Contamination routes and control of Listeria monocytogenes in Food Production

Listeria monocytogenes is the causative agent of the severe foodborne infection listeriosis. The number of listeriosis cases in recent years has increased in many European countries, including Finland. Contamination of the pathogen needs to be minimized and growth to high numbers in foods prevented in order to reduce the incidence of human cases. The aim of this study was to evaluate contamination routes of L. monocytogenes in the food chain and to investigate methods for control of the pathogen in food processing. L. monocytogenes was commonly found in wild birds, the pig production chain and in pork production plants. It was found most frequently in birds feeding at landfill site, organic farms, tonsil samples, and sites associated with brining. L. monococytogenes in birds, farms, food processing plant or foods did not form distinct genetic groups, but populations overlapped. The majority of genotypes recovered from birds were also detected in foods, food processing environments and other animal species and birds may disseminate L. monocytogenes into food chain. Similar genotypes were found in different pigs on the same farm, as well as in pigs on farms and later in the slaughterhouse. L. monocytogenes contamination spreads at farm level and may be a contamination source into slaughterhouses and further into meat. Incoming raw pork in the processing plant was frequently contaminated with L. monocytogenes and genotypes in raw meat were also found in processing environment and in RTE products. Thus, raw material seems to be a considerable source of contamination into processing facilities. In the pork processing plant, the prevalence of L. monocytogenes increased in the brining area, showing that the brining was an important contamination site. Recovery of the inoculated L. monocytogenes strains showed that there were strain-specific differences in the ability to survive in lettuce and dry sausage. The ability of some L. monocytogenes strains to survive well in food production raises a challenge for industry, because these strains can be especially difficult to remove from the products and raises a need to use an appropriate hurdle concept to control most resistant strains. Control of L. monocytogenes can be implemented throughout the food chain. Farm-specific factors affected the prevalence of L. monocytogenes and good farm-level practices can therefore be utilized to reduce the prevalence of this pathogen on the farm and possibly further in the food chain. Well separated areas in a pork production plant had low prevalences of L. monocytogenes, thus showing that compartmentalization controls the pathogen in the processing line. The food processing plant, especially the brining area, should be subjected to disassembling, extensive cleaning and disinfection to eliminate persistent contamination by L. monocytogenes, and replacing brining with dry-salting should be considered. All of the evaluated washing solutions decreased the populations of L. monocytogenes on precut lettuce, but did not eliminate the pathogen. Thus, the safety of fresh-cut produce cannot rely on washing with disinfectants, and high-quality raw material and good manufacturing practices remain important. L. monocytogenes was detected in higher levels in sausages without the protective culture than in sausages with this protective strain, although numbers of L. monocytogenes by the end of the ripening decreased to the level of < 100 MPN/g in all sausages. Protective starter cultures provide an appealing hurdle in dry sausage processing and assist in the control of L. monocytogenes.

Learning Challenges in Biogas Production for Sustainability : An activity theoretical study of a network from a swine industry chain

