Regulation of Kinin receptor Expression in Heart Failure with a Focus on Vascular Endothelium

Accumulating evidence show that kinins, notably bradykinin (BK) and kallidin, have cardioprotective effects. To these include reduction of left ventricular hypertrophy (LVH) and progression of heart failure. The effects are mediated through two G protein-coupled receptors- bradykinin type-2 receptor (BK-2R) and bradykinin type -1 receptor (BK-1R). The widely accepted cardioprotective effects of BK-receptors relate to triggering the production and release of vasodilating nitric oxide (NO) by endothelial cells. They also exert anti-proliferative effects on fibroblasts and anti-hypertrophic effects on myocytes, and thus may play an essential role in the cardioprotective response to myocardial injury. The role for BK-1Rs in HF is based on experimental animal models, where the receptors have been linked to cardioprotective- but also to cardiotoxic -effects. The BK-1Rs are induced under inflammatory and ischemic conditions, shown in animal models; no previous reports, concerning BK-1Rs in human heart failure, have been presented. The expression of BK-2Rs is down-regulated in human end-stage heart failure. Present results showed that, in these patients, the BK-1Rs were up-regulated, suggesting that also BK-1Rs are involved in the pathogenesis of human heart failure. The receptors were localized mainly in the endothelium of intramyocardial coronary vessels, and correlated with the increased TNF-α expression in the myocardial coronary vessels. Moreover, in cultured endothelial cells, TNF-α was a potent trigger of BK-1Rs. These results suggest that cytokines may be responsible for the up-regulation of BK-1Rs in human heart failure. A linear relationship between BK-2R mRNA and protein expression in normal and failing human left ventricles implies that the BK-2Rs are regulated on the transcriptional level, at least in human myocardium. The expression of BK-2Rs correlated positively with age in normal and dilated hearts (IDC). The results suggest that human hearts adapts to age-related changes, by up-regulating the expression of cardioprotective BK-2Rs. Also, in the BK-2R promoter polymorphism -58 T/C, the C-allele was accumulated in cardiomyopathy patients which may partially explain the reduced number of BK-2Rs. Statins reduce the level of plasma cholesterol, but also exert several non-cholesterol-dependent effects. These effects were studied in human coronary arterial endothelial cells (hCAEC) and incubation with lovastatin induced both BK-1 and BK-2Rs in a time and concentration-dependent way. The induced BK-2Rs were functionally active, thus NO production and cGMP signaling was increased. Induction was abrogated by mevalonate, a direct HMG-CoA metabolite. Lovastatin is known to inhibit Rho activation, and by a selective RhoA kinase inhibitor (Y27632), a similar induction of BK-2R expression as with lovastatin. Interestingly a COX-2-inhibitor (NS398) inhibited this lovastatin-induction of BK-2Rs, suggesting that COX-2 inhibitors may affect the endothelial BK-2Rs, in a negative fashion. Hypoxia is a common denominator in HF but also in other cardiovascular diseases. An induction of BK-2Rs in mild hypoxic conditions was shown in cultured hCAECs, which was abolished by a specific BK-2R inhibitor Icatibant. These receptors were functionally active, thus BK increased and Icatibant inhibited the production of NO. In rat myocardium the expression of BK-2R was increased in the endothelium of vessels, forming at the border zone, between the scar tissue and the healthy myocardium. Moreover, in in vitro wound-healing assay, endothelial cells were cultured under hypoxic conditions and BK significantly increased the migration of these cells and as Icatibant inhibited it. These results show, that mild hypoxia triggers a temporal expression of functionally active BK-2Rs in human and rat endothelial cells, supporting a role for BK-2Rs, in hypoxia induced angiogenesis. Our and previous results show, that BK-Rs have an impact on the cardiovascular diseases. In humans, at the end stage of heart failure, the BK-2Rs are down-regulated and BK-1Rs induced. Whether the up-regulation of BK-1Rs, is a compensatory mechanism against the down-regulation of BK-2Rs, or merely reflects the end point of heart failure, remains to bee seen. In a clinical point of view, the up-regulation of BK-2Rs, under hypoxic conditions or statin treatment, suggests that, the induction of BK-2Rs is protective in cardiovascular pathologies and those treatments activating BK-2Rs, might give additional tools in treating heart failure.

