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  • Yun, Jinhyeon (Helsingin yliopisto, 2015)
    It is well-known that prepartum sows have a strong instinct to build a nest before parturition. Under commercial conditions, however, the farrowing crate, widely used in modern pig husbandry, restricts this innate behaviour due to the lack of space, materials or both. Restriction of nest-building (NB) behaviour could generate an increase in physiological stress, resulting in a decrease in endogenous hormones, especially oxytocin (OT), which is recognized for its effect on reproductive and behavioural characteristics in mammals. The role of OT in modulation of maternal behaviour, parturition, and lactation has been demonstrated in a wide range of species, including pigs. It is also known that OT is related to the stress reaction, that it reduces anxiety and that it plays a part in emotional reactions in social situations. This study evaluated the effects of provision of space and abundant nesting materials on actual NB behaviour and circulating OT concentrations in prepartum sows. In addition, it also investigated whether facilitating prepartum NB behaviour could improve postpartum maternal characteristics during early lactation, possibly because of elevated OT concentrations in sows. All sows included in the experiment, approximately seven days before the expected parturition date, were housed in: 1) CRATE: the farrowing crate closed (210 × 80 cm), with provision of a bucketful of sawdust, 2) PEN: the farrowing crate opened, with provision of a bucketful of sawdust, and 3) NEST: the farrowing crate opened, with provision of abundant nesting materials. All sows were confined to the farrowing crates, without additional supply of nesting materials, after the first piglet was delivered until seven days post-parturition. Sow blood samples were collected for hormonal assays via indwelling ear vein catheters on days -3, -2, -1, +1, +2, +4 and +7 from parturition, twice a day. Pigs were video-recorded to observe prepartum NB, postpartum nursing and carefulness behaviour. Blood samples from piglets were collected to determine immunoglobulin concentrations. The longest duration of NB behaviour was observed in NEST sows, followed by PEN and CRATE sows respectively (III), and this duration tended to be correlated with prepartum OT (III). Both prepartum OT and prolactin (PRL) concentrations were greater in NEST than in CRATE and PEN sows (III). An interaction was recorded between prepartum OT and PRL concentrations (III). During the periods from days -3 to +7, NEST also brought about an increase in OT concentrations compared with CRATE and PEN (I), and PRL concentrations in NEST sows were also greater than for CRATE sows during those periods (I). From days one to seven of lactation, PRL concentrations were positively correlated with OT (I). Sows in NEST tended to have higher serum NEFA concentrations from days -3 to +7 (II). Piglet growth during early lactation was slower in CRATE than in PEN (II). Post-natal mortality, including piglet deaths resulting from crushing or other factors, indicated no differences between the three treatments (II). During early lactation, piglet serum IgG and IgM concentrations in NEST tended to be greater than in the other treatments (II). The incidence of carefulness behaviour for NEST sows was greater than for the other treatment sows in early lactation (I), and was correlated with OT during the seven days after parturition (I) and with total duration of prepartum NB behaviour (III). The average duration of successful nursing bouts in early lactation was longer for CRATE than for the other sows (I), and negatively correlated with total duration of prepartum NB behaviour (III). In conclusion, it appears that NB behaviour in prepartum sows could be enhanced by the provision of nesting materials. This, coupled with elevated OT and PRL concentrations, could result in improved sow metabolic status, successful colostrum intake measured via neonatal piglet serum IgG and IgM concentrations, and maternal carefulness behaviour during early lactation. This may have potential to increase piglet survival and growth performance during lactation.
  • Derman, Yağmur (Helsingin yliopisto, 2015)
    The capability of Clostridium botulinum to survive food processing and grow in foods is a substantial hazard to human health. Clostridium botulinum can thrive under stress conditions and the wide genetic variation among C. botulinum strains reflects differences in their growth properties. However, the strain variation in growth, molecular mechanisms, and genetic markers behind the stress tolerance of C. botulinum are poorly understood. This study focused on the variation in growth of Group II C. botulinum strains at temperatures out of the optimal range and characterized the role of the genes that are involved in the stress response of Group I ATCC 3502 and Group II Beluga strains. At extreme temperatures, Group II C. botulinum strains showed significant variation. Some strains showed better growth at 37 °C than their generally accepted optimum growth temperature of 30 °C. Although not absolute, the minimum growth temperatures of 24 Group II strains in our experimental setting were higher than expected. The AFLP typing indicated a weak association between genetic background and growth at extreme temperatures. In addition, clustering analysis showed that the type B and F strains are closely related, since they were clustered together, and the type E strains formed a separate cluster. Our data indicated that Group II C. botulinum strains show considerable differences in their growth properties, and this should be taken into account in designing safety measures against this foodborne pathogen. The study implied that csp genes play important roles in NaCl, pH, and ethanol stress and motility of C. botulinum ATCC 3502. The growth of the cspB mutant was impaired in relation to the wild-type strain under all stress conditions tested, suggesting a universal role in stress response. Additionally, functional cspC was required for efficient growth during NaCl, pH, and ethanol stress. The cspA did not take part in NaCl, pH, and ethanol stress responses, and inactivation of this gene enhanced growth, which suggested a possible growth repressor role. In addition, cspB was not involved in motility, but cspA and cspC were crucial for flagellation and motility. The two-component system CBO2306/CBO2307 of Group I C. botulinum ATCC 3502 and CLO3403/CLO3404 of the Group II Beluga strain were induced up to 4.4- and 3.4-fold, respectively, after cold shock, but not at optimum growth temperatures. This indicated that both systems were involved in rapid response to cold shock. This argument was further confirmed by inactivation of the CBO2306/CBO2307 or CLO3403/CLO3404 genes, which resulted in impaired ability of the mutant strains to grow at low temperatures compared with the wild-type strains. In addition, the histidine kinase encoding clo3403 was required for motility. The cbo2802 that encodes the DEAD-box RNA helicase in C. botulinum ATCC 3502 was cold-inducible. The RT-qPCR analysis showed a 7.6-fold rapid increase in the relative expression of cbo2802 after cold shock. The significance of cbo2802 in the growth at suboptimal temperature of C. botulinum ATCC 3502 was further characterized by disruption of this gene. The mutants without functional cbo2802 showed impaired growth and restricted motility at suboptimal temperature. This study concluded that strains of Group II C. botulinum pose considerable variation in growth properties, which may be of critical importance in predictive modeling of growth in foods. The major cold-shock protein-encoding gene cspB seems to play a universal role in stress response and cspC a role in NaCl, pH, ethanol stress, and motility. The cspA gene is not involved in the stress response of C. botulinum ATCC 3502, but is important for flagella formation. The CBO2306/CBO2307 in Group I C. botulinum ATCC 3502 and CLO3403/CLO3404 in the Group II Beluga strain are part of the cold stress response machinery. Moreover, clo3403 is required for motility. We report for the first time in C. botulinum that DEAD-box RNA helicase is crucial for growth at suboptimal temperature and motility.
