Epidemiological studies indicate long-term risks of ionizing radiation on the heart, even at moderate doses. In this study, we investigated the inflammatory, thrombotic and fibrotic late responses of the heart after low-dose irradiation (IR) with specific emphasize on the dose rate. Hypercholesterolemic ApoE-deficient mice were sacrificed 3 and 6 months after total body irradiation (TBI) with 0.025, 0.05, 0.1, 0.5 or 2 Gy at low (1 mGy/min) or high dose rate (150 mGy/min). The expression of inflammatory and thrombotic markers was quantified in frozen heart sections (CD31, E-selectin, thrombomodulin, ICAM-1, VCAM-1, collagen IV, Thy-1, and CD45) and in plasma samples (IL6, KC, MCP-1, TNFα, INFγ, IL-1β, TGFβ, INFγ, IL-10, sICAM-1, sE-selectin, sVCAM-1 and fibrinogen) by fluorescence analysis and ELISA. We found that even very low irradiation doses induced adaptive late responses, such as increases of capillary density and changes in collagen IV and Thy-1 levels indicating compensatory regulation. Slight decreases of ICAM-1 levels and reduction of Thy 1 expression at 0.025–0.5 Gy indicate anti-inflammatory effects, whereas at the highest dose (2 Gy) increased VCAM-1 levels on the endocardium may represent a switch to a pro-inflammatory response. Plasma samples partially confirmed this pattern, showing a decrease of proinflammatory markers (sVCAM, sICAM) at 0.025–2.0 Gy. In contrast, an enhancement of MCP-1, TNFα and fibrinogen at 0.05–2.0 Gy indicated a proinflammatory and prothrombotic systemic response. Multivariate analysis also revealed significant age-dependent increases (KC, MCP-1, fibrinogen) and decreases (sICAM, sVCAM, sE-selectin) of plasma markers. This paper represents local and systemic effects of low-dose irradiation, including also age- and dose rate-dependent responses in the ApoE-/- mouse model. These insights in the multiple inflammatory/thrombotic effects caused by low-dose irradiation might facilitate an individual evaluation and intervention of radiation related, long-term side effects but also give important implications for low dose anti-inflammatory radiotherapy.

Psiguria Arn. is a small genus of Neotropical vines in the Cucurbitaceae that grows in both wet and dry tropical forests from southern Mexico to Paraguay, and on Caribbean islands. The genus is estimated to be very young with natural history characteristics that have contributed to confusing species circumscriptions. The unique relationship of plants in the group with their butterfly pollinators makes Psiguria an interesting and important genus in tropical ecosystems. Both molecular and morphological approaches were used to investigate the monophyly of Psiguria, to elucidate the number of species in the genus, to discover sister relationships, and to identify characteristics for delineating species. Toward that end, an intensive screening of 141 primer combinations in search of phylogenetically informative low-copy nuclear markers was conducted along with a molecular phylogenetic analysis and a complete taxonomic revision of Psiguria. From the screening study, three potentially phylogenetically informative low-copy nuclear markers were discovered for Psiguria, 11 were found to be potentially useful in rosids, and 32 in other angiosperms. DNA sequences for eight chloroplast intergenic spacers (ndhF-rpL32, ndhC-trnV, rps16-trnQ, trnS-trnG, psbZ-trnM, psbM-trnD, rpoB-trnC, and psbE-petL), ITS, and the nuclear serine/threonine phosphatase intron were obtained for 70 samples of Psiguria plus 14 outgroups. Phylogenetic analyses support the monophyly of Psiguria and a sister relationship between P. umbrosa and P. warscewiczii. In the final chapter, two reviews on the genus are presented – one encapsulating the nomenclatural history, and one summarizing 35 years of ecological and natural history studies. In addition, morphological characters were databased, descriptions were written, and maps of geographic distribution were produced for all species. Considering both molecular and morphological data, six species of Psiguria are defined. To distinguish those species missing identifiable morphological characters, a set of DNA barcodes was developed. At least four chloroplast regions are required to differentiate species (ndhC-trnV, rps16- trnQ, rpoB-trnC, and ndhF-rpL32). Because of the absence of many morphological characters, two taxonomic keys are presented – one using male flowers, and the other using the set of DNA barcodes along with consistent leaf characteristics and geographic distribution. / text