Process Analytical Technology Approach on Fluid Bed Granulation and Drying : Identifying Critical Relationships and Constructing the Design Space

Fluid bed granulation is a key pharmaceutical process which improves many of the powder properties for tablet compression. Dry mixing, wetting and drying phases are included in the fluid bed granulation process. Granules of high quality can be obtained by understanding and controlling the critical process parameters by timely measurements. Physical process measurements and particle size data of a fluid bed granulator that are analysed in an integrated manner are included in process analytical technologies (PAT). Recent regulatory guidelines strongly encourage the pharmaceutical industry to apply scientific and risk management approaches to the development of a product and its manufacturing process. The aim of this study was to utilise PAT tools to increase the process understanding of fluid bed granulation and drying. Inlet air humidity levels and granulation liquid feed affect powder moisture during fluid bed granulation. Moisture influences on many process, granule and tablet qualities. The approach in this thesis was to identify sources of variation that are mainly related to moisture. The aim was to determine correlations and relationships, and utilise the PAT and design space concepts for the fluid bed granulation and drying. Monitoring the material behaviour in a fluidised bed has traditionally relied on the observational ability and experience of an operator. There has been a lack of good criteria for characterising material behaviour during spraying and drying phases, even though the entire performance of a process and end product quality are dependent on it. The granules were produced in an instrumented bench-scale Glatt WSG5 fluid bed granulator. The effect of inlet air humidity and granulation liquid feed on the temperature measurements at different locations of a fluid bed granulator system were determined. This revealed dynamic changes in the measurements and enabled finding the most optimal sites for process control. The moisture originating from the granulation liquid and inlet air affected the temperature of the mass and pressure difference over granules. Moreover, the effects of inlet air humidity and granulation liquid feed rate on granule size were evaluated and compensatory techniques used to optimize particle size. Various end-point indication techniques of drying were compared. The ∆T method, which is based on thermodynamic principles, eliminated the effects of humidity variations and resulted in the most precise estimation of the drying end-point. The influence of fluidisation behaviour on drying end-point detection was determined. The feasibility of the ∆T method and thus the similarities of end-point moisture contents were found to be dependent on the variation in fluidisation between manufacturing batches. A novel parameter that describes behaviour of material in a fluid bed was developed. Flow rate of the process air and turbine fan speed were used to calculate this parameter and it was compared to the fluidisation behaviour and the particle size results. The design space process trajectories for smooth fluidisation based on the fluidisation parameters were determined. With this design space it is possible to avoid excessive fluidisation and improper fluidisation and bed collapse. Furthermore, various process phenomena and failure modes were observed with the in-line particle size analyser. Both rapid increase and a decrease in granule size could be monitored in a timely manner. The fluidisation parameter and the pressure difference over filters were also discovered to express particle size when the granules had been formed. The various physical parameters evaluated in this thesis give valuable information of fluid bed process performance and increase the process understanding.

Levottomat Tuhkimot : Etnografinen tutkimus nuorten tyttöjen suhtautumisesta väkivaltaan Levottomat Tuhkimot -tyttöväkivaltaprojektin keskusteluryhmässä

In recent years girls’ increased violence has been a topical issue. Including the voices of the girls’ themselves engaged to violent offending is therefore essential. The goal for this research was to make sense of girls’ stance toward violence in the project Restless Cinderellas’ discussion group for violent girls. The methodological approach was ethnographic and the data was collected by using the method of participant observation. Girls’ stance toward violence was explored by asking the data following questions: 1. What is the purpose of violent behaviour? 2. Which issues motivate for violence? 3. What alternatives exist for violent behaviour? In a thick description of research material this study also aimed to chart if there was any change in girls’ attitudes due to participation on violence preventative discussion group. The ethnographic data was extensive and consisted of observations, start, - middle, - and end point group discussion records, fieldnotes, self report-questionnaires and feedback documentary. The four girls that participated on this study were 14 -15- years old by the time of research. They came from one elementary school in the capital region. They were chosen to the group based on NFG’s youth workers interview. The girls were allocated to the group by the school’s student welfare group because of their violent behaviour on school time. The analysis was qualitative and the concepts were created through theory triangulation. These concepts were exploited to exam girls’ violent attitudes. The study shows that violence was used to pitch for justice, lesson and respect. The factors that motivated on using violence were experienced insult, betrayal and concatenation of violence. Alternatives for violence were the circle of success and value consciousness. In the dialectics between the research documentaries some changes in girls’ attitudes heaved into sight. However, the study could not show reliable connection between the changes and participation on discussion group.