  • Herva, Tuomas (Helsingin yliopisto, 2015)
    Animal welfare (AW) is an issue of growing concern in Finland as well as in other developed countries. A public debate has focused on the potential AW problems resulting from current production systems. Possibilities to find mutual benefit for animals, farmers, industry and society have received less attention. According to the reviewed literature the inconsistency of determination and perception of AW appeared to be a major barrier to enhance AW. Farmers should be confident that their measures to promote AW satisfy public opinion and are ecomically sustainable. The main objective of the study was a thorough understanding of relationships between AW and beef production economics to find barriers and opportunities for enhanced AW. A version of the Animal Needs Index (ANI/TGI 35L), modified for Finnish beef production, called A-index was used for AW assessments. The A-Index was modified and evaluated based on Test Theory. On-field associations between A-index and production parameters were determined on 180 farms and over 12 000 bulls using statistical multilevel models. Economic evaluation of AW was based on comparison between cold and warm housing using the confirmed association between AW and production results. AW was associated with good production results. A-Index and the best subset of items used as welfare score (WFS) were covering different aspects of AW. The association between the used measures and production results, reflecting AW in certain degree, can be considered as a proof of the criterion validity of A-Index and WFS. Cold housing with enhanced welfare and bedding based on own straw at a reasonable price was economically favourable. Profitability of cold housing was sensitive to fluctuation in bedding price. Developing a reasonably priced market for bedding material would be a major way to enhance AW. Rubber covered slats were found to be a profitable way to enhance AW in warm housing. A reform of the subsidy system was suggested to be needed to fulfil the aims of the subsidy regime to support AW.
  • Kirk, David (Helsingin yliopisto, 2015)
    Clostridium botulinum presents a risk to food safety through the production of endospores. These spores are highly heat-resistant and may withstand temperatures used in food processing. Despite this, the process of spore formation is poorly understood in C. botulinum. This study aimed to analyse in Group I C. botulinum ATCC 3502 the role of sigma (σ) factors σF, σE, σG, and σK. The role of these σ factors is well known in other spore formers, activating in an ordered cascade to regulate gene transcription during sporulation. To study gene expression during sporulation in C. botulinum ATCC 3502, we identified a suitable normalisation reference gene for reverse-transcription real-time PCR (RT-qPCR). Mutants of sigF, sigE, sigG, and sigK were examined on the transcriptional level during sporulation, and each strain was characterised for growth and spore formation. Furthermore, the role of σK in stress tolerance was investigated under cold, NaCl, and pH stresses. Transcriptional analysis, from exponential to stationary phases of growth, of eight candidate reference genes was performed. The candidate genes were 16S ribosomal RNA (rrn), the ATP metabolism enzymes adenosine kinase (adK) and glutamate dehydrogenase (gluD), the DNA-binding protein gyrase (gyrA), and ribosome-related proteins alanyl-tRNA synthetase (alaS), GTP-binding Era (era), RNA polymerase β subunit (rpoC) and 30S ribosomal protein S10 (rpsJ). Of these candidates, only 16S rrn was stable during the study period. 16S rrn was used as the normalisation reference gene for RT-qPCR analysis of spo0A, sigF, sigE, sigG, and sigK expression during the same growth period. Expression of spo0A was highest during exponential growth, suggesting a role in early sporulation. Induction of sigF, sigE, and sigG expression occurred on entry into stationary growth, indicating a role in sporulation. Expression of sigK appeared biphasic, being expressed in both exponential and stationary phases, suggesting σK may play a dual role in sporulation. The genes of σF, σE, σG, and σK were mutated using the ClosTron tool. RT-qPCR analysis of the sigF and sigE sense mutants suggested that the sporulation pathway was disrupted in the early stages. This was confirmed by electron microscopy, which showed that all sigF and sigE mutants were unable to form spores. They halted sporulation after asymmetric cell division, stage II of the seven-stage sporulation cycle. The sigG sense mutant showed delayed transcription of the sporulation pathway and both sigG mutants possessed a thin spore coat but no cortex. This indicated that σG may be responsible for cortex, but not coat, formation in C. botulinum. The sigK sense mutant did not express the early-sporulation genes spo0A and sigF. Both sigK mutants appeared to halt sporulation early. Sporulation was restored by complementing the sigK mutation in trans. These results suggested that σK plays an essential role in early sporulation of C. botulinum ATCC 3502, and adds further weight to the possibility of a dual role in sporulation overall in this strain. Expression of sigK was assessed in C. botulinum ATCC 3502 after cold, osmotic (NaCl), and acidic shock. After cold and osmotic shock, expression of sigK was induced. Both sense and antisense sigK mutants were then grown under stress conditions of low temperature, high NaCl, and low pH. Under low temperature and high NaCl conditions, but not in low pH, growth of the mutant strains was negatively affected compared to parent strain growth, suggesting that σK may play a role in tolerance to low temperature and high salinity stress conditions.
  • Hang, Ingrid (Helsingin yliopisto, 2015)
    Considerable evidence suggests that dietary macronutrients impact upon activities and conditions in the gastrointestinal tract (GIT) including: functions and processes, digestive enzymes secretion, microbial ecology and bacteria-derived metabolism. Knowledge about the modulation of canine intestinal microbiota, bacteria-derived metabolic products,intestinal inflammatory status and adaptive exocrine pancreatic secretion in response to macronutrients is limited. However, such information is necessary to investigate further the complex interplay between host and intestinal microbiota in response to changes of diet. The reasearch for this PhD thesis focused upon the changes of the intestinal microbiota,bacteria-ferivedmetabolicproducts,anintestinalinflammatorymarker and pancreatic enzyme profiles of five healthy Beagle dogs in response to being fed three different diets: high-carbohydrate starch (HCS), high-protein greaves-meal (HPGM), or a balanced dry commercial (DC) diet. Every diet was crossed-over and fed to each dog for three 21-day periods. The microbial deoxyribonucleic acid (DNA) was profiled according to its percentageoftheguanine-cytosinecontent(%G+C)inordertodetectthefluctuations in intestinal microbiota. Thereafter, 16S ribosomal ribonucleic acid (16S rRNA) gene amplicons were obtained from the most abundant %G+C peaks and analysed by sequence analysis. The DC diet sample was associated with high abundances of representatives of the orders Clostridiales, Lactobacillales, Coriobacteriales and Bacteroidales. Sequence diversity was highest for the DC diet samples and included representatives of the orders Lactobacillales and Bacteroidales, which were not detected in samples obtained for the HPGM and HCS diets. The HPGM and HCS diets also had reduced numbers of representatives of the family Lachnospiraceae; specifically Clostridium cluster XIVa. The HCS diet favoured the proliferation of representatives of the order Erysipelotrichales, specifically the Clostridium cluster XVIII, whereas the HPGM diet favoured representatives of the order Fusobacteriales. Bacterial metabolism and intestinal inflammatory status were assessed by determining dry matter, pH, ammonia, short-chained fatty acids (SCFAs), and faecal canine calprotectin concentrations. Faecal ammonia concentrations decreased with the HCS diet. All dogs fed the HPGM diet developed diarrhoea, which led to differences in faecal consistency scores and increased faecal pH. Moreover, decreases in propionic and acetic acids coupled with increases in branched-chain fatty acids and valeric acid caused changes in faecal total SCFAs. Faecal canine calprotectin concentration was also higher for the HPGM diet than with the other diets and correlated positively with valeric acid concentrations.8 Dietary effects on digestive enzyme composition in the serum, in jejunal fluid, and in the faeces were studied by determining the following factors: amylase activity, the concentrations of canine trypsin-like immunoreactivity (cTLI), canine pancreatic lipase immunoreactivity (cPLI), and canine pancreatic elastase (cE1) concentrations with the two radioimmunoassays (RIAs) for determining cTLI and cPLI concentrationswere specifically validated for jejunalfluid and faecal specimen analysis. Both RIAs were linear, accurate, precise, and reproducible. Dog specific serum enzyme concentrations did not differ between diets. Feeding the HCS diet was associated with decreased amylase activities and cPLI concentrations in the lower jejunum, when compared to the corresponding cPLI activities of the HPGM and the DC diet. The HPGM diet decreased the concentrations of cPLI and cE1 in faecal samples, but not in the jejunal fluid. In conclusion, all bacterial clusters discovered in this research represent the normal GIT microbiota of canines. The HPGM diet favoured Fusobacterium and this Gram-negative bacterial genus may be associated with the observed elevated inflammation status. The latter was deduced from the observed diarrhoea and elevated levels of canine faecal calprotectin in all dogs fed the HPGM diet. It seems likely that these research results could be associated with the quality and increased or decreased amounts of dietary protein or carbohydrate being available for fermentation by the intestinal microbiota. The limited capacity of pancreatic enzymes to adapt adequately by a change inprofile inresponse to changes indietary components seems to be an essential factor, which influences the nutrient levels available for the intestinal microbiota.