Asexual species which never incorporate novel genetic material from other lineages will go extinct faster than sexually reproducing species, because adaptive variability may be lower and a larger number of harmful mutations may accumulate. One form of asexuality, androgenesis, results in offspring that are clones of the father. Both androgenetic and sexual species are found in the clam genus Corbicula. I used genetic data to explore why there are multiple species of androgenetic Corbicula, and whether genetic exchange occurs between species. I found that in North American locations where two invasive, androgenetic species co-occur, restriction digest mapping of rDNA failed to detect recent nuclear exchange. However, in these same locations, mitochondrial markers were shared between species. In places where only one species was found, mitochondrial markers were unique to that species. This suggests androgenetic clams are able to parasitize eggs of closely related species. Whereas maternal mitochondria are retained in the fertilized egg, maternal nuclear chromosomes are expelled, and the mother incubates male clones of another species. To look at possible gene exchange over the long term, I compared phylogenetic tree topologies of one mitochondrial and two nuclear markers from multiple sexual and androgenetic species. Since several androgenetic species share similar or identical alleles, androgenesis seems to have evolved relatively recently in Corbicula. However, since different androgenetic species also have divergent alleles not shared between species, genetic capture of maternal nuclear DNA from other species may rarely occur. This rare capture of genetic material from other species may permit the long-term persistence of androgenesis in Corbicula. / text

Hyperglycemia is prevalent in critical care, as patients experience stress-induced hyperglycemia, even with no history of diabetes. Hyperglycemia has a significant impact on patient mortality and other negative clinical outcomes such as severe infection, sepsis and septic shock. Tight glycemic control can significantly reduce these negative outcomes by reducing hyperglycemic episode, but achieving it remains clinically elusive, particularly with regard to what constitutes tight control and what protocols are optimal in terms of results and clinical effort. The model used in this thesis is validated using an independent data and readily be used for different clinical interventions. Moreover, this model also able to accurately predict clinical intervention outcomes given that the model prediction error is very small, which is better than any other reported model. In particular, model-based glycemic control methods is used to capture patient-specific physiological dynamics, such as insulin sensitivity, SI. To date, sepsis diagnosis has been a great challenge despite advancement in technologies and medical research. Critically, septic patients are often classified by practitioners according to their experience before standard test results can be assessed, as to avoid delay in treatment. Moreover, several scoring systems have also been widely used to represent sepsis condition and better standardization of sepsis definition across different centers. In this thesis, insulin sensitivity, SI, a model-based metric is used to identify sepsis condition based on the finding that SI represents metabolic condition of a patient. Additionally, several clinical and physiological variables obtained during patient’s stay in critical care are also investigated using mathematical computation and statistical analysis to identify relevant metric which can be accurately use for sepsis interventions. Even though information on SI, clinical and physiological variables of a patient are insufficient to determine the sepsis status, these informations have brought to a different perspective of diagnosing sepsis. Microcirculation dysfunction is very common in sepsis. Tracking of microcirculation state among septic patient enable better tracking of patient state particularly sepsis status. The tracking can potentially be done by using a pulse oximeter that can extract additional information related to oxygen extraction level. The processed signals are therefore represent relative absorption of oxyhemoglobin and reduced hemoglobin that can be used to assess microcirculation status. In addition, this thesis focus on the real challenge of early treatment of sepsis and sepsis diagnosis where several potential metabolic markers are investigated. Microcirculation conditions are assessed using a non-invasive method that is generally used in typical ICU settings. In particular, the concept and method used to assess microcirculation and metabolic conditions are developed in this thesis. Finally, the work presented in this thesis can act as a starting point for many other glycemic control problems in other environments. These areas include cardiac critical care and neonatal critical care that share most similarities to the environment studied in this thesis, to general diabetes where the population is growing exponentially world wide. Eventually, this added knowledge can lead clinical developments from protocol simulations to better clinical decision making.

