Membrane proteins and proteomics : un amour possible?

Membrane proteins constitute 20-30 % of the human genome and make up 60 % of all drug targets. They play important roles in key cellular functions such as small molecules transport, cell-cell interactions and cell signaling. Membrane proteins and proteomics have been notoriously difficult to combine. Most proteomic methods commonly used for analysis of soluble proteins cannot be used for membrane proteins, mainly due to their amphipathic nature. In this thesis, a method is described for quantitative proteomic analysis of membrane protein enriched samples. In paper I the method is applied to small cell lung cancer cell lines to elucidate Doxorubicin resistance mechanisms. We demonstrate that the microsomal preparation and iTRAQ labeling is reproducible regarding protein content and composition. The rationale using narrow range peptide isoelectric focusing separation is demonstrated by its ability to: i) lowering the complexity of the sample by two thirds while keeping a high proteome coverage (96%), ii) providing high separation efficiency and iii) allowing for peptide validation and possibly identifications of post transcriptional modifications. The work from paper I encouraged us to further explore the properties of narrow range peptide IEF as a separation strategy. We and others have shown that using IEF as the first dimension of separation is a highly suitable method when analyzing complex samples. It provides an orthogonal separation strategy to reversed phase chromatography as well as giving the opportunity to reduce false positives and false negatives generated in the database search, using the pI values of the peptides. In paper II, we wanted to explore the possibilities of using a combination of narrow range peptide IEF and reversed phase chromatography to increase the information content of a proteomic analysis. In the final two papers of this thesis, the method described in paper I is subsequently applied to clinical material. In paper III, the membrane protein fractions of benign and malignant adrenocortical tumors are compared. The mitochondrial membrane protein GRIM-19, a negative regulator of STAT3, is identified as down-regulated in the malignant tissue. The possible role of GRIM-19 down-regulation in the tumorigenesis is discussed. In paper IV, the membrane-associated protein population of alveolar macrophages isolated from Sarcoidosis patients is compared with healthy controls. Affected pathways are described and discussed

Clinical and epidemiological studies on prognostic and predicitve factors in cutaneous melanoma

Background Cutaneous malignant melanoma (CMM) is one of the most rapidly increasing cancers in Sweden, as in many other western countries. In clinical practice, the histopathological evaluation has remained the basis for staging the CMMs and, thus, providing important information on prognosis and on therapeutic recommendations. Interobserver variation regarding the histopathological evaluation is known to exist. However, few studies have investigated how clinical, biological and social mechanisms interact and influence the prognosis in CMM. For patients with advanced disease progress of the treatment options has been achieved for tumours carrying BRAFV600 mutations by the development of specific small-molecule BRAF inhibitors. The development of these targeted therapies thus mandates determination of BRAF mutation status. This thesis aims to describe the interobserver variability in evaluating histopathological prognostic factors, to assess the association between education as well as cohabitation status and prognosis in patients with a primary CMM, and also to analyze the patterns of BRAFV600E protein expression in primary and metastatic CMMs. Methods In Paper I, a total of 234 cases of invasive CMMs from the Stockholm–Gotland Healthcare Region in Sweden were included in the study. In Papers II and III, 27,235 patients diagnosed with a primary invasive CMM between 1990 and 2007 were identified from the Swedish Melanoma Register. Data were linked to nationwide, population based, health and census registers with a follow-up through 2010. In Paper IV, a total of 200 CMMs were stained by immunohistochemistry (IHC) using a BRAFV600E specific monoclonal antibody (VE1). Results Overall, interobserver variability between a general pathologist and an expert review was 79% in Paper I. The best agreement was found for tumour thickness, but 15.5% of the tumours were re-classified after review in a sub-set of thin (≤ 1 mm) CMMs 15.5% were re-classified. In Paper II, we found significantly elevated odds ratios (OR) of higher disease stage at diagnosis among lower education groups after adjustments for other prognostic factors (OR stage II vs. I = 1.6; 95% confidence interval (CI) = 1.5-1.7. OR stage III–IV vs. I = 2.3; 95% CI = 1.8-2.9). The risk of dying of CMM, was significantly increased in patients with low education after the final adjustments for all clinical and histopathological prognostic factors (hazard ratio (HR) low vs. high = 1.13; 95% CI=1.01-1.27; p = 0.04). In Paper III, after adjustments for established prognosticators and education, the ORs of higher clinical stage at diagnosis were significantly increased among men living alone vs. men living with a partner (OR stage II vs. stage I = 1.42; 95% CI = 1.29-1.57. OR stage III-IV vs. stage I = 1.43; 95% CI = 1.14-1.79). The ORs for higher stage among women living alone were also increased (OR stage II vs. stage I = 1.15; 95% CI = 1.04-1.28. OR stage III-IV vs. stage I = 1.04; 95% CI = 0.79-1.37). After adjustments for all potential and established prognostic factors, HR for CMM death for men living alone vs. living with a partner was 1.33 (95% CI = 1.19-1.49; p<0.0001), indicating a residual adverse effect on survival not accounted for by these parameters. In Paper IV, the VE1 IHC staining intensity varied between primary CMMs and matched metastases in 47% of the cases, as well as between separate metastases. We found a sensitivity of the VE1 antibody of 97% and a specificity of 80% for detection of BRAFV600E mutations. A comparable sensitivity was obtained for primary CMMs and metastases when analyzed separately. However, the specificity was lower among primary CMMs (71%) compared to metastases (93%). Conclusions Our results imply that the recommendation of surgical excision margins and/or sentinel node biopsy changed in subgroups of thin CMMs after a CMM-expert pathology review. Moreover, the results emphasize the need for improved early detection strategies directed towards specific patient groups to improve the CMM-specific survival. IHC using the VE1 antibody should be used in combination with genomic testing in primary CMMs specifically in cases with weak/moderate staining to accurately predict BRAFV600E status, whereas in metastases with strong VE1 staining no further mutation testing seems to be required