This study is about the challenges of learning in the creation and implementation of new sustainable technologies. The system of biogas production in the Programme of Sustainable Swine Production (3S Programme) conducted by the Sadia food processing company in Santa Catarina State, Brazil, is used as a case example for exploring the challenges, possibilities and obstacles of learning in the use of biogas production as a way to increase the environmental sustainability of swine production. The aim is to contribute to the discussion about the possibilities of developing systems of biogas production for sustainability (BPfS). In the study I develop hypotheses concerning the central challenges and possibilities for developing systems of BPfS in three phases. First, I construct a model of the network of activities involved in the BP for sustainability in the case study. Next, I construct a) an idealised model of the historically evolved concepts of BPfS through an analysis of the development of forms of BP and b) a hypothesis of the current central contradictions within and between the activity systems involved in BP for sustainability in the case study. This hypothesis is further developed through two actual empirical analyses: an analysis of the actors senses in taking part in the system, and an analysis of the disturbance processes in the implementation and operation of the BP system in the 3S Programme. The historical analysis shows that BP for sustainability in the 3S Programme emerged as a feasible solution for the contradiction between environmental protection and concentration, intensification and specialisation in swine production. This contradiction created a threat to the supply of swine to the food processing company. In the food production activity, the contradiction was expressed as a contradiction between the desire of the company to become a sustainable company and the situation in the outsourced farms. For the swine producers the contradiction was expressed between the contradictory rules in which the market exerted pressure which pushed for continual increases in scale, specialisation and concentration to keep the production economically viable, while the environmental rules imposed a limit to this expansion. Although the observed disturbances in the biogas system seemed to be merely technical and localised within the farms, the analysis proposed that these disturbances were formed in and between the activity systems involved in the network of BPfS during the implementation. The disturbances observed could be explained by four contradictions: a) contradictions between the new, more expanded activity of sustainable swine production and the old activity, b) a contradiction between the concept of BP for carbon credits and BP for local use in the BPfS that was implemented, c) contradictions between the new UNFCCC1 methodology for applying for carbon credits and the small size of the farms, and d) between the technologies of biogas use and burning available in the market and the small size of the farms. The main finding of this study relates to the zone of proximal development (ZPD) of the BPfS in Sadia food production chain. The model is first developed as a general model of concepts of BPfS and further developed here to the specific case of the BPfS in the 3S Programme. The model is composed of two developmental dimensions: societal and functional integration. The dimension of societal integration refers to the level of integration with other activities outside the farm. At one extreme, biogas production is self-sufficient and highly independent and the products of BP are consumed within the farm, while at the other extreme BP is highly integrated in markets and networks of collaboration, and BP products are exchanged within the markets. The dimension of functional integration refers to the level of integration between products and production processes so that economies of scope can be achieved by combining several functions using the same utility. At one extreme, BP is specialised in only one product, which allows achieving economies of scale, while at the other extreme there is an integrated production in which several biogas products are produced in order to maximise the outcomes from the BP system. The analysis suggests that BP is moving towards a societal integration, towards the market and towards a functional integration in which several biogas products are combined. The model is a hypothesis to be further tested through interventions by collectively constructing the new proposed concept of BPfS. Another important contribution of this study refers to the concept of the learning challenge. Three central learning challenges for developing a sustainable system of BP in the 3S Programme were identified: 1) the development of cheaper and more practical technologies of burning and measuring the gas, as well as the reduction of costs of the process of certification, 2) the development of new ways of using biogas within farms, and 3) the creation of new local markets and networks for selling BP products. One general learning challenge is to find more varied and synergic ways of using BP products than solely for the production of carbon credits. Both the model of the ZPD of BPfS and the identified learning challenges could be used as learning tools to facilitate the development of biogas production systems. The proposed model of the ZPD could be used to analyse different types of agricultural activities that face a similar contradiction. The findings could be used in interventions to help actors to find their own expansive actions and developmental projects for change. Rather than proposing a standardised best concept of BPfS, the idea of these learning tools is to facilitate the analysis of local situations and to help actors to make their activities more sustainable.