Five atherosclerotic carotid and femoral plaques were sliced longitudinally. Each section was analysed for the concentrations of neopterin, α-tocopherol, TBARS, DOPA, dityrosine, protein carbonyl, protein and cholesterol. The spatial concentrations of the oxidative and inflammatory markers were diverse across and between the individual plaques suggested by the lack of consistent correlations and trends. The only correlation that occurred twice within the individual plaques was a positive correlation between α-tocopherol and cholesterol levels. In the combined plaque analysis which included data from eight previously studied plaques, neopterin, protein carbonyl and protein concentrations all had significant positive correlations and α-tocopherol concentrations positively correlated to cholesterol and negatively to TBARS. Thus overall the level of protein may influence protein carbonyl concentration and α-tocopherol may provide an antioxidant effect towards lipid peroxidation. Furthermore, the plaques were divided into three zones, pre-bifurcation, bifurcation and post-bifurcation, associated with shear stress levels. The neopterin concentrations were significantly high within the pre- and post-bifurcation region and the opposite trend occurred with the to peroxyl radical driven TBARS levels. The protein and cholesterol content in the postbifurcation was high, possibly due to the low and/or oscillatory shear stress occurring at these sites. The overall composition of the plaque, either thrombosed, heavily calcified or neither, also identified significant trends in marker concentrations between the plaques. The calcified plaques had significantly low levels of protein, cholesterol, α-tocopherol, DOPA and dityrosine whereas the thrombosed plaques had significantly high protein, α-tocopherol and dityrosine concentrations. The medication and symptoms presented by the patient had no major influence of the overall concentration of the markers within the plaques. Therefore even though individually the plaques have varied biochemical compositions, common influences were dictate the spatial and overall concentration of the markers within and across the plaques. Further potential markers were investigated for detection within plaque. AAS and GGS for replacement of the protein carbonyl assay as a more specific marker for protein oxidation, as well as the oxysterol 7-ketocholesterol detected simultaneously during α-tocopherol analysis. The 7-ketocholesterol would increase the information on lipid oxidation occurring in the plaque without increasing the volume of the limited homogenate required for the analysis. Investigation was also carried out on the mechanism of protein oxidation in human plasma that may provide mechanisms and interactions to protein oxidation within plaques.

Today, some kind of laboratory results is required for around 70% of the diagnostics and follow-ups for diseases. In many of the cases the time from sampling to a result is very critical. Therefore the discussion of how to improve this situation has begun. For many analyses serum has been the routine choice for a long time but now it is disputed. After blood collection in a serum tube it is essential to wait 30-60 minutes before centrifugation and analysis of the sample, a long time for someone in an acute state. Other problems like post centrifugation clots of fibrin causing false results or time-consuming reruns of the sample have also been reported. These problems have initiated the laboratory in Hudiksvall’s hospital to find out an alternative to the common serum sampling.In this report, the differences between serum and lithium heparin plasma for 31 analyses has been evaluated. Paired blood samples, one serum and one plasma, were collected for routine, hormonal and for tumor markers analyses and analyzed in a Cobas c501, e411 or e601 (ROCHE). The results of the analyzed samples were compared to each other by statistical analysis.The results prove that serum and lithium heparin plasma is equal for ALT, GGT, NT-proBNP, FT3, FT4, cobalamin, LH, prolactin, TSH, CA19-9, CEA and PSA. The results also prove that serum and lithium heparin plasma is not equal for 19 other analyses. Therefore, a shift between different types of sampling is not to be recommended without further evaluations.

There is a pressing need to develop strategies to reduce the emergence of macrocyclic lactone anthelmintic resistance in sheep flocks. Management practices aimed at maintaining anthelmintic susceptible nematodes in refugia while achieving a satisfactory level of production may prove to be useful. However, sensitive molecular tests are required to monitor the subtle effects of these practices on the frequency of resistance alleles within nematode populations. To-date, conventional studies of candidate genes coding for the known methods of action of macrocyclic lactone anthelmintics have produced a complex picture, highlighting the relevance of different approaches to the identification of resistance markers. This thesis describes the development of a single nematode parent genetic crossing method and discusses its application to identify molecular markers for anthelmintic resistance. Parasitological and molecular verification of successful inbreeding of the MHco3 strain of H. contortus derived from the progeny of a genetic cross between single nematode parents is described. The single parent genetic crossing method has enabled the production of diverse inbred lines of the MHco3 H. contortus and may prove useful for genome assembly, or for the development of a genetic map. The study has afforded insights to the biology of H. contortus and effects of host immunity on nematode parasites. New information is presented concerning the period during which adult female nematodes continue to shed fertilised eggs after removal of males, the development of unfertilised H. contortus eggs, and the population genetics of mixed infections of two different strains of H. contortus. Novel backcrossing experiments initially between a macrocyclic lactone resistant (MHco4 or MHco10) and a susceptible (MHco3) strain of H. contortus and then between ivermectin treated backcross generations and the parental susceptible strain are described. The resources provided by these experiments should enable comparative genomic analysis and conventional molecular biology to identify resistance genes derived from the parental resistant strains in fourth backcross generations that are the same as a parent ivermectin susceptible population, apart from the presence of alleles linked to anthelmintic resistance, derived from parent resistant strains.

