39 resultados para post-dynamic softening
Resumo:
Ei saatavilla
Resumo:
As a Novice Teacher at Comprehensive School: The authentic experiences of the beginning teachers during their first year of teaching The aim of this study is to explicate the novice year of teaching in the light of teachers´ authentic experiences. The subject of this investigation is the teachers´ subjective world of experience during their first academic year of teaching and the sharing of these experiences in collaborative consulting meetings. The themes discussed in the meetings were introduced into the collaborative group by the novice teachers themselves, and the progress of discussion was con-trolled by them. The research data was gathered in a consultative working group where the way a novice teacher starts to interpret, analyze and identify his/her own complex and dynamic teaching situations was observed. The research data gathered in this way illuminates novice teachers´ world of experience and mental picture as well as the unconscious sides of school life. In a theoretical frame of reference, the work of a teacher is identified, according to systemic scientific thought, as a dynamic triangle in which the basic elements are the personality of the teacher, the role of the teacher and the school as an organization. These basic elements form a whole within which the teacher works. The dynamics of this triangle in a teacher’s work are brought to light through the study of the phenomena of groups and group dynamics since a teacher works either as a member of a group (working community), as a leader of a group (teaching situations) or in a network (parent – teacher cooperation). Therefore, tension and force are always present in teaching work. The main research problem was to explain how a novice teacher experiences his/her first working year as a teacher. The participants (n=5) were teaching at five different comprehensive schools in the city of Helsinki. This was their first long-term post as a teacher. The research data consists of seven collaborative consulting meetings, as well as recordings and transcripts of the meetings. A classificatory framework was developed for data analysis which enabled a sys-tematic qualitative content analysis based on theory and material. In addition to the consulting meetings, the teachers were interviewed at the beginning and at the end of the process of collecting the research material. The interviews were used to interpret the meanings of the content analysis based on raw data. The findings show that there is a gap between teacher education and the reality of school life, which causes difficulties for a novice teacher during his/her first teaching year. The gap is rather a global educational problem than a national one, and therefore it is independent of cultural factors. Novice teachers desire a well-structured theory of teacher education and a clear programme where the themes and content delve deeper and deeper into the subject matter during the study years. According to the novice teachers, teacher education frequently consists of sporadic and unconnected study and class situations. An individual content weakness of teacher education is the lack of insufficient initiation into evaluation processes. The novice teachers suggest that a student must be provided good-quality and competent guidance during the study years and during his or her induction. There should be a well-organized, structured and systematic induction program for novice teachers. The induction program should be overseen by an organization so that the requirements of a qualified induction can be met. The findings show that the novice teachers find the first year of teaching at comprehensive school emotionally loaded. The teachers experienced teaching as difficult work and found the workload heavy. Nevertheless, they enjoyed their job because, as they said, there were more pleasant than unpleasant things in their school day. Their main feeling at school was the joy of success in teaching. The novice teachers felt satisfaction with their pupils. The teachers experienced the more serious feelings of anger and disgust when serious violence took place. The most difficult situations arose from teaching pupils who had mental health problems. The toughest thing in the teacher´s work was teaching groups that are too heterogeneous. The most awkward problems in group dynamics happened when new immigrants, who spoke only their own languages, were integrated into the groups in the middle of the school year. Teachers wanted to help children who needed special help with learning but restated at the same time that the groups being taught shouldn’t be too heterogeneous. The teachers wished for help from school assistants so that they could personally concentrate more on teaching. Not all the parents took care of their children according to the comprehensive school law. The teachers found it hard to build a confidential relationship between home and school. In this study, novice teachers found it hard to join the teaching staff at school. Some of the teachers on staff were very strong and impulsive, reacting loudly and emotionally. These teachers provoked disagreement, conflicts, power struggles and competition among the other teachers. Although the novice teachers of the study were all qualified teachers, three of them were not interested in a permanent teaching job. For these teachers teaching at a primary school was just a project, a short period in their working life. They will remain in the teaching profession as long as they are learning new things and enjoying their teaching job. This study is an independent part of the research project on Interplay – Connecting Academic Teacher Education and Work, undertaken by the Department of Applied Sciences of Education at the University of Helsinki. Key words: novice teacher, emotions, groups and group dynamics, authority, co-operation between home and school, teacher community, leadership at school, induction, consulting
Resumo:
This academic work begins with a compact presentation of the general background to the study, which also includes an autobiography for the interest in this research. The presentation provides readers who know little of the topic of this research and of the structure of the educational system as well as of the value given to education in Nigeria. It further concentrates on the dynamic interplay of the effect of academic and professional qualification and teachers' job effectiveness in secondary schools in Nigeria in particular, and in Africa in general. The aim of this study is to produce a systematic analysis and rich theoretical and empirical description of teachers' teaching competencies. The theoretical part comprises a comprehensive literature review that focuses on research conducted in the areas of academic and professional qualification and teachers' job effectiveness, teaching competencies, and the role of teacher education with particular emphasis on school effectiveness and improvement. This research benefits greatly from the functionalist conception of education, which is built upon two emphases: the application of the scientific method to the objective social world, and the use of an analogy between the individual 'organism' and 'society'. To this end, it offers us an opportunity to define terms systematically and to view problems as always being interrelated with other components of society. The empirical part involves describing and interpreting what educational objectives can be achieved with the help of teachers' teaching competencies in close connection to educational planning, teacher training and development, and achieving them without waste. The data used in this study were collected between 2002 and 2003 from teachers, principals, supervisors of education from the Ministry of Education and Post Primary Schools Board in the Rivers State of Nigeria (N=300). The data were collected from interviews, documents, observation, and questionnaires and were analyzed using both qualitative and quantitative methods to strengthen the validity of the findings. The data collected were analyzed to answer the specific research questions and hypotheses posited in this study. The data analysis involved the use of multiple statistical procedures: Percentages Mean Point Value, T-test of Significance, One-Way Analysis of Variance (ANOVA), and Cross Tabulation. The results obtained from the data analysis show that teachers require professional knowledge and professional teaching skills, as well as a broad base of general knowledge (e.g., morality, service, cultural capital, institutional survey). Above all, in order to carry out instructional processes effectively, teachers should be both academically and professionally trained. This study revealed that teachers are not however expected to have an extraordinary memory, but rather looked upon as persons capable of thinking in the right direction. This study may provide a solution to the problem of teacher education and school effectiveness in Nigeria. For this reason, I offer this treatise to anyone seriously committed in improving schools in developing countries in general and in Nigeria in particular to improve the lives of all its citizens. In particular, I write this to encourage educational planners, education policy makers, curriculum developers, principals, teachers, and students of education interested in empirical information and methods to conceptualize the issue this study has raised and to provide them with useful suggestions to help them improve secondary schooling in Nigeria. Though, multiple audiences exist for any text. For this reason, I trust that the academic community will find this piece of work a useful addition to the existing literature on school effectiveness and school improvement. Through integrating concepts from a number of disciplines, I aim to describe as holistic a representation as space could allow of the components of school effectiveness and quality improvement. A new perspective on teachers' professional competencies, which not only take into consideration the unique characteristics of the variables used in this study, but also recommend their environmental and cultural derivation. In addition, researchers should focus their attention on the ways in which both professional and non-professional teachers construct and apply their methodological competencies, such as their grouping procedures and behaviors to the schooling of students. Keywords: Professional Training, Academic Training, Professionally Qualified, Academically Qualified, Professional Qualification, Academic Qualification, Job Effectiveness, Job Efficiency, Educational Planning, Teacher Training and Development, Nigeria.
Resumo:
Infection by Epstein-Barr virus (EBV) occurs in approximately 95% of the world s population. EBV was the first human virus implicated in oncogenesis. Characteristic for EBV primary infection are detectable IgM and IgG antibodies against viral capsid antigen (VCA). During convalescence the VCA IgM disappears while the VCA IgG persists for life. Reactivations of EBV occur both among immunocompromised and immunocompetent individuals. In serological diagnosis, measurement of avidity of VCA IgG separates primary from secondary infections. However, in serodiagnosis of mononucleosis it is quite common to encounter, paradoxically, VCA IgM together with high-avidity VCA IgG, indicating past immunity. We determined the etiology of this phenomenon and found that, among patients with cytomegalovirus (CMV) primary infection a large proportion (23%) showed antibody profiles of EBV reactivation. In contrast, EBV primary infection did not appear to induce immunoreactivation of CMV. EBV-associated post-transplant lymphoproliferative disease (PTLD) is a life threatening complication of allogeneic stem cell or solid organ transplantation. PTLD may present with a diverse spectrum of clinical symptoms and signs. Due to rapidity of PTLD progression especially after stem cell transplantation, the diagnosis must be obtained quickly. Pending timely detection, the evolution of the fatal disease may be halted by reduction of immunosuppression. A promising new PTLD treatment (also in Finland) is based on anti-CD-20 monoclonal antibodies. Diagnosis of PTLD has been demanding because of immunosuppression, blood transfusions and the latent nature of the virus. We set up in 1999 to our knowledge first in Finland for any microbial pathogen a real-time quantitative PCR (qPCR) for detection of