Production process of Scots pine.

The accompanying collective research report is the result of the research project in 1986­90 between The Finnish Academy and the former Soviet Academy of Sciences. The project was organized around common field work in Finland and in the former Soviet Union and theoretical analyses of tree growth determining processes. Based on theoretical analyses, dynamic stand growth models were made and their parameters were determined utilizing the field results. Annual cycle affects the tree growth. Our theoretical approach was based on adaptation to local climate conditions from Lapland to South Russia. The initiation of growth was described as a simple low and high temperature accumulation driven model. Linking the theoretical model with long term temperature data allowed us to analyze what type of temperature response produced favorable outcome in different climates. Initiation of growth consumes the carbohydrate reserves in plants. We measured the dynamics of insoluble and soluble sugars in the very northern and Karelian conditions. Clear cyclical pattern was observed but the differences between locations were surprisingly small. Analysis of field measurements of CO2 exchange showed that irradiance is the dominating factor causing variation in photosynthetic rate in natural conditions during summer. The effect of other factors is so small that they can be omitted without any considerable loss of accuracy. A special experiment carried out in Hyytiälä showed that the needle living space, defined as the ratio between the shoot cylindric volume and needle surface area, correlates with the shoot photosynthesis. The penetration of irradiance into Scots pine canopy is a complicated phenomenon because of the movement of the sun on the sky and the complicated structure of branches and needles. A moderately simple but balanced forest radiation regime submodel was constructed. It consists of the tree crown and forest structure, the gap probability calculation and the consideration of spatial and temporal variation of radiation inside the forest. The common field excursions in different geographical regions resulted in a lot of experimental data of regularities of woody structures. The water transport seems to be a good common factor to analyse these properties of tree structure. There are evident regressions between cross-sectional areas measured at different locations along the water pathway from fine roots to needles. The observed regressions have clear geographical trends. For example, the same cross-sectional area can support three times higher needle mass in South Russia than in Lapland. Geographical trends can also be seen in shoot and needle structure. Analysis of data published by several Russian authors show, that one ton of needles transpire 42 ton of water a year. This annual amount of transpiration seems to be independent of geographical location, year and site conditions. The produced theoretical and experimental material is utilised in the development of stand growth model that describes the growth and development of Scots pine stands in Finland and the former Soviet Union. The core of the model is carbon and nutrient balances. This means that carbon obtained in photosynthesis is consumed for growth and maintenance and nutrients are taken according to the metabolic needs. The annual photosynthetic production by trees in the stand is determined as a function of irradiance and shading during the active period. The utilisation of the annual photosynthetic production to the growth of different components of trees is based on structural regularities. Since the fundamental metabolic processes are the same in all locations the same growth model structure can be applied in the large range of Scots pine. The annual photosynthetic production and structural regularities determining the allocation of resources have geographical features. The common field measurements enable the application of the model to the analysis of growth and development of stands growing on the five locations of experiments. The model enables the analysis of geographical differences in the growth of Scots pine. For example, the annual photosynthetic production of a 100-year-old stand at Voronez is 3.5 times higher than in Lapland. The share consumed to needle growth (30 %) and to growth of branches (5 %) seems to be the same in all locations. In contrast, the share of fine roots is decreasing when moving from north to south. It is 20 % in Lapland, 15 % in Hyytiälä Central Finland and Kentjärvi Karelia and 15 % in Voronez South Russia. The stem masses (115­113 ton/ha) are rather similar in Hyytiälä, Kentjärvi and Voronez, but rather low (50 ton/ha) in Lapland. In Voronez the height of the trees reach 29 m being in Hyytiälä and Kentjärvi 22 m and in Lapland only 14 m. The present approach enables utilization of structural and functional knowledge, gained in places of intensive research, in the analysis of growth and development of any stand. This opens new possibilities for growth research and also for applications in forestry practice.