The purpose of this thesis was to investigate overtraining (OT) and burnout (BO) in young athletes. Very little data on the incidence of OT in young athletes is available, hence the purpose of the 1st study was to investigate the prevalence and symptomology of NFOR (non-functional overreaching) and OT in young English athletes practicing different sports and competing across all competitive levels. Data from 376 young athletes (age 15.00 ± 1.97 y) indicated that 29 % had experienced at least one episode of NFOR/OT, and that NFOR/OT was significantly higher at national and international competitive levels (p < 0.01). Presenting symptomology was similar to that reported in adults, with both training and non-training stressors identified as important associates: losses of appetite during periods of hard training, frequent injuries and feelings of a lack of recovery from training, combined with apathy, feeling intimidated by opponents, and being “moody” were the most frequently reported physical and psychological symptoms, respectively. Training load, the commonly believed cause of NFOR/OT, had no significant association with NFOR/OT incidence; however competitive level and gender were significant predictors of NFOR/OT, albeit of a small explained variance (~4%). This study demonstrated that NFOR/OT is evident in young athletes and that the associated factors are multifactorial. The 2nd study monitored prospectively, 4 national-level female swimmers during an 11-month competitive season. Two swimmers (16.00 ± 1.41 y) were diagnosed as OT based on performance decrements (mean decrement of 9 %). One of the OT swimmers (OT2) presented with the classical psychophysiological profile, i.e. high monthly training volumes, low IgA concentration, depressed maximal lactates and high self-reported distress. Conversely the other OT swimmer (OT1) only presented with high Training Distress Scale (TDS) scores. These findings show that both, OT is a complex problem to diagnose and that it’s approach needs to be individualized. The 3rd study investigated the acute psycho-physiological responses to a 6-day training camp in 4 young female swimmers (15.00 ± 1.21 y), of which one was OT and another burnt out (OT1 swimmer from study 2). Both mal-adapted athletes showed performance decrements of ~8 % that lasted for more than 6 months. The OT swimmer, unlike her BO friend, showed a depressed IgA concentration, an unresponsive cortisol, reduced maximal lactate production, and high psychological distress, measured by the TDS. Both swimmers reported slower reaction times on the Stroop test, with the BO swimmer evidencing the worst performance. Finally, the BO swimmer reported very high scores on the Athlete Burnout Questionnaire (ABQ; reduced sense of accomplishment = 4.3; emotional/physical exhaustion = 2.6; sport devaluation = 3.7). This study showed that the psychophysiological profile of an OT swimmer may differ considerably from a BO athlete, with the ABQ being potentially the most efficient tool to diagnose BO. Once more, the individuality of the profiles reinforces the importance of investigating this phenomenon on a case by case basis. The final study used Interpretative Phenomenological Analysis to investigate the psychosocial nature of OT and BO in a 15 year-old female swimmer (OT1 and BO from studies 2 and 3, respectively) and revealed how multiple sources of training and non-training stressors all combined to negatively affect the athlete. The swimmer revealed a past in which she experienced rapid success at an early age and a training mentality of “the more, the better” which was promoted by parents, coaches and herself. Her strong unidimensional identity – centred on swimming – provided few recreational or social opportunities outside the sport. She also reported communication difficulties with her coaches, unwelcome changes in coaching staff, periods of separation from her family, and an over-involved mother. The findings of this thesis suggest that NFOR/OT and BO are issues that many young athletes have to contend with during their sporting careers. The multifactorial nature of these conditions mean that any screening, prevention or recovery interventions must address the problem from a holistic standpoint and as such, Ken Wilber’s (1998) Integral Model is proposed as a suitable framework through which this condition may be investigated in young athletes.