Mikrobiell dynamik hos svensk, industriellt bryggd kombucha - och variationer mellan startkulturer

Kombucha is an acidic, slightly alcoholic beverage made by fermenting sweetened tea. Kombucha is rising in popularity worldwide as a result of the alleged health benefits. Common microbes involved has been found to vary, but include yeasts, acetic acid bacteria, and occasionally lactic acid bacteria. The aim of this thesis was to investigate how the microbial population differed between three starter cultures from one industrial brewery of kombucha, what microbes could be found in one cellulosic biofilm, and how the microbial population of one batch of industrially brewed combucha altered from day 2 to 21 of fermentation, using samples obtained from a Swedish brewery. This was done by plating sample dilutions on differential media and counting the colony forming units, as well as by partial amplification and sequencing of the 16S and 26S RNA gene respectively. Concentrations of sugars and end products were measured by high performance liquid chromatography. The starter liquids were found to consist of similar microorganisms but were not identical. The microbial population originating from the cellulosic pellicle was not as diverse as the most diverse starter liquid but had a greater microbial density. In the batch of industrially brewed kombucha, Brettanomyces anomalus was the dominating yeast throughout. Hanseniaspora mollemarum was found in samples from the first half of the fermentation period, and Zymomonas mobilis in the second. Sucrose equivalents decreased from 51.8 to 32.2 g L-1, while ethanol increased from 3.5 to 15.4 g L.1. The final concentration of acetic acid was found to be 3.0 g L-1. No acetic acid bacteria or lactic acid bacteria were identified, and no lactate was formed.Kombucha är en syrlig, något alkoholhaltig dryck tillverkad genom att fermentera te. Kombuchas popularitet ökar världen över, på grund av påstådda hälsofördelar. Mikrober delaktiga i fermentationen kan variera mycket, men innefattar ofta jäst, ättiksyrebakterier, och ibland mjölksyrebakterier. Syftet med denna avhandling var att undersöka hur den mikrobiella populationen skiljde sig åt mellan tre startkulturer från ett industriellt bryggeri av kombucha, vilka mikrober som kunde hittas i en biofilm, samt hur den mikrobiella populationen i en omgång industriellt bryggd kombucha förändrades från dag 2 till dag 21 av fermentationen, med prover inhämtade från ett svenskt bryggeri. Detta gjordes genom att sprida prover på differentierade medier och räkna koloniformande enheter, samt genom att partiellt amplifiera och sekvensera 16S och 26S RNA genen för jäst respektive bakterier. Koncentrationer av socker och slutprodukter mättes med högupplösande vätskekromatografi. Startvätskorna visade sig bestå av liknande, men inte identiska populationer av mikrober. Den mikrobiella sammansättningen från biofilmen var inte lika artrik som den mest artrika startvätskan, men hade en högre mikrobiell densitet. I omgången industriellt bryggd kombucha dominerade Brettanomyces anomalus vid alla tidpunkter. Hanseniaspora mollemarum kunde identifieras i prover från den första halvan av fermentationen, och Zymomonas mobilis i den andra. Sukrosekvivalenter minskade från 51.8 till 32.2 g L-1, medan etanol ökade från 3.5 till 15.4 g L-1. Den slutliga koncentrationen av ättiksyra uppmättes till 3.0 g L-1. Inga ättiksyrabakterier eller mjölksyrabakterier identifierades, och ingen mjölksyra kunde uppmätas

