MALDI-TOF. Slutsatsen i detta projekt är att samtliga av dessa tre metoder har potential som molekylär diagnostikmetod. Dock krävs mer forskning kring H. contortus resistensmekanismer, storskalig sekvensering samt kartläggning av transkriptom och proteomikstudier innan teknikerna kan appliceras som standardiserade diagnostikmetoder.

In the human body, the main function of the healthy thyroid gland is the regulation of the metabolism and hormone production. Included in the thyroid are organized structured and uniformly shaped follicles ranging from 50-500 μm in diameter. Pathologies lead to morphological changes of these follicles, affecting the density and size, but can also lead to an absence. In this study optical coherence tomography (OCT) was used to examine pathological thyroid tissue by extracting structural information of the follicles from image segmentation. However, OCT images usually include a high amount of speckle noise which affects the segmentation outcome. Due to that, the OCT images need to be improved. The aim of this thesis was to investigate the appropriate filtering methods to enhance the images and thus improve the segmentation outcome. The images of pathological thyroid tissues with a size of 0:5-1 cm where scanned by a spectral domain OCT system (Telesto II, Thorlabs GmbH, Germany) using a center wavelength of 1300nm. The obtained 2D and 3D images were saved as .oct file as well as implemented and visualized in a MATLAB graphical user interface (GUI) for further processing. For image improvement, four filtering enhancement methods were applied to the 2D images such as the enhanced resolution imaging (ERI), adaptive Wiener filter, discrete wavelet transform (DWT) and multi-frame wavelet transform (WT). The processed images were further converted to grayscale and binary images for intensity-based segmentation. The output of all methods were compared and evaluated using signal-to-noise ratio (SNR), contrast-to-noise ratio (CNR), enhanced number of looks (ENL), edge profiles and outcome of the segmented images. It was demonstrated that the complex DWT (cDWT) with a higher threshold and the multi-frame WT using the haar wavelet showed enhanced results over the other filtering methods. The computed SNR could be increased up to 52% and the ENL value up to 4802%, applying the multi-frame WT, while the CNR could be increased up to 106% for cDWT. The lowest obtained gradient was equal to an intensity decrease of -61% and -68% for multi-frame WT and cDWT, respectively. The filtering method could increase the smoothness of the image while the edge sharpness could be kept. The segmentation could detect both small and large follicles. ERI did not show any improvement in the segmentation but could enhance the structural detail of the image. Larger neighbourhoods of the adaptive Wiener filter showed a highly blurred image and led to merged follicles in the image segmentation. The wavelet filters DWT and multi-frame WT gave most satisfying results since high and low frequencies were divided into subbands, where individual information on vertical, horizontal and diagonal edges was stored. Applied cDWT had an even higher amount of subbands, so that more information on signal and speckle noise could be specified. Due to this fact, it was possible to achieve a decreased noise level while edge sharpness where maintained. Using a multi-frame image an increased SNR was obtained, as the intensity information stayed constant over the individual frames while the noise information changed. Wavelet based filtering showed higher improved results in comparison to the adaptive Wiener filter or the ERI in the 2D domain. By applying filtering methods in higher dimensions such as 3D or even 4D, better results in noise reduction are expected. Improved settings for the individual filtering methods as well as enhancement in segmentation are part of the future work.

The objective of this study was to investigate the mechanisms and factors controlling growth and proliferation in serum-free Spodoptera frugiperda Sf9 cultures as well as the implications of these factors for protein production in the baculovirus expression system. The physiology of recently thawed, low passage (Lp) Sf9 cultures, were compared to high passage (Hp) cultures at p&gt;100. Lp cells passed a switch in proliferation kinetics after 30-40 passages, characterized by a shorter lag-phase and an increased maximum specific proliferation rate, µN,max, from 0.03/h to 0.04/h. Conditioned medium (CM), 10 kDa CM filtrate and 10 kDa CM concentrate promoted proliferation of Lp Sf9 cells, but had no effect after the switch. Sf9 cell cycle dynamics were characterized by an initial G2/M arrest, which synchronized the cells and this feature was more pronounced for Hp than for Lp cells. CM addition decreased the initial arrest for Lp cultures, but did not affect Hp cells. Late in the culture, a final G2/M accumulation occurred. An octaploid population emerged during G2/M arrests. Further, a 49 kDa proform and a 39 kDa active form of Sf9 cathepsin L was identified from gelatine zymography of Sf9 CM, on basis of inhibitor profile and substrate range. Removal of procathepsin L during the course of an Sf9 culture had a negative effect on Sf9 proliferation. Procathepsin L was also identified in Trichoplusia ni High five CM. High five CM promoted growth of Sf9 cells, but when procathepsin L was removed no effect was observed. It is suggested that these observations are due to an autocrine system controlling proliferation. One Sf9 mitogen might be a &lt;10 kDa peptide, while the effect of 10 kDa CM concentrate may originate from procathepsin L. A hypothesis is therefore that procathepsin L acts as a mitogen in Sf9 cultures, perhaps in concert with the &lt;10 kDa peptide. The volumetric product yield (P) in baculovirus infected Sf9 cells increased linearly up to 68-75 h of culture. Beyond this point almost no product was detected. Medium renewal at infection prolonged the productivity phase until 117 h, but generated only a 10% increase in P. The specific product formation rate (YP/N) was highest at µN,max. YP/N of Lp cells decreased by 30-50% when 20% CM or 10 kDa CM filtrate was added, whereas addition of CM to cells having passed the switch on growth kinetics did not affect productivity. Further, Hp cells exhibited a two-fold higher YP/N than Lp cells, when infected during the initial 48 h of culture. This coincided with a high degree of synchronization. Yeastolate limitation was used to achieve artificial synchronization of an Lp culture, and YP/N could thereby be maintained high during a prolonged time, resulting in a 69% increased P. This suggests that a decreasing degree of synchronization during the course of a culture partly explains the cell-density dependent drop in productivity in Sf9 cells. Finally, ~10 kDa gel filtration fractions from Sf9 and High five CM were found to be bactericidal. Exposure of a Bacillus megaterium culture for eight min to an Sf9 CM fraction killed 99% of the population, and 60 min exposure killed 35% of an Escherichia coli population. In both cases cell lysis was observed. B. megaterium incubated in an High five CM fraction lost 97% viability in 40 min. The effect of the High five CM fraction most probably originated from a lysozyme precursor protein, whereas the Sf9 executor remains unknown. / QC 20100908