Nephrin and the Pathogenesis of Nephropathy - Emphasis on Type I Diabetes

End-stage renal disease is an increasingly common pathologic condition, with a current incidence of 87 per million inhabitants in Finland. It is the end point of various nephropathies, most common of which is the diabetic nephropathy. This thesis focuses on exploring the role of nephrin in the pathogenesis of diabetic nephropathy. Nephrin is a protein of the glomerular epithelial cell, or podocyte, and it appears to have a crucial function as a component of the filtration slit diaphragm in the kidney glomeruli. Mutations in the nephrin gene NPHS1 lead to massive proteinuria. Along with the originally described location in the podocyte, nephrin has now been found to be expressed in the brain, testis, placenta and pancreatic beta cells. In type 1 diabetes, the fundamental pathologic event is the autoimmune destruction of the beta cells. Autoantibodies against various beta cell antigens are generated during this process. Due to the location of nephrin in the beta cell, we hypothesized that patients with type 1 diabetes may present with nephrin autoantibodies. We also wanted to test whether such autoantibodies could be involved in the pathogenesis of diabetic nephropathy. The puromycin aminonucleoside nephrosis model in the rat, the streptozotocin model in the rat, and the non-obese diabetic mice were studied by immunochemical techniques, in situ -hybridization and the polymerase chain reaction -based methods to resolve the expression of nephrin mRNA and protein in experimental nephropathies. To test the effect of antiproteinuric therapies, streptozotocin-treated rats were also treated with aminoguanidine or perindopril. To detect nephrin antibodies we developed a radioimmunoprecipitation assay and analyzed follow-up material of 66 patients with type 1 diabetes. In the puromycin aminonucleoside nephrosis model, the nephrin expression level was uniformly decreased together with the appearance of proteinuria. In the streptozotocin-treated rats and in non-obese diabetic mice, the nephrin mRNA and protein expression levels were seen to increase in the early stages of nephropathy. However, as observed in the streptozotocin rats, in prolonged diabetic nephropathy the expression level decreased. We also found out that treatment with perindopril could not only prevent proteinuria but also a decrease in nephrin expression in streptozotocin-treated rats. Aminoguanidine did not have an effect on nephrin expression, although it could attenuate the proteinuria. Circulating antibodies to nephrin in patients with type 1 diabetes were found, although there was no correlation with the development of diabetic nephropathy. At diagnosis, 24% of the patients had these antibodies, while at 2, 5 and 10 years of disease duration the respective proportions were 23%, 14% and 18%. During the total follow-up of 16 to 19 years after diagnosis of diabetes, 14 patients had signs of nephropathy and 29% of them tested positive for nephrin autoantibodies in at least one sample. In conclusion, this thesis work could show changes of nephrin expression along with the development of proteinuria. The autoantibodies against nephrin are likely generated in the autoimmune process leading to type 1 diabetes. However, according to the present work it is unlikely that these autoantibodies are contributing significantly to the development of diabetic nephropathy.