  • Mölsä, Sari (Helsingin yliopisto, 2014)
    Cranial cruciate ligament (CCL) disease is one of the most common causes of lameness in dogs. Surgical treatment is recommended to stabilize the stifle joint, alleviate pain, and delay the progression of osteoarthritis (OA). A variety of surgical techniques has been introduced and can be broken down into the more traditional intracapsular ligament replacement and extracapsular suture techniques and the newer neutralizing dynamic osteotomy techniques. Although an enormous amount of literature is available concerning this disease, surprisingly few studies have assessed surgical outcome with objective evaluation methods and comparison between groups. Previous studies have demonstrated significant improvement of limb function after CCL surgery. However, OA has been shown to progress in most patients, and this may cause deterioration in the function of the surgically treated limb over time. Long-term follow-up studies that include objective evaluation methods designed for assessing orthopedic outcome are needed. This thesis aimed to evaluate the long-term surgical outcome and signs of chronic pain after CCL surgery. Also, outcomes between the intracapsular, extracapsular and osteotomy techniques were compared. A multimodal approach, including an owner questionnaire and assessment of chronic pain using the validated Helsinki Chronic Pain Index (HCPI), orthopedic and radiographic examinations, force plate analysis, and as a new evaluation method, a physiotherapeutic examination was included. To provide reference values, clinically healthy Rottweilers and Labrador Retrievers were examined. A long-term retrospective follow-up with a mean time interval of at least 2.7 years between the surgery and evaluation was conducted. Of the 206 surgically treated dogs that were evaluated by their owners, 31.1% had a HCPI value ≥ 12, indicating pain. Of the 47 dogs evaluated by a veterinarian 31.9% showed a pain response to flexion/extension of the surgically treated joint. In addition to the unilaterally treated CCL rupture, contralateral stifle joint pathology and other orthopedic problems were frequently diagnosed during the evaluation. When symmetry of weight bearing was evaluated in dogs with no other orthopedic findings (n=21),approximately 30% of dogs had decreased dynamic and static weight bearing in the surgically treated limbs. Also, goniometric angles of the surgically treated limbs remained inferior to those of healthy limbs, and impairment of active range of motion was frequently observed. The owner assessments revealed no significant differences between the surgical techniques in long-term outcome. However, differences were seen in clinical evaluation of dynamic and static weight bearing between intracapsular and osteotomy technique groups. Although the retrospective study design and low sample size have to be acknowledged and may cause bias to the results, osteotomy techniques may offer long-term limb function that is superior to that achieved with the intracapsular technique. The low number of dogs treated with the extracapsular technique did not allow comparison of dynamic or static weight bearing to other technique groups.
  • Lilja-Maula, Liisa (Helsingin yliopisto, 2014)
    Canine idiopathic pulmonary fibrosis (CIPF) is an incurable fibrosing lung disease occurring mainly in West Highland White Terriers (WHWTs). The clinical picture of CIPF has many similarities with human idiopathic pulmonary fibrosis (IPF). Signs include dry cough, exercise intolerance, and respiratory difficulties. Prognosis for CIPF and human IPF is poor, and only limited treatment options are available. Histopathological CIPF shares features of both human usual interstitial pneumonia (UIP), the pathological counterpart of human IPF, and other human idiopathic interstitial pneumonia, the non-specific interstitial pneumonia (NSIP). Chronic bronchitis (CB) is the main differential diagnosis for CIPF, but antemortem differentiation can be challenging, as surgical lung biopsies are seldom taken and no clinically useful biomarkers are currently available. The natural history of CIPF has not been previously studied and little is known about the molecular pathophysiology of CIPF. This thesis describes the clinical course, survival, and evaluation of exercise intolerance using the 6-minute walk test in CIPF WHWTs. Bronchoalveolar lavage fluid (BALF) protein expression was studied to find potential biomarkers for CIPF, and aspects of transforming growth factor (TGF)-β related molecular pathways in pathogenesis of CIPF were investigated. In addition, results of TGF-β signaling activity and its known extracellular matrix (ECM) regulatory proteins, latent TGF-β binding protein (LTBP)-1 and fibrillin-2, in CIPF were compared with findings in human IPF/UIP and NSIP. The follow-up study showed that CIPF has a significant negative impact on life expectancy of diseased dogs. The median CIPF-specific survival after onset of clinical signs in WHWTs was 2.7 years, but individual survival varied considerably from only a few months to over 4 years. This variance indicates that CIPF may have either a slow or a rapid disease progression, as also seen in human IPF. In addition, during the disease course acute exacerbations (AEs) occurred. The 6-minute walk distance proved to be an easy and noninvasive parameter to evaluate lung function and level of exercise intolerance in CIPF WHWTs. No significant prognostic factors were identified. The quantitative comparison of BALF proteomes obtained from CIPF WHWTs, CB dogs, and healthy dogs revealed similar changes for CIPF and CB, which suggests a common response to disease processes in these otherwise different lung diseases. Specific biomarkers for CIPF were not identified. The immunohistochemical stainings suggested that increased TGF-β signaling and its ECM regulatory proteins LTBP-1 and fibrillin-2 are part of the molecular pathophysiology of CIPF, as also seen in human IPF/UIP and NSIP. We demonstrated strong expression of activin B, a member of the TGF-β superfamily, in the altered alveolar epithelium of WHWTs with CIPF. Furthermore, activin B was detected in BALF of CIPF WHWTs, most notably in samples from dogs with AE, but not in BALF of healthy WHWTs. This novel finding suggests that activin B is part of the CIPF pathophysiology and might act as a potential marker of alveolar epithelial damage. Our findings add important new knowledge about the natural history and molecular pathophysiology of CIPF in WHWTs and show similarities between CIPF and human IPF. Better understanding of the complex pathogenesis of CIPF and human IPF is crucial for developing novel diagnostic tools and treatment strategies for these yet incurable diseases.