Colorectal neoplasia (CRN) continues to be a leading cause of morbidity and mortality in the developed world and with westernisation, similar trends are now emerging in the developing world. Although secondary prevention through screening programmes has reduced mortality, uptake remains poor due to the invasive nature of colonoscopy, which also exerts increased costs to the health care system. Primary prevention remains the ultimate aim to reduce the morbidity and mortality associated with CRN. In this regard, chemoprevention strategies through regular use of aspirin and other NSAIDS have showed great promise but the associated significant side-effects of these drugs has prevented their routine clinical application for this purpose. Hence there is an urgent need for the identification of safer alternatives for primary prevention of CRN. In parallel to this search, better understanding of the molecular pathogenesis of CRN to identify biomarkers that aid in stratification of at risk individuals would also help. In this regard, the role of chronic inflammation and the influence of host genetics in the pathogenesis of CRN has been the focus of extensive research in recent years. However there is a lack of studies which have investigated these associations in an exclusively screened population, which confers some advantages for this type of investigation. Firstly, most of the screened subjects are relatively healthy, asymptomatic and with no significant co-morbidities, the factors which could otherwise influence the levels of inflammatory markers. Secondly, the screened population is in the 50 to74 year age group which represents the group with a high prevalence of CRN and hence increasing the possibility of finding associations which would be more relevant and generalisable. Thirdly, the selected controls match the cases in all important respects, apart from having CRN, thus increasing the validity of the findings in this population. The Grampian region was one of the first in the UK to participate in the National Colorectal Cancer Screening Programme and this resource gave the ideal opportunity to conduct research involving an exclusively screened population. Utilising this cohort, the current thesis addressed three important aspects of the association between inflammation and CRN. Firstly the investigation of the association of inflammatory genotype, inflammatory phenotype and CRN risk. Secondly the impact of environmental factors, specifically dietary antiinflammatory salicylic acid intakes on CRN risk. And finally assessing if inflammation, and hence in the long term risk of CRN, could be attenuated through a comprehensive anti-inflammatory dietary supplementation in the form of a randomised dietary intervention clinical trial. The study of the association of polymorphisms in key inflammatory genes (IL1B- 31, IL8-251, IL6-174, TNFα-308, IL10-1082, IL10-592, PTGS2-765, and IL1RN VNTR) and CRN risk showed some significant findings. A novel finding was that the homozygous IL1B-31C*C genotype was associated with statistically significant increased risk of CRN, OR 1.63 (95% CI 1.06-2.50) whilst the IL8-251 A*A genotype increased the propensity of having high risk lesions by two-fold (OR 2.04; 95% CI 1.02-4.07). The study of circulating inflammatory marker levels in subjects in whom the CRN was in-situ showed that increased CRP levels were associated with increased risk of CRN, OR 1.55 (95% CI 1.00-2.39). Increased levels of IL8 were associated with increased risk of having a high risk lesion, OR 2.57 (95% CI 1.03-6.44). In a sub group of subjects, it was observed that levels IL8 and CRP decreased following polypectomy (mean IL8 20.3 pg/ml to 14.9 pg/ml, p=0.05 and mean CRP 5.99 mg/l to 3.82 mg/l, p=0.07) raising an important question regarding the sequence of the inflammation-neoplasia cascade, “Is inflammation the cause or the effect of neoplasia?” The study of the association of dietary salicylic acid (SA) and CRN using the newly constructed SA database showed that high levels of total SA (aspirin and dietary SA) intakes were associated with a 75% and moderate levels with a 67% decreased risk of CRN. But dietary SA on its own showed no significant effect on CRN risk probably because of low intake levels in the current cohort. Applying the SA database to populations with higher dietary SA intake would help to further explore its association with CRN risk. The randomised clinical trial examining the effect of a combined antiinflammatory dietary supplement (curcumin, omega-3 PUFA and polyphenols rich fruit smoothie) on markers of inflammation in subjects who had adenomatous colorectal polyps removed showed that the inflammatory marker levels in the control group who just continued their habitual diet remained stable without any statistically significant changes at 6 weeks compared to the baseline. Whereas following 6 weeks of dietary intervention, there was marginally significant increase in IL8 and IL1B levels. One of the possible mechanisms for increase in pro-inflammatory marker levels in the intervention group was the weight gain seen in the intervention group. In the intervention group, the post-intervention mean weight (86.80kgs) was significantly higher than the pre-intervention mean weight (85.38 kgs). In summary, the findings from these investigations suggest that a proinflammatory genotype (IL1B-31C*C and IL8-251 A*A) and elevated circulating inflammatory marker levels (CRP and IL8) are associated with increased risk of CRN. And along with the findings that regular NSAID use and total dietary SA are associated with decreased risk of CRN, our data point to inflammation as an underlying pathogenetic mechanism in CRN. The pilot clinical trial has demonstrated that a clinical trial with combined dietary supplementation is feasible, but challenging. The anti-inflammatory dietary intervention strategy employed to reduce the inflammatory markers did not achieve the desired effect and hence more research is required to establish the ideal delivery strategy of the anti-inflammatory dietary agents. Once this is established, dietary chemoprevention of CRN as a safe alternative should be a realistic achievable goal in the future.

This research evaluated the potential of activation and attachment, as sequential companion biomarkers of phagocytosis by earthworm, Lumbricus terrestris, immunoactive coelomocytes for use in immunotoxicology. The potential was assessed by exposing earthworms to sublethal concentrations of CuSO4 and Arochlor 1254®, chemicals used as reference or standard immunotoxicants.