Primary total hip arthroplasty for primary osteoarthritis in Finland : A national register based analysis

The first aim of the current study was to evaluate the survival of total hip arthroplasty (THA) in patients aged 55 years and older on a nation-wide level. The second aim was to evaluate, on a nation wide-basis, the geographical variation of the incidence of primary THA for primary OA and also to identify those variables that are possibly associated with this variation. The third aim was to evaluate the effects of hospital volume: on the length of stay, on the numbers of re-admissions and on the numbers of complications of THR on population-based level in Finland. The survival of implants was analysed based on data from the Finnish Arthroplasty Register. The incidence and hospital volume data were obtained from the Hospital Discharge Register. Cementless total hip replacements had a significantly reduced risk of revision for aseptic loosening compared with cemented hip replacements. When revision for any reason was the end point in the survival analyses, there were no significant differences found between the groups. Adjusted incidence ratios of THA varied from 1.9- to 3.0-fold during the study period. Neither the average income within a region nor the morbidity index was associated with the incidence of THA. For the four categories of volume of total hip replacements performed per hospital, the length of the surgical treatment period was shorter for the highest volume group than for the lowest volume group. The odds ratio for dislocations was significantly lower in the high volume group than in the low volume group. In patients who were 55 years of age or older, the survival of cementless total hip replacements was as good as that of the cemented replacements. However, multiple wear-related revisions of the cementless cups indicate that excessive polyethylene wear was a major clinical problem with modular cementless cups. The variation in the long-term rates of survival for different cemented stems was considerable. Cementless proximal porous-coated stems were found to be a good option for elderly patients. When hip surgery was performed on with a large repertoire, the indications to perform THAs due to primary OA were tight. Socio-economic status of the patient had no apparent effect on THA rate. Specialization of hip replacements in high volume hospitals should reduce costs by significantly shortening the length of stay, and may reduce the dislocation rate.

Total hip arthroplasty in young patients : with special references to patients under 55 years of age and to patients with developmental dysplasia of the hip

There is an ongoing controversy as to which methods in total hip arthroplasty (THA) could provide young patients with best long-term results. THA is an especially demanding operation in patients with severely dysplastic hips. The optimal surgical treatment for these patients also remains controversial. The aim of this study was to evaluate the long-term survival of THA in young patients (<55 years at the time of the primary operation) on a nation-wide level, and to analyze the long-term clinical and radio-graphical outcome of uncemented THA in patients with severely dysplastic joints. Survival of 4661 primary THAs performed for primary osteoarthritis (OA), 2557 primary THAs per-formed for rheumatoid arthritis (RA), and modern uncemented THA designs performed for primary OA in young patients, were analysed from the Finnish Arthroplasty Register. A total of 68 THAs were per-formed in 56 consecutive patients with high congenital hip dislocation between 1989-1994, and 68 THAs were performed in 59 consecutive patients with severely dysplastic hips and a previous Schanz osteotomy of the femur between 1988-1995 at the Orton Orthopaedic Hospital, Helsinki, Finland. These patients underwent a detailed physical and radiographical evaluation at a mean of 12.3 years and 13.0 years postoperatively, respectively. The risk of stem revision due to aseptic loosening in young patients with primary OA was higher for cemented stems than for proximally porous-coated or HA-coated uncemented stems implanted over the 1991-2001 period. There was no difference in the risk of revision between all-poly cemented-cups and press-fit porous-coated uncemented cups implanted during the same period, when the end point was defined as any revision (including exchange of liner). All uncemented stem designs studied in young patients with primary OA had >90% survival rates at 10 years. The Biomet Bi-Metric stem had a 95% (95% CI 93-97) survival rate even at 15 years. When the end point was defined as any revision, 10 year survival rates of all uncemented cup designs except the Harris-Galante II decreased to <80%. In young patients with RA, the risk of stem revision due to aseptic loosening was higher with cemented stems than with proximally porous-coated uncemented stems. In contrast, the risk of cup revision was higher for all uncemented cup concepts than for all-poly cemented cups with any type of cup revision as the end point. The Harris hip score increased significantly (p<0.001) both in patients with high con-genital hip dislocation and in patients with severely dysplastic hips and a previous Schanz osteotomy, treated with uncemented THA. There was a negative Trendelenburg sign in 92% and in 88% of hips, respectively. There were 12 (18%) and 15 (22%) perioperative complications. The rate of survival for the CDH femoral components, with revision due to aseptic loosening as the end point, was 98% (95% CI 97-100) at 10 years in patients with high hip dislocation and 92% (95% CI, 86-99) at 14 years in patients with a previous Schanz osteotomy. The rate of survival for press-fit, porous-coated acetabular components, with revision due to aseptic loosening as the end point, was 95% (95% CI 89-100) at 10 years in patients with high hip dislocation, and 98% (95% CI 89-100) in patients with a previous Schanz osteotomy. When revision of the cup for any reason was defined as the end point, 10 year sur-vival rates declined to 88% (95% CI 81-95) and to 69% (95% CI, 56-82), respectively. For young patients with primary OA, uncemented proximally circumferentially porous- and HA-coated stems are the implants of choice. However, survival rates of modern uncemented cups are no better than that of all-poly cemented cups. Uncemented proximally circumferentially porous-coated stems and cemented all-poly cups are currently the implants of choice for young patients with RA. Uncemented THA, with placement of the cup at the level of the true acetabulum, distal advancement of the greater trochanter and femoral shortening osteotomy provided patients with high congenital hip dislocation good long-term outcomes. Most of the patients with severely dysplastic hips and a previous Schanz osteotomy can be successfully treated with the same method. However, the subtrochanteric segmental shortening with angular correction gives better leg length correction for the patients with a previous low-seated unilateral Schanz osteotomy.