  • Jalanka, Jonna (Helsingin yliopisto, 2014)
    The human gastrointestinal microbiota is a complex ecosystem harbouring trillions of bacteria from hundreds of species. Co-evolution has resulted in a mutually beneficial relationship in which gut bacteria make essential contributions to human health, moreover alterations in the microbiota composition and function have been associated to several disease states. In order to identify such changes in patients, it is essential to characterize the microbiota of healthy subjects. In this thesis the intestinal microbiota of healthy adults were thoroughly investigated and shown to display high subject-specificity and stability over time. Moreover, significant correlations between the microbiota and mild intestinal symptoms were identified, including the association of abdominal pain and bloating with the reduction of health-associated bifidobacteria. The intestinal microbiota of healthy subjects was benchmarked against two conditions that were hypothesized to perturb the microbial composition; bowel cleansing, a normal procedure prior colonoscopy and irritable bowel syndrome (IBS), a common disorder estimated to affect 10% of Finnish population. Adequate bowel cleansing by lavage has been shown to be safe for patients, but its long-term effects on the intestinal microbiota, and especially the potential alterations arising from different dosing regimes have not been addressed previously. We showed that there is a short but drastic reduction of the intestinal microbiota after lavage, however the microbiota recovers back to its original form within two weeks after the treatment. Additionally, we found that the recovery rate is dependent on the dosing of the purgative agent, suggesting that two smaller volumes of the purgative should be preferred over a single larger dose. There is growing evidence of the involvement of the intestinal microbiota in the pathophysiology of different IBS subtypes. However, the microbial component in post-infectious IBS patients has previously remained uncharacterised. In parallel to the characterization of intestinal microbiota of PI-IBS patients, the aim of this work was to address the associations between the intestinal microbiota and patient's clinical characteristics. We identified a bacterial signature of 27 taxa and the abundances of implicated bacteria correlated with clinical markers as well as expression levels of several host gene pathways, all suggesting an impaired gut epithelial barrier function in IBS. Observation of these specific associations between the host and intestinal microbiota may provide novel insights into the origin and mechanistic background of intestinal symptoms in IBS as well as enables novel stratification of the IBS patient material with a different aetiology. In summary, this thesis characterised the intestinal microbiota of healthy individuals and showed how perturbations such as IBS and bowel cleansing disrupt the composition, and detected associations between the microbiota and health parameters of the host. The results have clinical relevance by providing novel and a much needed tool for segregating the IBS patient material objectively as well as providing grounds for choosing the split-dosing regime of the purgative agent as an optimal bowel cleansing method. Furthermore, associations between the microbiota and health markers of the host were detected that will give grounds for future research on the aetiology of IBS.
  • Zhang, Zhen (Helsingin yliopisto, 2014)
    Clostridium botulinum is an infamous pathogen that produces botulinum neurotoxin, the causative agent of potentially fatal, neuroparalytic disease in humans and animals called botulism. Despite decades of research, much remains unknown regarding neurotoxin gene location, toxigenesis, and physiology of C. botulinum. First, it is unclear whether mobile genetic elements can mediate transmission of type E neurotoxin gene. Second, the regulatory circuits of botulinum neurotoxin synthesis remain largely unknown. Third, the physiological mechanism of C. botulinum cold stress tolerance is of great importance in cold chain food processing and preservation, but remains poorly understood. The aim of this study was to investigate the location of the type E botulinum neurotoxin gene, identify the potential regulators of neurotoxin synthesis in Group I C. botulinum type A1 strain ATCC 3502, and to characterize the regulon of the two-component signal transduction system (TCS) CBO0366/CBO0365 and its role in cold stress tolerance in C. botulinum ATCC 3502. A group of 36 C. botulinum type E strains was examined by pulsed-field gel electrophoresis and Southern hybridization with probes specific for type E neurotoxin gene (botE) and orfX1, a member of type E neurotoxin gene cluster. The results suggest that three C. botulinum strains encode botE and orfX1 in large plasmids of about 146 kb in size. In C. botulinum ATCC 3502, TCS CBO0787/CBO0786 was found to negatively regulate botulinum neurotoxin expression. Inactivation of cbo0787 encoding a sensor histidine kinase, or of cbo0786 encoding a response regulator, resulted in significantly elevated neurotoxin gene expression levels and increased neurotoxin production. Recombinant CBO0786 regulator was shown to bind to the conserved -10 site of the core promoters of the ntnh-botA and ha operons, which encode the toxin structural and accessory proteins. Increasing concentration of CBO0786 inhibited BotR-directed transcription from the ntnh-botA and ha promoters, demonstrating direct transcriptional repression of the ntnh-botA and ha operons by CBO0786. CodY, a global regulator conserved in low-G+C Gram-positive bacteria, was shown to positively regulate the botulinum neurotoxin gene expression. Inactivation of codY resulted in decreased neurotoxin gene expression levels and reduced neurotoxin synthesis. Complementation of the codY mutation in trans rescued neurotoxin synthesis, and overexpression of codY in trans caused elevated neurotoxin production. Recombinant CodY was found to bind to probes possessing the neurotoxin gene promoters, but strongly interacted only with a 30-bp region in the promoter of ntnh-botA operon, suggesting regulation of the neurotoxin gene transcription through direct interaction. GTP enhanced the binding affinity of CodY to the ntnh-botA promoter, suggesting CodY-dependent neurotoxin regulation is associated with nutritional status. A total of about 150 genes regulated by TCS CBO0366/CBO0365, were identified by DNA microarray. Inactivation of CBO0365 regulon genes encoding acetone-butanol-ethanol fermentation pathway, phosphate transport and arsenical resistance did not affect the growth of C. botulinum ATCC 3502 at 37 °C, but significantly impacted growth after a temperature downshift to 17 °C. These results suggest TCS CBO0366/CBO0365-regulated acetone-butanol-ethanol fermentation, phosphate transport and arsenical resistance play an important role in cold tolerance in C. botulinum ATCC 3502.
  • Lähteinen, Tanja (Helsingin yliopisto, 2014)
    Lactobacilli are important members of the commensal microbiota of both man and animals, contributing to the health and well-being of the host. Dietary supplementation with lactobacilli to enhance the health and productivity of the host has generated much interest in the production animal sector, especially after the prohibition of in-feed antimicrobials. In the swine rearing industry, disturbances in gastro-intestinal (GI) health, such as diarrhea, are very common, causing significant financial losses and compromising animal welfare. The use of lactobacilli as dietary probiotics to maintain and restore a balanced intestinal microbiota during various stressful situations, like the weaning of piglets, might help to prevent the development of GI infections. In addition to their use as probiotics, lactobacilli are also considered as good candidates for antigen carriers in vaccine applications. In particular, those strains carrying a surface (S) layer are attractive vaccine vectors, as the production of the antigenic epitope in each protein subunit as part of the S-layer lattice would enable the expression of a large number of antigens on the surface of the bacterial cell. In this thesis, the probiotic potential of swine originating lactobacilli for use in swine production was characterized by using both in vitro and vivo methods. The screening assays for selected traits commonly considered important for a putative probiotic, such as acid and bile tolerance, adherence to host structures, ability to inhibit pathogens as well as immunostimulatory patterns, showed that these properties vary considerably, even between strains of the same species. This finding emphasizes the need for careful selection of the putative probiotic strain. Based on these in vitro screening tests, six Lactobacillus strains were selected for use in a multispecies bacterial supplementation, which was assessed in a feeding trial performed in recently weaned piglets. Additionally, a Lactobacillus strain possessing an S layer, namely Lactobacillus brevis ATCC 8287, was used in the feeding trial as a monostrain supplementation. Although the ability of the supplemented strains to survive and colonize within the porcine gut appeared to be limited, both of the supplementations did induce some alterations on the mRNA levels of selected cytokines in the intestinal mucosa, and more pronounced effects were evident with the multispecies supplementation. While these supplementations may be suitable for use as probiotics in swine, additional studies will be needed to explore the effects of the strains on piglet health and immune status in more detail. The genomic characterization of the S-layer protein-carrying L. amylovorus strains revealed that several slp like genes are carried by each of the strains; these genes were named slpA, slpB and slpC. In all of the strains, the major Slp protein was encoded by the slpA gene. Unexpectedly, none of the major S-layer proteins was found to solely mediate adhesion of the strains to IPEC-1 cells. Interestingly, the adhesion efficiency of the strains to IPEC-1 cells did not strictly correlate with their ability to inhibit adhesion of an Escherichia coli strain to the same cells. Thus, apart from competition for binding sites, other mechanisms are also involved in the ability of lactobacilli to inhibit pathogen adhesion.