Australien und China : boomende Wirtschaftsbeziehungen, ambivalente Öffentlichkeit

Studies indicate that between 6-9% of all children has some type of voice problems. Voice problems in children may have different etiology and can affect all ages. In Sweden, there is a lack of a standardized questionnaire addressed to children with voice problems and their legal guardians. The purpose of this study was to translate and validate the Pediatric Voice Handicap Index (pVHI) in Swedish children aged 6 to 10 years. The study was carried out through an internet-based assessment form for children referred to speech and language pathologist/ phoniatrican due to voice disorders in Sweden and a matched control group of children without voice problems. The experimental group consisted of 13 children with voice problems and the control group of 29 children. The questionnaire was filled out by the guardian in cooperation with the child. It contained 23 claims separated into three domains: functional, physical and emotional. The results of each child’s assessment were the total pVHI, with a maximum of 92 points. A comparison between results presented in the original pVHI showed great similarities regarding mean values within the three domains the total pVHI and the experimental group’s estimation of over- all voice problems on a VAS- scale. The internal validity of the translation was high with a Cronbach´s alpha at .95- .96 within the different domains. Significant differences were found between the experimental group and the control group in each domain. There was also a significant difference between the experimental group and the control group regarding total pVHI. The results indicate that the translation of pVHI is easy to fill out and close to the original. Also, it discriminates between children with and without voice disorders indicating good sensitivity and specificity, which is central in a broader use.Studier visar att mellan 6- 9 % av alla barn har någon form av röstproblem. Röstproblem hos barn kan ha olika etiologi och kan drabba alla åldrar. I Sverige har ett standardiserat skattningsformulär riktat till barn med röstproblem och dess målsmän saknats. Syftet med föreliggande studie var att översätta och validera pediatric Voice Handicap Index (pVHI) för svenska barn mellan 6 till 10 år. Studien utfördes genom ett internetbaserat skattningsformulär till barn med målsmän som sökt hjälp för röstbesvär hos logoped/foniater i Sverige samt en matchad kontrollgrupp utan uttalade röstproblem. Experimentgruppen bestod av 13 barn med röstproblem och kontrollgruppen av 29 barn. Skattningsformuläret fylldes i av målsmän i samråd med barnet. Det innehöll 23 påståenden fördelade inom tre domäner; funktionell, kroppslig och emotionell. Sammantagna resultatet av varje barns skattning utgjorde ett total pVHI där 92 var maxpoäng. Vid jämförelser mellan resultaten i den ursprungliga pVHI och föreliggande studie återfanns stora likheter på medelvärden inom de tre domänerna, för total pVHI och för experimentgruppens skattning av omfattning av deras röstproblem på en VAS-skala. Den interna validiteten av den översatta svenska versionen var hög med ett Cronbach's alfa på .95 till .96 inom de olika domänerna. Signifikant skillnad återfanns mellan experimentgrupp och kontrollgrupp inom varje domän. Det var även signifikant skillnad mellan experimentgruppen och kontrollgruppens total- pVHI. Resultaten i föreliggande studie tyder på att översättningen av pVHI var nära den ursprungliga versionen. Dessutom diskriminerar översättningen mellan barn med och utan röstproblem vilket indikerar på god sensitivitet och specificitet, något som är centralt för en bredare användning

Calf or grass - What would the cow choose?