Transcending “the Impossible” : From Dead End to Expansive Learning

This dissertation investigates changes in bank work and the experience of impossibility attached to these by workers at the local level from the viewpoint of work-related well-being and collective learning. A special challenge in my work is to conceptualize the experience of impossibility as related to change, and as a starting point and tool for development work. The subject of the dissertation, solving the impossible as a collective learning process, came up as a central theme in an earlier project: Work Units between the Old and the New (1997 – 1999). Its aim was to investigate how change is constructed as a long-term process, starting from the planning of the change until its final realization in everyday banking work. I studied changes taking place in the former Postipankki (Postal Bank), later called Leonia. The three-year study involved the Branch Office of Martinlaakso, and was conducted from the perspective of well-being in a change process. The sense of impossibility involved in changes turned out to be one of the most crucial factors impairing the sense of well-being. The work community that was the target of my study did not have the available tools to construct the change locally, or to deal with the change-related impossibility by solving it through a mutual process among themselves. During the last year of the project, I carried out an intervention for development in the Branch Office, as collaboration between the researchers and the workers. The purpose of the intervention was to resolve such perceived change-related impossibility as experienced repeatedly and considered by the work community as relevant to work-related well-being. The documentation of the intervention – audio records from development sessions, written assignments by workers and assessment or evaluation interviews – constitute the essential data for my dissertation. The earlier data, collected and analysed during the first two years, provides a historical perspective on the process, all the way from construction of the impossibility towards resolving and transcending it. The aim of my dissertation is to understand the progress of developmental intervention as a shared, possibly expansive learning process within a work community and thus to provide tools for perceiving and constructing local change. I chose the change-related impossibility as a starting point for development work in the work community and as a target of conceptualization. This, I feel, is the most important contribution of my dissertation. While the intervention was in progress, the concept of impossibility started emerging as a stimulating tool for development work. An understanding of such a process can be applied to development work outside banking work as well. According to my results, it is pivotal that a concept stimulating development is strongly connected with everyday experiences of and speech about changes in work activity, as well as with the theoretical framework of work development. During this process, development work on a local level became of utmost interest as a case study for managing change. Theoretically, this was conceptualized as so-called second-order work and this concept accompanies us all the way through the research process. Learning second-order work and constructing tools based on this work have proved crucial for promoting well-being in the change circumstances in a local work unit. The lack of second-order work has led to non-well-being and inability to transcend the change-related sense of impossibility in the work community. Solving the impossible, either individually or situationally, did not orient the workers towards solving problems of impossibility together as a work community. Because the experience of the impossibility and coming to terms with transcending it are the starting point and the target of conceptualization in this dissertation, the research provides a fresh viewpoint on the theoretical framework of change and developmental work. My dissertation can facilitate construction of local changes necessitated by the recent financial crisis, and thus promote fluency and well-being in work units. It can also support change-related well-being in other areas of working life.