  • Ahonen, Saija (Helsingin yliopisto, 2014)
    The availability of the genome sequence and genomic tools combined with the unique phenotypic diversity and breed structure has increased interest in dog as a large, physiologically relevant animal model for human genetic research. Dogs suffer from hundreds of hereditary conditions of which many resemble closely disorders in humans. Investigation of the genetic background of these conditions is important to understand the disease mechanisms and pathways for therapeutic options, to identify novel candidate genes for corresponding human conditions, and to inform the breeding programs. Remarkable resources have been established in the University of Helsinki for canine genetic research, including one of the largest canine DNA banks. This study has focused on degenerative eye diseases of unknown genetic cause. We combined efforts in basic and clinical veterinary research with a unique network of collaboration with dog breeders and owners to establish clinically relevant study cohorts for two types of retinal degeneration in Papillon and Phaléne, and in Swedish Vallhund, SV breeds and two different types of glaucoma in Dandie Dinmont Terriers, DDT and Norwegian Elkhounds, NE. Genome wide approaches with the latest genomic tools, including SNP arrays and exome sequencing were then utilized to identify causative loci and genes. We report breakthroughs in all projects and highlight shared molecular etiologies in canine and human vision disorders. The cause of the retinal degeneration in Papillons and Phalénes was found in the CNGB1 gene, which is important for the rod cell function and has been linked before to human retinal degenerations. A unique type of multifocal retinal degeneration was described in SV with association to the upregulation of the MERTK gene. MERTK mutations cause also retinal degeneration in humans. DDTs were found to present a primary closed angle glaucoma (PACG) with severe pectinate ligament dysplasia (PLD). The disease was mapped to a region on canine chromosome 8, which is syntenic to human chromosome 14 with multiple glaucoma loci. However, further studies are required to identify the causative mutation. Finally, we found that the primary glaucoma in NEs is caused by a missense mutation in the ADAMTS10 gene. This gene has been associated with a Weill-Marchesani syndrome (WMS) with ocular and non-ocular abnormalities in humans. This study provides new candidate genes, and mechanisms for human eye disorders, and has identified models for potential therapeutic trials. At the same time breeding programs benefit from novel gene tests to eradicate the blinding conditions from the studied breeds.
  • Hakanen, Janne (Helsingin yliopisto, 2014)
    The development of the forebrain is dependent on controlled regulation of neural stem/progenitor cells, since the vast majority of all cells in the forebrain are generated by them. The cellular processes influencing forebrain development include cell proliferation, migration, differentiation, and apoptosis, which are regulated in a spatiotemporal manner by genetic programs and interactive protein networks in a given environment. In this thesis, the influence of Netrin1 (NTN1), Human papilloma virus (HPV) E6/E7, and actin-bundling protein with BAIAP2 homology (ABBA)proteins on neural stem/progenitor cells cellular processes was studied and evaluated. In vitro methods were used to uncover the cellular processes regulated and affected by these proteins. In addition, in vivo methods were used to assess their possible impact on forebrain development in mice. NTN1 belongs to a conserved family of laminin-related molecules and it is present in the developing and adult mouse brain. NTN1 has been thought to act as a diffusible long or short-range guidance cue, which influences growing axons and migrating cells in either a chemoattractive or repulsive manner, and NTN1 deficiency in the brain causes defects in axon guidance and cell migration. The main issue of this thesis was to determine the developmental impact of NTN1 in the formation of two forebrain structures, the olfactory bulb and the corpus callosum (CC). Olfactory bulb processes arriving odour information from the nasal cavity and transmit this information forward to various brain regions. During development, the different cell types of olfactory bulb are generated in distinct germinal regions of the brain. Projection neurons are mainly generated by the olfactory bulb stem/progenitor cells whereas majority of the interneurons are produced by the progenitors, which migrate from the forebrain germinal zones into the olfactory bulb via rostral migratory stream (RMS). The origin of non-neural cells, astrocytes and oligodendrocytes, is not as well understood as neurons. We observed that NTN1 has a significant impact on the beginning of stem/progenitor cells migration from the forebrain germinal zones into the olfactory bulb. In more detail, the migration of Ntn1 expressing stem/progenitor cells was delayed, which led to an accumulation of these cells in RMS, and to a substantial reduction of GABAergic interneurons and oligodendrocytes in the olfactory bulb. Thus, the results suggest that NTN1 acts mainly as a detachment/release factor for the Ntn1 expressing stem/progenitor cells in the beginning of their migration and this function is mediated in either cell-autonomous or paracrine manner. CC is the largest axon tract in the forebrain and it integrates motor, sensory and cognitive performances between cerebral hemispheres. In mice, the cerebral hemispheres have to fuse in the midline before the commissural axons can cross the midline and form the CC. This interhemispheric fusion normally includes the removal of leptomeningeal cells found between the hemispheres and disruption of pial basal lamina, which is produced and maintained by the leptomeningeal cells. In Ntn1 deficient mice the leptomeningeal cells were not removed and the pial basal lamina remained intact in the hemispheric midline. Thus, the results suggest that NTN1 is required for the interhemispheric fusion, which precedes midline crossing of commissural axons and is a prerequisite for the formation of the CC. ABBA belongs to the Bin amphiphysin Rvs167 (BAR) protein superfamily, which regulates plasma membrane morphology by directly influencing plasma membrane assembly or through rearrangement of the actin cytoskeleton. The functional and expression studies revealed that ABBA participates in the regulation of cell protrusions by enhancing actin dynamics and connecting plasma membrane deformation to actin cytoskeleton. ABBA was present in radial glia-like cells and radial glial cell extensions near meningeal pial basal lamina during CNS development. During early development of brain, interaction between radial glial end-feet and pial basal lamina is required to sustain radial migration and integrity of pial basal lamina. Thus, ABBA may have an important role in formation of radial glial extensions and their connections in CNS. Exquisite balance between proliferation, self-renew and differentiation of stem/progenitor cells is necessary to maintain appropriate tissue homeostasis. HPV16 E6/E7 oncoprotein mediated degradation of p53 and pRb family of proteins seemed to facilitate a defective coupling of proliferation, self-renewal, and differentiation processes inside neural stem/progenitor cells. In addition, these processes also seemed to be susceptible to the environmental signals. Hence, a defective coupling of proliferation, self-renewal, and differentiation processes in neural stem/progenitor cells may lead to an imbalance in normal tissue homeostasis and abnormal growth of tissue in the brain. In summary, even though NTN1, HPV E6/E7, and ABBA proteins affected different cellular processes of neural stem/progenitor cells, all these cellular processes participate in the development of forebrain conformation and, therefore, the future functionality of it. In addition, defects in regulation of neural stem/progenitor cells cellular processes may lead to congenital neural disorders in mice and humans. Thus, these results also increase our overall comprehension of the developmental cellular processes behind the different neural congenital disorders and diseases.