The influence of lone-pair repulsions on C–C bond lengths: a critical evaluation of the experimental and theoretical evidence

X-Ray structural data, as well as semiempirical and ab initio molecular orbital calculations, reveal no systematic and substantial difference between the C–C bond lengths of cis and trans 1,2-diketones. Additional results on various conformations of 1,2-diimines and 1,2-dithiones follow the same pattern. Therefore, lone-pair repulsions cannot be implicated in the observed lengthening of C–C bonds in isatin and several related molecules. Conjugation in these systems occurs peripherally avoiding the participation of the central C–C bond. Negative hyperconjugative interaction between the oxygen lone pairs and the adjacent C–C σ* orbital is suggested to be the principal reason for the relatively long C–C bond in diketones. This effect is found in both the cis and trans conformations.

Molecular Characterization of Viruses Causing the Cassava Brown Streak Disease Epidemic in Eastern Africa

Cassava brown streak disease (CBSD) was described for the first time in Tanganyika (now Tanzania) about seven decades ago. Tanganyika (now Tanzania) about seven decades ago. It was endemic in the lowland areas of East Africa and inland parts of Malawi and caused by Cassava brown streak virus (CBSV; genus Ipomovirus; Potyviridae). However, in 1990s CBSD was observed at high altitude areas in Uganda. The causes for spread to new locations were not known.The present work was thus initiated to generate information on genetic variability, clarify the taxonomy of the virus or viruses associated with CBSD in Eastern Africa as well as to understand the evolutionary forces acting on their genes. It also sought to develop a molecular based diagnostic tool for detection of CBSD-associated virus isolates. Comparison of the CP-encoding sequences of CBSD-associated virus isolates collected from Uganda and north-western Tanzania in 2007 and the partial sequences available in Genbank revealed occurrence of two genetically distinct groups of isolates. Two isolates were selected to represent the two groups. The complete genomes of isolates MLB3 (TZ:Mlb3:07) and Kor6 (TZ:Kor6:08) obtained from North-Western (Kagera) and North-Eastern (Tanga) Tanzania, respectively, were sequenced. The genomes were 9069 and 8995 nucleotides (nt), respectively. They translated into polyproteins that were predicted to yield ten mature proteins after cleavage. Nine proteins were typical in the family Potyviridae, namely P1, P3, 6K1, CI, 6K2, VPg, NIa-Pro, NIb and CP, but the viruses did not contain HC-Pro. Interestingly, genomes of both isolates contained a Maf/HAM1-like sequence (HAM1h; 678 nucleotides, 25 kDa) recombined between the NIb and CP domains in the 3’-proximal part of the genomes. HAM1h was also identified in Euphorbia ringspot virus (EuRSV) whose sequence was in GenBank. The HAM1 gene is widely spread in both prokaryotes and eukaryotes. In yeast (Saccharomyces cerevisiae) it is known to be a nucleoside triphosphate (NTP) pyrophosphatase. Novel information was obtained on the structural variation at the N-termini of polyproteins of viruses in the genus Ipomovirus. Cucumber vein yellowing virus (CVYV) and Squash vein yellowing virus (SqVYV) contain a duplicated P1 (P1a and P1b) but lack the HC-Pro. On the other hand, Sweet potato mild mottle virus (SPMMV), has a single but large P1 and has HC-Pro. Both virus isolates (TZ:Mlb3:07 & TZ:Kor6:08) characterized in this study contained a single P1 and lacked the HC-Pro which indicates unique evolution in the family Potyviridae. Comparison of 12 complete genomes of CBSD-associated viruses which included two genomes characterized in this study, revealed genetic identity of 69.0–70.3% (nt) and amino acid (aa) identities of 73.6–74.4% at polyprotein level. Comparison was also made among 68 complete CP sequences, which indicated 69.0-70.3 and 73.6-74.4 % identity at nt and aa levels, respectively. The genetic variation was large enough for dermacation of CBSD-associated virus isolates into two distinct species. The name CBSV was retained for isolates that were related to CBSV isolates available in database whereas the new virus described for the first time in this study was named Ugandan cassava brown streak virus (UCBSV) by the International Committee on Virus Taxonomy (ICTV). The isolates TZ:Mlb3:07 and TZ:Kor6:08 belong to UCBSV and CBSV, respectively. The isolates of CBSV and UCBSV were 79.3-95.5% and 86.3-99.3 % identitical at nt level, respectively, suggesting more variation amongst CBSV isolates. The main sources of variation in plant viruses are mutations and recombination. Signals for recombination events were detected in 50% of isolates of each virus. Recombination events were detected in coding and non-coding (3’-UTR) sequences except in the 5’UTR and P3. There was no evidence for recombination between isolates of CBSV and UCBSV. The non-synonomous (dN) to synonomous (dS) nucleotide substitution ratio (ω) for the HAM1h and CP domains of both viruses were ≤ 0.184 suggesting that most sites of these proteins were evolving under strong purifying selection. However, there were individual amino acid sites that were submitted to adaptive evolution. For instance, adaptive evolution was detected in the HAM1h of UCBSV (n=15) where 12 aa sites were under positive selection (P< 0.05) but not in CBSV (n=12). The CP of CBSV (n=23) contained 12 aa sites (p<0.01) while only 5 aa sites in the CP gene of UCBSV were predicted to be submitted to positive selection pressure (p<0.01). The advantages offered by the aa sites under positive selection could not be established but occurrence of such sites in the terminal ends of UCBSV-HAMIh, for example, was interpreted as a requirement for proteolysis during polyprotein processing. Two different primer pairs that simultaneously detect UCBSV and CBSV isolates were developed in this study. They were used successfully to study distribution of CBSV, UCBSV and their mixed infections in Tanzania and Uganda. It was established that the two viruses co-infect cassava and that incidences of co-infection could be as high as 50% around Lake Victoria on the Tanzanian side. Furthermore, it was revealed for the first time that both UCBSV and CBSV were widely distributed in Eastern Africa. The primer pair was also used to confirm infection in a close relative of cassava, Manihot glaziovii (Müller Arg.) with CBSV. DNA barcoding of M. glaziovii was done by sequencing the matK gene. Two out of seven M. glaziovii from the coastal areas of Korogwe and Kibaha in north eastern Tanzania were shown to be infected by CBSV but not UCBSV isolates. Detection in M. glaziovii has an implication in control and management of CBSD as it is likely to serve as virus reservoir. This study has contributed to the understanding of evolution of CBSV and UCBSV, which cause CBSD epidemic in Eastern Africa. The detection tools developed in this work will be useful in plant breeding, verification of the phytosanitary status of materials in regional and international movement of germplasm, and in all diagnostic activities related to management of CBSD. Whereas there are still many issues to be resolved such as the function and biological significance of HAM1h and its origin, this work has laid a foundation upon which the studies on these aspects can be based.