Prevention of type 2 diabetes with lifestyle intervention - emphasis on dietary composition and identification of high-risk individuals

Type 2 diabetes is an increasing, serious, and costly public health problem. The increase in the prevalence of the disease can mainly be attributed to changing lifestyles leading to physical inactivity, overweight, and obesity. These lifestyle-related risk factors offer also a possibility for preventive interventions. Until recently, proper evidence regarding the prevention of type 2 diabetes has been virtually missing. To be cost-effective, intensive interventions to prevent type 2 diabetes should be directed to people at an increased risk of the disease. The aim of this series of studies was to investigate whether type 2 diabetes can be prevented by lifestyle intervention in high-risk individuals, and to develop a practical method to identify individuals who are at high risk of type 2 diabetes and would benefit from such an intervention. To study the effect of lifestyle intervention on diabetes risk, we recruited 522 volunteer, middle-aged (aged 40 - 64 at baseline), overweight (body mass index > 25 kg/m2) men (n = 172) and women (n = 350) with impaired glucose tolerance to the Diabetes Prevention Study (DPS). The participants were randomly allocated either to the intensive lifestyle intervention group or the control group. The control group received general dietary and exercise advice at baseline, and had annual physician's examination. The participants in the intervention group received, in addition, individualised dietary counselling by a nutritionist. They were also offered circuit-type resistance training sessions and were advised to increase overall physical activity. The intervention goals were to reduce body weight (5% or more reduction from baseline weight), limit dietary fat (< 30% of total energy consumed) and saturated fat (< 10% of total energy consumed), and to increase dietary fibre intake (15 g / 1000 kcal or more) and physical activity (≥ 30 minutes/day). Diabetes status was assessed annually by a repeated 75 g oral glucose tolerance testing. First analysis on end-points was completed after a mean follow-up of 3.2 years, and the intervention phase was terminated after a mean duration of 3.9 years. After that, the study participants continued to visit the study clinics for the annual examinations, for a mean of 3 years. The intervention group showed significantly greater improvement in each intervention goal. After 1 and 3 years, mean weight reductions were 4.5 and 3.5 kg in the intervention group and 1.0 kg and 0.9 kg in the control group. Cardiovascular risk factors improved more in the intervention group. After a mean follow-up of 3.2 years, the risk of diabetes was reduced by 58% in the intervention group compared with the control group. The reduction in the incidence of diabetes was directly associated with achieved lifestyle goals. Furthermore, those who consumed moderate-fat, high-fibre diet achieved the largest weight reduction and, even after adjustment for weight reduction, the lowest diabetes risk during the intervention period. After discontinuation of the counselling, the differences in lifestyle variables between the groups still remained favourable for the intervention group. During the post-intervention follow-up period of 3 years, the risk of diabetes was still 36% lower among the former intervention group participants, compared with the former control group participants. To develop a simple screening tool to identify individuals who are at high risk of type 2 diabetes, follow-up data of two population-based cohorts of 35-64 year old men and women was used. The National FINRISK Study 1987 cohort (model development data) included 4435 subjects, with 182 new drug-treated cases of diabetes identified during ten years, and the FINRISK Study 1992 cohort (model validation data) included 4615 subjects, with 67 new cases of drug-treated diabetes during five years, ascertained using the Social Insurance Institution's Drug register. Baseline age, body mass index, waist circumference, history of antihypertensive drug treatment and high blood glucose, physical activity and daily consumption of fruits, berries or vegetables were selected into the risk score as categorical variables. In the 1987 cohort the optimal cut-off point of the risk score identified 78% of those who got diabetes during the follow-up (= sensitivity of the test) and 77% of those who remained free of diabetes (= specificity of the test). In the 1992 cohort the risk score performed equally well. The final Finnish Diabetes Risk Score (FINDRISC) form includes, in addition to the predictors of the model, a question about family history of diabetes and the age category of over 64 years. When applied to the DPS population, the baseline FINDRISC value was associated with diabetes risk among the control group participants only, indicating that the intensive lifestyle intervention given to the intervention group participants abolished the diabetes risk associated with baseline risk factors. In conclusion, the intensive lifestyle intervention produced long-term beneficial changes in diet, physical activity, body weight, and cardiovascular risk factors, and reduced diabetes risk. Furthermore, the effects of the intervention were sustained after the intervention was discontinued. The FINDRISC proved to be a simple, fast, inexpensive, non-invasive, and reliable tool to identify individuals at high risk of type 2 diabetes. The use of FINDRISC to identify high-risk subjects, followed by lifestyle intervention, provides a feasible scheme in preventing type 2 diabetes, which could be implemented in the primary health care system.