  • Rönnqvist, Maria (Helsingin yliopisto, 2014)
    Human noroviruses (HuNoVs) are a leading cause of foodborne gastroenteritis worldwide and spread easily among humans via the faecal-oral route. A low infective dose, a high viral load in the vomit and faeces of infected persons, a lack of long-term immunity following previous infection, and a high environmental stability of the viruses all enhance the spreading of HuNoV in the population. The aim of this doctoral thesis is to investigate the prevalence of HuNoVs on environmental surfaces and to observe and measure virus transfer during manual food preparation. A method for the detection of HuNoV is optimized and used in the laboratory and also in field studies, both in a resort and in food preparation premises. Finally, ultraviolet light irradiation (UV) is tested as a means to inactivate the HuNoVs from environmental surfaces. HuNoV and its surrogate murine norovirus (MuNoV) were detected from environmental surfaces by swabbing, after which the viruses were eluted from the swabs and their genomes were extracted by a commercial kit. HuNoV and MuNoV genomes were detected using reverse transcription quantitative polymerase chain reaction (RT-QPCR) method using specific primers and probes. The effects of UV on the viruses were investigated both by viability assays (MuNoV) and by RT-QPCR (MuNoV and HuNoV). An enzymatic pre-PCR treatment was conducted before RT-QPCR detection to distinguish infective viruses from non-infective viruses. Out of the four swab materials tested for swabbing HuNoV on surfaces, the recovery rates of the viruses were highest for swabs made of microfiber and polyester. When stored at 4⁰C, HuNoV persisted well in swabs, whereas at 22⁰C, viruses persisted better on swabs moistened by phosphate buffered saline (PBS, pH 7.2) than by glycine buffer pH 9.5. HuNoV and MuNoV transferred easily from the hands to the gloves when gloving. The viruses were also repeatedly transferred to the first recipient surface (left hand, cucumber, and knife) during the sandwich preparation process. Virus-contaminated gloves were estimated to spread HuNoV to the food servings more efficiently than a single contaminated cucumber can during handling. In a resort, where a gastroenteritis outbreak had taken place, HuNoV was detected in 10/36 swabs (27.8%) taken from environmental surfaces and further genotyped as a new variant, GII.4 Sydney_2012. In the field study that was conducted in three food-processing companies with no recently reported outbreaks of gastroenteritis, 5/90 swabs (5.6%) in 2010, 4/168 swabs (2.4%) in 2011, and 7/82 swabs (8.5%) in 2012 were found to be HuNoV GII-positive. The positives were detected in a production line and from the food handlers break room and restroom areas. UV was observed as a potential inactivation method for HuNoV: a loss of infectivity and a 4 log10 reduction of HuNoV surrogate MuNoV were observed when the virus-containing surfaces were exposed to UV dose of 60 mJ/cm2 or higher. Methods based on genome detection seemed to overestimate HuNoV persistence even when samples were pre-treated before the RT-QPCR was conducted. As seen in the studies included in the thesis, HuNoV is transmitted very easily from hands to food and environmental surfaces. Proper hand hygiene combined with effective measures to inactivate HuNoV from surfaces, such as UV, is needed to manage the transmission of the virus. Adequate monitoring of the environment for virus contamination in potential fountainheads of gastroenteritis outbreaks, such as in hospitals and restaurants serving RTE foods, could prevent or restrict HuNoV outbreaks.
  • Liljavirta, Jenni (Helsingin yliopisto, 2014)
    The majority of research studies on immunological mechanisms have been conducted in human or rodent models and the results are generalized in respect of other vertebrates. However, the generation of B cells varies considerably between species. B cells are produced in the bone marrow of rodents and humans throughout life. The initial antibody diversity is produced by the random assembly of a great variety of antibody encoding gene segments. The repertoire of these gene segments is limited in many domestic species such as cattle, sheep, chicken, rabbit and pig and de novo B lymphopoiesis takes place only during fetal and/ or neonatal life. This results in a basal B cell population but one that is capable of being further expanded by other mechanisms. In this thesis, the specific diversification mechanisms in the bovine are explored. Knowledge of immunoglobulin genes is essential in order to distinguish various biological mechanisms, which are involved in antibody repertoire diversification. After the bovine genome was sequenced in 2009, the characterization of the immunoglobulin loci became possible. Previously, the bovine immunoglobulin light chain loci were characterized by only a small number of functional gene segments that had been found. Subsequently, we were able to analyse the heavy chain locus and found a total number of 62 heavy chain variable gene segments, of which 10-20 were verified as functional genes. Many livestock species and chicken rely on gut-associated lymphoid tissue for further expanding their fetal/neonatal antibody repertoire to compensate for the limited effective recombinatorial diversity. In cattle, the ileal Peyer s patch (IPP) is considered to be the major organ for B cell proliferation. In this research, the fingerprints of somatic hypermutation (SHM) in the IPP were analysed. SHM is dependent on activation-induced cytidine deaminase (AID), which contributes to modifying the immunoglobulin variable regions. SHM is conventionally considered to be a secondary diversification mechanism, which is activated with external antigens. AID-mediated SHM was shown to diversify the antibody repertoire in the fetal IPP, before the exposure of exogenous antigen encounters. Junctional diversity is produced by the random additions and excisions of nucleotides between immunoglobulin gene segments that occur during the somatic recombination. Non-templated nucleotides are added by terminal deoxynucleotidyl transferase (TdT). Sequence investigation in this study indicated that TdT-mediated junctional diversity contributes to the diversification of the antibody repertoire in bovine fetuses. Extensive junctional diversity mainly in the heavy chain sequences in bone marrow, ileum and spleen but also to a lesser extent in the light chains was detected in this study. The following model for bovine preimmune repertoire diversification was suggested in this study. First, the restricted immunoglobulin germline repertoire is diversified by junctional diversity in fetal bone marrow. Second, a small population of the B cell clones migrates to the fetal/neonatal IPP. In the IPP, further diversification by AID-mediated SHM takes place, which is associated with extensive proliferation. Third, these clones migrate to other peripheral organs where they are subjected to secondary, antigen-induced modifications. The IPP starts to involute in young animals and the animal survives for the rest of its life by proliferating and differentiating its B cell clones from the peripheral repertoire.