The growing interest for keeping dairy cows with their calves for an extended period after calving is putting pressure on the scientific community to investigate the effects of cow-calf contact systems on the animals ' welfare. The main aims of this study were to investigate the dairy cows ' motivation for accessing their calves over a fresh pasture, and to evaluate if their motivation decreased with increasing calf age. Twenty-two Swedish Red and Swedish Holstein cow-calf pairs were enrolled at calving. The dams were housed in a robotic barn with free access to outdoor pasture, where the calves were kept during pasture season. The behaviours of the dams were recorded on three separate test days occurring every two weeks, starting when the calves were on average 10 weeks old. During test days, the calves were confined to a heavily grazed area, while the dams had free access both to the calf area and to an adjacent fresh pasture. Which of the areas the dams entered first upon returning outdoors from the barn, and what behaviours they performed in which area were registered using 10-min scan sampling during 8 h per day. The dams spent more time outdoors on Test day I (76.8 +/- 3.09%; LSMeans +/- SEM) compared to Day II (60.9 +/- 3.86%; P=0.006), while Day III (66.8 +/- 4.38%) did not differ from Day I (P=0.15) or Day II (P=0.55). On Test day II, they chose the calf area 54 +/- 10.05% of the times they returned to pasture, which was significantly more often than during Day I (18.2 +/- 4.96%; P=0.01), while no difference was found between Day III (37.1 +/- 9.86%) and Day I (P=0.17) or Day II (P=0.5). There was no effect of breed on total time spent outdoors, but Holstein dams tended to spend more outdoor time in the calf area (36.4 +/- 5.28%) than Swedish Red dams (24.2 +/- 3.95%; P=0.09). Upon returning outdoors, Holstein dams also chose the calf area over fresh pasture more often (46.0 +/- 7.19%) than did Swedish Red dams (25.2 +/- 6.21%; P=0.05). Primiparous cows tended to choose the calf area over fresh pasture more often (46.9 +/- 6.11%) than multiparous cows (24.5 +/- 7.25%; P=0.06). Exploratory analyses suggest that the effects of test day were more affected by the ambient weather than by the age of the calves. The study results provide some further information about factors influencing maternal motivation to reunite with their calves on pasture. However, further research is needed so that cow-calf contact systems can be designed to enable good welfare

Assessing short-term economic consequences of cow-calf contact systems in dairy production using a stochastic partial budgeting approach

Introduction: While early separation of dairy calves after birth has been debated from an ethical and animal welfare lens, the economic evidence surrounding alternative cow-calf contact (CCC) systems is scarce.Methods: To address this knowledge gap, we assessed the economic consequences of CCC systems using data from the Agriwise database as well as parameters from published literature in a stochastic partial budget model. The implementation of CCC is very diverse between farms, so in our study we therefore selected a limited number of CCC systems to assess. The examined CCC systems were: (i) dam rearing with limited contact at milking (15 minutes twice a day for 115 days) with no manual milk feeding; (ii) dam rearing with 21-day full contact, after which calves are manually fed 8 kg of whole milk for 94 days; and (iii) mixed calf rearing with using both dams and foster cows with full contact; calves are initially kept with their dams and then moved to foster cows at 9 days of age.Results: We found that adoption of CCC systems was associated with decreases in contribution margins in the range of 1 to 5.4%, as compared to a rearing system where the calves were separated from their dams after one day and were manually fed 8 kg of milk for 12 weeks. These results illustrated that the costs associated with CCC systems may be fairly high under certain circumstances and may prohibit farmers from adopting this practice. Sensitivity analysis suggested that differences in milk sales, assumptions on changes in labor requirements, and changes in daily calf weight gain depending on CCC system were the main variables that governed the net impact on the contribution margins.Discussion: We did not include building costs in the analyses assuming that barn structures may not change in the short-term. The study focused only on short-term pecuniary associations between changes in CCC systems and contribution margins. To strengthen the economic decision-making around CCC systems, future research should consider non-monetary impacts of different CCC systems, as well as long-term economic impacts of these production strategies

Proposed methods for estimating loss of saleable milk in a cow-calf contact system with automatic milking