A quantum chemical investigation of the metal centres in cytochrome c oxidase

Quantum effects are often of key importance for the function of biological systems at molecular level. Cellular respiration, where energy is extracted from the reduction of molecular oxygen to water, is no exception. In this work, the end station of the electron transport chain in mitochondria, cytochrome c oxidase, is investigated using quantum chemical methodology. Cytochrome c oxidase contains two haems, haem a and haem a3. Haem a3, with its copper companion, CuB, is involved in the final reduction of oxygen into water. This binuclear centre receives the necessary electrons from haem a. Haem a, in turn, receives its electrons from a copper ion pair in the vicinity, called CuA. Density functional theory (DFT) has been used to clarify the charge and spin distributions of haem a, as well as changes in these during redox activity. Upon reduction, the added electron is shown to be evenly distributed over the entire haem structure, important for the accommodation of the prosthetic group within the protein. At the same time, the spin distribution of the open-shell oxidised state is more localised to the central iron. The exact spin density distribution has been disputed in the literature, however, different experiments indicating different distributions of the unpaired electron. The apparent contradiction is shown to be due to the false assumption of a unit amount of unpaired electron density; in fact, the oxidised state has about 1.3 unpaired electrons. The validity of the DFT results have been corroborated by wave function based coupled cluster calculations. Point charges, for use in classical force field based simulations, have been parameterised for the four metal centres, using a newly developed methodology. In the procedure, the subsystem for which point charges are to be obtained, is surrounded by an outer region, with the purpose of stabilising the inner region, both electronically and structurally. Finally, the possibility of vibrational promotion of the electron transfer step between haem a and a3 has been investigated. Calculating the full vibrational spectra, at DFT level, of a combined model of the two haems, revealed several normal modes that do shift electron density between the haems. The magnitude of the shift was found to be moderate, at most. The proposed mechanism could have an assisting role in the electron transfer, which still seems to be dominated by electron tunnelling.

The Population Consequences of Sex and Conflict

Natural selection generally operates at the level of the individual, or more specifically at the level of the gene. As a result, individual selection does not always favour traits which benefit the population or species as a whole. The spread of an individual gene may even act to the detriment of the organism in which it finds. Thus selection at the level of the individual can affect processes at the level of the organism, group or even at the level of the species. As most behaviours ultimately affect births, deaths and the distribution of individuals, it seems inevitable that behavioural decisions will have an impact on population dynamics and population densities. Behavioural decisions can often involve costs through allocation of energy into behavioural strategies, such as the investment into armaments involved in fighting over resources or increased mortality due to injury or increased predation risk. Similarly, behaviour may act o to benefit the population, in terms of higher survival and increased fecundity. Examples include increased investment through parental care, choosing a mate based on the nuptial gifts they may supply and choosing territories in the face of competition. Investigating the impact of behaviour on population ecology may seem like a trivial task, but it is likely to have important consequences at different levels. For example, antagonistic behaviour may occasionally become so extreme that it increases the risk of extinction, and such extinction risk may have important implications for conservation. As a corollary, any such behaviour may also act as a macroevolutionary force, weeding out populations with traits which, whilst beneficial to the individuals in the short term, ultimately result in population extinction. In this thesis, I examine how behaviours, specifically conflict and competition over a resource and aspects of behaviour involved in sexual selection, can affect population densities, and what the implications are for the evolution and ecology of the populations in question. It is found that both behaviours related to individual conflict and mating strategies can have an effect at the level of the population, but that various factors, such as a feedback between selection and population densities or macroevolution caused by species extinctions, may act to limit the intensity of conflicts that we observe in nature.