Development of radiosynthesis methods for 18F-labelled radiopharmaceuticals : General considerations and production of a dopamine transporter radioligand

Positron emission tomography (PET) is a molecular imaging technique that utilises radiopharmaceuticals (radiotracers) labelled with a positron-emitting radionuclide, such as fluorine-18 (18F). Development of a new radiotracer requires an appropriate radiosynthesis method: the most common of which with 18F is nucleophilic substitution with [18F]fluoride ion. The success of the labelling reaction is dependent on various factors such as the reactivity of [18F]fluoride, the structure of the target compound in addition to the chosen solvent. The overall radiosynthesis procedure must be optimised in terms of radiochemical yield and quality of the final product. Therefore, both quantitative and qualitative radioanalytical methods are essential in developing radiosynthesis methods. Furthermore, biological properties of the tracer candidate need to be evaluated by various pre-clinical studies in animal models. In this work, the feasibility of various nucleophilic 18F-fluorination strategies were studied and a labelling method for a novel radiotracer, N-3-[18F]fluoropropyl-2beta-carbomethoxy-3beta-4-fluorophenyl)nortropane ([18F]beta-CFT-FP), was optimised. The effect of solvent was studied by labelling a series of model compounds, 4-(R1-methyl)benzyl R2-benzoates. 18F-Fluorination reactions were carried out both in polar aprotic and protic solvents (tertiary alcohols). Assessment of the 18F-fluorinated products was studied by mass spectrometry (MS) in addition to conventional radiochromatographic methods, using radiosynthesis of 4-[18F]fluoro-N-[2-[1-(2-methoxyphenyl)-1-piperazinyl]ethyl-N-2-pyridinyl-benzamide (p-[18F]MPPF) as a model reaction. Labelling of [18F]beta-CFT-FP was studied using two 18F-fluoroalkylation reagents, [18F]fluoropropyl bromide and [18F]fluoropropyl tosylate, as well as by direct 18F-fluorination of sulfonate ester precursor. Subsequently, the suitability of [18F]beta-CFT-FP for imaging dopamine transporter (DAT) was evaluated by determining its biodistribution in rats. The results showed that protic solvents can be useful co-solvents in aliphatic 18F-fluorinations, especially in the labelling of sulfonate esters. Aromatic 18F-fluorination was not promoted in tert-alcohols. Sensitivity of the ion trap MS was sufficient for the qualitative analysis of the 18F-labelled products; p-[18F]MPPF was identified from the isolated product fraction with a mass-to-charge (m/z) ratio of 435 (i.e. protonated molecule [M+H]+). [18F]beta-CFT-FP was produced most efficiently via [18F]fluoropropyl tosylate, leading to sufficient radiochemical yield and specific radioactivity for PET studies. The ex vivo studies in rats showed fast kinetics as well as the specific uptake of [18F]beta-CFT-FP to the DAT rich brain regions. Thus, it was concluded that [18F]beta-CFT-FP has potential as a radiotracer for imaging DAT by PET.

On measurement of top polarization as a probe of t(t)over-bar production mechanisms at the LHC

In this note we demonstrate the use of top polarization in the study of t (t) over bar resonances at the LHC, in the possible case where the dynamics implies a non-zero top polarization. As a probe of top polarization we construct an asymmetry in the decay-lepton azimuthal angle distribution (corresponding to the sign of cos phi(l)) in the laboratory. The asymmetry is non-vanishing even for a symmetric collider like the LHC, where a positive z axis is not uniquely defined. The angular distribution of the leptons has the advantage of being a faithful top-spin analyzer, unaffected by possible anomalous tbW couplings, to linear order. We study, for purposes of demonstration, the case of a Z' as might exist in the little Higgs models. We identify kinematic cuts which ensure that our asymmetry reflects the polarization in sign and magnitude. We investigate possibilities at the LHC with two energy options: root s = 14TeV and root s = 7TeV, as well as at the Tevatron. At the LHC the model predicts net top quark polarization of the order of a few per cent for M-Z' similar or equal to 1200GeV, being as high as 10% for a smaller mass of the Z' of 700GeV and for the largest allowed coupling in the model, the values being higher for the 7TeV option. These polarizations translate to a deviation from the standard-model value of azimuthal asymmetry of up to about 4% (7%) for 14 (7) TeV LHC, whereas for the Tevatron, values as high as 12% are attained. For the 14TeV LHC with an integrated luminosity of 10 fb(-1), these numbers translate into a 3 sigma sensitivity over a large part of the range 500 less than or similar to M-Z' less than or similar to 1500GeV.

A standard reference frame for the description of Nucleic Acid Base-Pair Geometry

A common point of reference is needed to describe the three-dimensional arrangements of bases and base-pairs in nucleic acid structures. The different standards used in computer programs created for this purpose give rise to con¯icting interpretations of the same structure.1 For example, parts of a structure that appear ``normal'' according to one computational scheme may be highly unusual according to another and vice versa. It is thus dif®cult to carry out comprehensive comparisons of nucleic acid structures and to pinpoint unique conformational features in individual structures