Cow-calf contact (CCC) systems, although beneficial in many respects, introduce additional challenges to collect reliable data on milk production, which is important to assess individual cow efficiency and dairy farm profitability. Apart from weighing calves before and after each feeding, the amount of saleable milk lost due to calf suckling is practically impossible to measure. Here, we assess 2 indirect methods for estimating loss of saleable milk when housing cows and calves together in a robotic milking unit. In our study, treatment (CCC) cows and calves were kept together full time until the calves were 127 ± 6.6 d old (mean ± SD). Control cows were separated from their calves within 12 h of birth and then kept in the same unit as the treatment cows but with no access to either their own or treatment calves. Milk yield recording of both groups was performed from calving until pasture release at 233 ± 20 d in milk. The first estimation method relied on observed postseparation milk yield data, which were fed into a modified Wilmink regression model to determine the best-fitting lactation curve for the preseparation period. The second method was based on the cows' daily energy intake postseparation, calculated by measuring the daily feed intake and analyzing the energy content of the ration. The calculated energy intake was used to determine the average ratio between energy intake and the observed milk yield the following day for each individual cow, assuming constant rates of mobilization and deposition of body fat. The obtained ratio was then used to calculate the expected daily milk yield based on daily energy intake data during the preseparation period. In this paper, we analyzed data from 17 CCC cows kept together with their calves and 16 control cows; both groups calved from September to October 2020 and were followed up until release to pasture in May 2021. Saleable milk yield was lower in CCC cows than in control cows, both before and after separation. The 2 methods were used on data for control cows and showed milk yield loss using the lactation curve method (average of −3.4 ± 2.8 kg/d) and almost no loss using energy intake data (average of −1.4 ± 2.7 kg/d). Milk yield loss for CCC cows was estimated at average 11.3 ± 4.8 and 7.3 ± 6.6 kg milk/d, respectively. The proposed lactation curve estimation method tends to overestimate milk yield loss, whereas the method based on energy intake is more accurate. However, collecting detailed energy intake data per individual cow requires additional effort and equipment, which is not always feasible on commercial farms. Further research is needed to improve milk loss estimation and to better understand trade-offs in CCC systems

Hålla kor och kalv tillsammans - vad vet vi från de svenska Ko-Kalv-projekten såhär långt?

Presentation on research findings from the finished cow-calf contact studies done in Sweden

The capability concept – On how to define and describe capability in relation to risk, vulnerability and resilience

Capabilities-based planning and capability assessment are high on the agendas of several countries and organisations as part of their risk management and emergency preparedness. Despite this, few definitions of capability exist, and they are not easily related to concepts such as risk, vulnerability and resilience. The aim of the present study was thus to broaden the scientific basis of the risk field to also include the concept of capability. The proposed definition is based on a recently developed risk framework, and we define capability as the uncertainty about and the severity of the consequences of an activity given the occurrence of the initiating event and the performed task. We provide examples of how the response capability for a fictive scenario can be described using this definition, and illustrate how our definition can be used to analyse capability assessments prepared according to the Swedish crisis management system. We have analysed the content of 25 capability assessments produced in 2011 by stakeholders on local, regional and national level. It was concluded that none addressed uncertainty to any appreciable extent, and only a third described capability in terms of consequences and task, making it difficult to relate these capability assessments to risk assessments

Solution-Spinning of a Collection of Micro- and Nanocarrier-Functionalized Polysaccharide Fibers

Continuous polysaccharide fibers and nonwovens—based on cellulose, hydroxypropyl cellulose, chitosan, or alginate—containing biopolymeric microcapsules (MC) or mesoporous silica nanoparticles (MSN) are prepared using a wet-spinning or solution blowing technique. The MCs are homogeneously distributed in the fiber matrices whereas the MSNs form discrete micron-sized aggregates as demonstrated using scanning electron-, fluorescence-, and confocal microscopy. By encapsulating the model compound pyrene, it is shown that 95% of the substance remains in the fiber during the formation process as compared to only 7% for the nonencapsulated substance. The material comprising the MC has a strong impact on the release behavior of the encapsulated pyrene as investigated using methanol extraction. MCs based on poly(l-lactic acid) prove to be practically impermeable with no pyrene released in contrast to MCs based on poly(lactic-co-glycolic acid) which allow for diffusion of pyrene through the MC and fiber as visualized using fluorescence microscopy