Redox-linked proton transfer by cytochrome c oxidase

The respiratory chain is found in the inner mitochondrial membrane of higher organisms and in the plasma membrane of many bacteria. It consists of several membrane-spanning enzymes, which conserve the energy that is liberated from the degradation of food molecules as an electrochemical proton gradient across the membrane. The proton gradient can later be utilized by the cell for different energy requiring processes, e.g. ATP production, cellular motion or active transport of ions. The difference in proton concentration between the two sides of the membrane is a result of the translocation of protons by the enzymes of the respiratory chain, from the negatively charged (N-side) to the positively charged side (P-side) of the lipid bilayer, against the proton concentration gradient. The endergonic proton transfer is driven by the flow of electrons through the enzymes of the respiratory chain, from low redox-potential electron donors to acceptors of higher potential, and ultimately to oxygen. Cytochrome c oxidase is the last enzyme in the respiratory chain and catalyzes the reduction of dioxygen to water. The redox reaction is coupled to proton transport across the membrane by a yet unresolved mechanism. Cytochrome c oxidase has two proton-conducting pathways through which protons are taken up to the interior part of the enzyme from the N-side of the membrane. The K-pathway transfers merely substrate protons, which are consumed in the process of water formation at the catalytic site. The D-pathway transfers both substrate protons and protons that are pumped to the P-side of the membrane. This thesis focuses on the role of two conserved amino acids in proton translocation by cytochrome c oxidase, glutamate 278 and tryptophan 164. Glu278 is located at the end of the D-pathway and is thought to constitute the branching point for substrate and pumped protons. In this work, it was shown that although Glu278 has an important role in the proton transfer mechanism, its presence is not an obligatory requirement. Alternative structural solutions in the area around Glu278, much like the ones present in some distantly related heme-copper oxidases, could in the absence of Glu278 support the formation of a long hydrogen-bonded water chain through which proton transfer from the D-pathway to the catalytic site is possible. The other studied amino acid, Trp164, is hydrogen bonded to the ∆-propionate of heme a3 of the catalytic site. Mutation of this amino acid showed that it may be involved in regulation of proton access to a proton acceptor, a pump site, from which the proton later is expelled to the P-side of the membrane. The ion pair that is formed by the ∆-propionate of heme a3 and arginine 473 is likely to form a gate-like structure, which regulates proton mobility to the P-side of the membrane. The same gate may also be part of an exit path through which water molecules produced at the catalytically active site are removed towards the external side of the membrane. Time-resolved optical and electrometrical experiments with the Trp164 to phenylalanine mutant revealed a so far undetected step in the proton pumping mechanism. During the A to PR transition of the catalytic cycle, a proton is transferred from Glu278 to the pump site, located somewhere in the vicinity of the ∆-propionate of heme a3. A mechanism for proton pumping by cytochrome c oxidase is proposed on the basis of the presented results and the mechanism is discussed in relation to some relevant experimental data. A common proton pumping mechanism for all members of the heme-copper oxidase family is moreover considered.

A model Integrating the Facilities Management Process with the Building End User’s Business Process (ProFacil)

The ProFacil model is a generic process model defined as a framework model showing the links between the facilities management process and the building end user’s business process. The purpose of using the model is to support more detailed process modelling. The model has been developed using the IDEF0 modelling method. The ProFacil model describes business activities from the generalized point of view as management-, support-, and core processes and their relations. The model defines basic activities in the provision of a facility. Examples of these activities are “operate facilities”, “provide new facilities”, “provide re-build facilities”, “provide maintained facilities” and “perform dispose of facilities”. These are all generic activities providing a basis for a further specialisation of company specific FM activities and their tasks. A facilitator can establish a specialized process model using the ProFacil model and interacting with company experts to describe their company’s specific processes. These modelling seminars or interviews will be done in an informal way, supported by the high-level process model as a common reference.