  • Hemmann, Karin (Helsingin yliopisto, 2014)
    Crib-biting in horses is an oral stereotypy. A crib-biting horse grasps a fixed object with its incisor teeth, contracts the lower neck muscles to retract the larynx caudally and draws air into the cranial oesophagus while emitting a characteristic grunt. The prevalence of crib-biting varies among equine populations from 1.8% to 15%. Free-ranging horses are not known to crib-bite. The aims of this study were to assess the differences in plasma concentrations of hormones related to stress or appetite between crib-biting and control horses, and to investigate the inheritance of crib-biting behaviour in horses. In comparing plasma hormone concentrations, the case control pairs were matched as closely as possible for breed, sex and age. Plasma concentrations of leptin, ghrelin, cortisol, β-endorphin and ACTH were determined (8+8 in study I, 14+14 in study II, cases and controls, respectively). The plasma leptin concentrations were lower in verified crib-biters than in the non-crib-biting control horses before receiving concentrates and after feeding. There was a negative correlation between the intensity of crib-biting and the plasma leptin concentration before concentrates and after feeding. In general, the plasma ghrelin concentration was higher in crib-biting horses than their controls. The plasma cortisol concentration was not affected by being a crib-biter; instead, an ordinary physiological circadian rhythm in the cortisol concentration was seen. No time or group effect was detected in plasma ACTH and β-endorphin concentrations. Phenotypic background information on the crib-biting behaviour surveyed through an owner-completed questionnaire was available from 106 crib-biting horses. According to the owners, crib-biting often started after feeding and was associated with suspected stress. Eight genes associated with stereotypic behaviours (Ghrelin, GHS-RIA, Leptin, DRD1, OPRM1, CDH2, Htr1B and SEMA6) were selected for the candidate gene assay performed in a cohort of privately owned horses (98 cases and 135 controls) comprising Finnhorses and half-breds. Validated SNPs were genotyped either by TaqMan assays or Sanger sequencing. Comparison of the allele and genotype frequencies between the cases and controls in each breed separately did not indicate an association with any of the studied genes in either of the breeds.The heritability (h2) of the trait in the Finnhorse population (111 cases, 285 controls) estimated by using linear sire model was 0.68. Our results suggest that leptin and ghrelin may be involved in mediating feeding- and reward-related signals in crib-biting horses. The expression of crib-biting probably involves the interaction between genetic predisposition and a particular environmental stimulus, and horses may consequently inherit a certain behavioural susceptibility that predisposes them to crib-biting behaviour.
  • Vainionpää, Mari (Helsingin yliopisto, 2014)
    Thermographic imaging has been studied and used widely in human and equine medicine, but published data from small animal medicine is still lacking. The primary aim of this study was to obtain basic knowledge of the method of thermographic imaging and to map possible areas of its use in companion animals. To determine the requirements for an optimal thermal camera, three cameras with different resolutions (80 x 80 pixels, 180 x 180 pixels and 320 x 240 pixels) were tested. A total of six thermographic images were taken from the hips of 49 dogs of 26 breeds. Two images were shot with each of the three thermal cameras. Two different persons took the thermographic images with the three cameras. Repeatability between thermographers and interpreters was studied. The usability of basic software for interpreting the thermographic images was examined by having three individuals interpret the thermographic images. The camera with the resolution of 320 x 240 pixels was considered the most suitable for thermographic imaging in dogs and, therefore, the rest of the studies were carried out using this camera. The impact of physical exercise on canine superficial temperature was studied with 47 racing greyhounds during two race days. Four superficial temperature points from the right and left hind leg were selected (Tendo calcaneus, Musculus gastrocnemius, Musculus gracilis and Musculus biceps femoris portio caudalis) to compare the changes in superficial temperatures before and after the race. The temperatures differed by 0 4 degrees at each selected point between the right and left legs after the completion of the race. However, no systematic asymmetry was detected between the dogs left and right side. The superficial temperatures were significantly colder when the ambient temperature was lower. Cats are known to be challenging subjects in clinical examinations and, therefore, more feline-friendly study methods are required. Thermographic imaging was used to detect temperature differences between both sides of 103 cats and, further, as an indicator of potentially painful processes. Both long-haired (n = 26) and short-haired (n = 77) cats were included. The cats that tolerated manual manipulation were also physically palpated. Owners filled in a questionnaire about the behaviour of their cat and estimated whether the cat was in any pain. The questionnaire responses, the owner s estimation of pain on a scale and thermographic imaging with palpation suggested that thermographic imaging is a potential tool of choice in clinical practice for detecting and screening cats potentially in pain. Certain drugs used in sedation and anaesthesia affect cardiovascular function. Peripheral temperature changes induced by distinct sedation protocols in dogs (n = 8) were identified by thermographic imaging from the digital and metatarsal foot pads. The obtained foot pad temperatures were compared to the rectal temperature of the same animal. The results indicated that superficial temperature changes caused by certain sedative drugs can be detected and monitored with thermographic imaging. Our studies suggested that thermographic imaging is a practical clinical method with dogs and cats. However, the effects of physical exercise and medications should be taken into consideration when interpreting thermographic images.
  • Jäntti, Maria (Helsingin yliopisto, 2014)
    Orexins (orexin-A and -B) are neuropeptides with multiple physiological functions, among which regulation of wakefulness and appetite are the best known. Neurons producing orexins are localized in the lateral hypothalamus, from where they send projections to many parts of the brain. Orexins exert their functions by activating two G-protein-coupled receptors, OX1 and OX2. Orexin receptor expression was first reported in the brain, but they are also expressed outside of the central nervous system (CNS). Upon activation, OX1 and OX2 receptors can couple to heterotrimeric G-proteins of different families, including Gq, Gs and Gi/o. Orexin receptor activation can evoke increases in intracellular Ca2+ concentrations via multiple mechanisms, including activation of phospholipase C and increased Ca2+ influx, and it also regulates adenylyl cyclase activity, both positively and negatively. Multiple kinases have also been reported in their signaling cascades, including protein kinases C and D, extracellular signal-regulated kinase, p38, Src, and phosphatidylinositol-3-kinase. Previous studies have suggested interactions between the orexin and endocannabinoid systems. The endocannabinoid system consists of endocannabinoids, which are neuromodulatory lipids produced on demand by neurons, their effectors (CB1 and CB2 cannabinoid receptors, as well as other receptors and channels), and finally the enzymes that degrade them. The cannabinoid and orexin systems have several overlapping functions, such as regulation of pain transmission, appetite, learning, and reward. Evidence for the existence of interactions between these systems has been gained from physiological, but also molecular studies. Even heteromerization of CB1 and OX1 receptors has been reported. In this thesis, the signaling of the OX1 receptor was further investigated, with special emphasis on lipid mediators. Recombinant cells (mainly CHO cells) were employed as model systems. We were able to directly demonstrate the activation of phospholipase D upon OX1 stimulation, and consequently add PLD to the signaling cascades mediating orexin responses. Phospholipase D activation was mediated by a novel protein kinase C isoform, most likely protein kinase Cδ. OX1 receptor activation also leads to the release of two other messengers: arachidonic acid and the endocannabinoid 2-arachidonoylglycerol. In this thesis, the release of these messengers and the pathways leading to their production, upon orexin receptor activation, were investigated in detail for the first time. Powerful arachidonic acid release by cytosolic phospholipase A2 (cPLA2) was observed in recombinant CHO cells. In contrast, 2-arachidonoylglycerol was released by a cascade involving activation of phospholipase C and diacyglycerol lipase; this was observed in CHO, neuro-2a, and HEK293 cells. By utilizing CHO cells in an artificial cell−cell communication assay, we saw that the released 2-arachidonoyl¬glycerol can act as a paracrine messenger, activating neighboring cells expressing CB1 cannabinoid receptors. 2-Arachidonoylglycerol similarly also acted as an autocrine messenger, and co-signaling of OX1 and CB1 receptors upon orexin stimulation of the receptor-co-expressing cells via the "2-arachidonoylglycerol loop" led to the potentiation of ERK activation. This implies that the significance of the previously reported OX1−CB1 interaction is more likely to originate from functional than physical interaction of the receptors. However, the idea of heteromerization between OX1 and CB1 receptors is interesting, and in the final study of the present series, we utilized the bioluminescence resonance energy transfer (BRET) method to investigate constitutive homo- and heteromerization between OX1, OX2, and CB1 receptors. According to our results, all receptor combinations readily form heteromeric complexes when expressed in CHO cells.
  • Palviainen, Mari (Helsingin yliopisto, 2014)
    Acute kidney injury (AKI) is a rapid loss of kidney function, which can be a consequence of ischemic, toxic or obstructive insult to the tubules, a reduction of the filtering capacity of the glomeruli or tubulointerstitial inflammation. The diagnosis and prognosis of AKI are problematic due to the lack of sufficiently specific and sensitive markers. The aims of these studies were to assess how kidney impairment impacts on the urinary proteome and to reveal the pathophysiological processes in kidney tissue caused by toxic insult. Changes in the urine proteome after toxic insult to the kidneys were studied by measuring urine enzyme activities, total protein and creatinine concentrations, and using proteomic methods. The usefulness of urinary enzyme activities in kidney impairment diagnosis was studied with in dogs bitten by Vipera berus berus (common European adder) and in sheep with ketoprofen-induced AKI. In both cases, the urinary enzyme activities were demonstrated to be promising markers of kidney impairment. Two-dimensional gel electrophoresis (2D-GE) and two-dimensional differential gel electrophoresis (2D-DIGE) were used to detect potential new urinary protein markers in diagnosing AKI resulting from intoxication in sheep and dogs. The detected differentially expressed proteins were identified using a peptide mass fingerprinting method with either a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF) or a liquid chromatograph hybrid quadrupole mass spectrometer (LC-MS/MS). As a result, several potential urinary markers of kidney impairment were identified: retinol binding protein 4, calbindin D28k, CD1d, complement C3 and complement C4 in our sheep model of AKI, and alpha-1-antirypsin, β-2-microglobulin, fetuin-B and superoxide dismutase (Cu-Zn) in dogs bitten by the common European adder. The pathophysiological process in kidney tissue in renal impairment was examined using immunohistochemical methods. The possible involvement of calbindin D28k, CD1d and complement (C) components in the pathophysiology of AKI in our sheep model was also investigated. All antigens were localized in kidney tubule epithelial cells and the tubular lumina of the exposed sheep, confirming acute tubular injury detected by histological stains. In summary, the measurement of urine enzyme activities proved to be an efficient method to evaluate kidney function in two domestic animal species. Several potential urinary markers were found, and warrant further investigation in clinical veterinary patients suffering from kidney impairment. The complement system was activated in kidney tissue in the ketoprofen-induced sheep model of AKI.
  • Virtanen, Sonja (Helsingin yliopisto, 2014)
    Yersinia enterocolitica is a foodborne zoonotic pathogen. Among domestic animals, pigs are considered the major reservoir of Y. enterocolitica bioserotype 4/O:3. The pathogen is found in pig carcasses and pluck sets at slaughterhouses. Carcass contamination at the slaughterhouse originates from pigs that are already infected on farms. Considerable variation exists in the prevalence of Y. enterocolitica between different pig farms. The aim of this study was to determine the factors in farm management that can be used to prevent the presence and spread of this pathogen within and between farms. Multiple-locus variable-number tandem repeat analysis (MLVA) has been developed and used for genotyping of Y. enterocolitica strains of human origin. This genotyping method was used here to investigate its discriminatory ability, advantages and limitations, and use in genotyping Y. enterocolitica strains isolated from pigs. Among Y. enterocolitica 4/O:3 strains that originated from humans, pigs, and pork products from four European countries, the use of MLVA was found to have high discriminatory power. Similar MLVA types were detected among humans and pigs, human clinical isolates from limited geographical locations indicating the presence of past unidentified epidemics and also from pigs that originated from the same farms. MLVA proved to be able to detect farm-specific genotypes, but mild variation was common in strains originating from the same farms. Sampling of the farms revealed the spread of similar MLVA types among farms that had previously transported pigs between each other. Pigs were found to be a major source of transmission of this pathogen between all production types, including farrowing, farrow-to-finish, and fattening farms. Piglets from certain breeding farms served as a major source of infection for fattening pigs. These piglets carried farrowing farm-specific MLVA types of Y. enterocolitica to the fattening farm, and the infection spread throughout the fattening unit. Farm management practices and their association with carriage and shedding of Y. enterocolitica in pigs were studied by a purpose-designed questionnaire for farms whose pigs were previously sampled at slaughterhouses. The use of municipal water, organic production, and purchase of feed from a certain feed company were found to be protective factors against the carriage of Y. enterocolitica. In contrast, snout-to-snout contacts between pens and buying feed from another company were discovered as risk factors for fecal shedding of the pathogen. In total, 30 farms were further visited and sampled for enteropathogenic Yersinia, and the management practices and conditions were recorded during each sampling visit. The use of municipal water, the use of an all-in all-out system in the units of weaned piglets and fattening pigs, buying piglets from no more than one supplier at a time, and generous use of bedding were associated with lower prevalence of Y. enterocolitica on farms.
  • Putula, Jaana (Helsingin yliopisto, 2014)
    Neuropeptides orexin-A and orexin-B, and their receptors OX1 and OX2 were first found as regulators of appetite, and later several other functions have been found as well. Orexin peptides and receptors have been researched, especially in the field of drug discovery; however many relevant biochemical properties remain partly unsolved, including the factors determining orexin ligand binding properties and ligand selectivity. The study of these factors was pursued in this thesis. Ligand selectivity was studied with chimaeric orexin receptors. We mapped some of the molecular determinants of orexin receptors that are needed for the selectivity of orexin agonists and OX1-specific antagonist. The second quarter of the orexin receptors seems to be the most important area both for agonist and antagonist selectivity. However, for antagonist selectivity, the third quarter also seems to have a role. Activated orexin receptors cause a strong elevation of intracellular calcium. However, reduction of extracellular calcium attenuates that increase and other orexin receptor signalling, mediated by certain phospholipases and kinases, for instance. It remains unknown, however, how calcium causes these effects. With [125I]-orexin-A, a clear decrease in binding was observed after reduction of the extracellular calcium concentration. Also, we saw a similar reduction in the activities of phospholipase C (PLC) and adenylyl cyclase (AC). The concentration-relationship of calcium was identical for radioligand binding, PLC activation, and AC stimulation, while AC inhibition was more strongly attenuated. When the driving force for calcium influx was reduced with high-K+ medium, the orexin-A-induced PLC activity was more strongly reduced than orexin-A binding. In addition, inhibition of the orexin receptor-operated calcium channels had a more pronounced effect on the PLC activity than on the binding. It is thus suggested that reduction of extracellular calcium concentration both inhibits orexin binding and attenuates enzymatic activity. Orexin-B has higher binding affinity for OX2 than OX1 receptor and [Ala11, D-Leu15]-orexin-B, an orexin-B variant, has been reported to display even higher OX2-selectivity. We observed that [Ala11, D-Leu15]-orexin-B showed much lower OX2-selectivity than originally reported. In addition, the selectivity of both forms of orexin-B was dependent on the cell line. These findings may be caused by biased agonism of the orexin receptor, meaning that the orexin receptor can be found in multiple conformations, each of which can interact differently with an agonist. This result extends our knowledge of orexin ligand binding properties, and the phenomenon should be considered, for instance, when novel agonists for orexin receptors are screened.