EBV DNA in blood serum/plasma. In addition, we set up an in situ hybridisation assay for EBV RNA in tissue sections. In collaboration with a group of haematologists at Helsinki University Central Hospital we retrospectively determined the incidence of PTLD among 257 allogenic stem cell transplantations (SCT) performed during 1994-1999. Post-mortem analysis revealed 18 cases of PTLD. From a subset of PTLD cases (12/18) and a series of corresponding controls (36), consecutive samples of serum were studied by the new EBV-qPCR. All the PTLD patients were positive for EBV-DNA with progressively rising copy numbers. In most PTLD patients EBV DNA became detectable within 70 days of SCT. Of note, the appearance of EBV DNA preceded the PTLD symptoms (fever, lymphadenopathy, atypical lymphocytes). Among the SCT controls, EBV DNA occurred only sporadically, and the EBV-DNA levels remained relatively low. We concluded that EBV qPCR is a highly sensitive (100%) and specific (96%) new diagnostic approach. We also looked for and found risk factors for the development of PTLD. Together with a liver transplantation group at the Transplantation and Liver Surgery Clinic we wanted to clarify how often and how severely do EBV infections occur after liver transplantation. We studied by the EBV qPCR 1284 plasma samples obtained from 105 adult liver transplant recipients. EBV DNA was detected in 14 patients (13%) during the first 12 months. The peak viral loads of 13 asymptomatic patients were relatively low (<6600/ml), and EBV DNA subsided quickly from circulation. Fatal PTLD was diagnosed in one patient. Finally, we wanted to determine the number and clinical significance of EBV infections of various types occurring among a large, retrospective, nonselected cohort of allogenic SCT recipients. We analysed by EBV qPCR 5479 serum samples of 406 SCT recipients obtained during 1988-1999. EBV DNA was seen in 57 (14%) patients, of whom 22 (5%) showed progressively rising and ultimately high levels of EBV DNA (median 54 million /ml). Among the SCT survivors, EBV DNA was transiently detectable in 19 (5%) asymptomatic patients. Thereby, low-level EBV-DNA positivity in serum occurs relatively often after SCT and may subside without specific treatment. However, high molecular copy numbers (>50 000) are diagnostic for life-threatening EBV infection. We furthermore developed a mathematical algorithm for the prediction of development of life-threatening EBV infection.
Resumo:
Costs of purchasing new piglets and of feeding them until slaughter are the main variable expenditures in pig fattening. They both depend on slaughter intensity, the nature of feeding patterns and the technological constraints of pig fattening, such as genotype. Therefore, it is of interest to examine the effect of production technology and changes in input and output prices on feeding and slaughter decisions. This study examines the problem by using a dynamic programming model that links genetic characteristics of a pig to feeding decisions and the timing of slaughter and takes into account how these jointly affect the quality-adjusted value of a carcass. The model simulates the growth mechanism of a pig under optional feeding and slaughter patterns and then solves the optimal feeding and slaughter decisions recursively. The state of nature and the genotype of a pig are known in the analysis. The main contribution of this study is the dynamic approach that explicitly takes into account carcass quality while simultaneously optimising feeding and slaughter decisions. The method maximises the internal rate of return to the capacity unit. Hence, the results can have vital impact on competitiveness of pig production, which is known to be quite capital-intensive. The results suggest that producer can significantly benefit from improvements in the pig's genotype, because they improve efficiency of pig production. The annual benefits from obtaining pigs of improved genotype can be more than €20 per capacity unit. The annual net benefits of animal breeding to pig farms can also be considerable. Animals of improved genotype can reach optimal slaughter maturity quicker and produce leaner meat than animals of poor genotype. In order to fully utilise the benefits of animal breeding, the producer must adjust feeding and slaughter patterns on the basis of genotype. The results suggest that the producer can benefit from flexible feeding technology. The flexible feeding technology segregates pigs into groups according to their weight, carcass leanness, genotype and sex and thereafter optimises feeding and slaughter decisions separately for these groups. Typically, such a technology provides incentives to feed piglets with protein-rich feed such that the genetic potential to produce leaner meat is fully utilised. When the pig approaches slaughter maturity, the share of protein-rich feed in the diet gradually decreases and the amount of energy-rich feed increases. Generally, the optimal slaughter weight is within the weight range that pays the highest price per kilogram of pig meat. The optimal feeding pattern and the optimal timing of slaughter depend on price ratios. Particularly, an increase in the price of pig meat provides incentives to increase the growth rates up to the pig's biological maximum by increasing the amount of energy in the feed. Price changes and changes in slaughter premium can also have large income effects. Key words: barley, carcass composition, dynamic programming, feeding, genotypes, lean, pig fattening, precision agriculture, productivity, slaughter weight, soybeans
Resumo:
Positron emission tomography (PET) is an imaging technique in which radioactive positron-emitting tracers are used to study biochemical and physiological functions in humans and in animal experiments. The use of PET imaging has increased rapidly in recent years, as have special requirements in the fields of neurology and oncology for the development of syntheses for new, more specific and selective radiotracers. Synthesis development and automation are necessary when high amounts of radioactivity are needed for multiple PET studies. In addition, preclinical studies using experimental animal models are necessary for evaluating the suitability of new PET tracers for humans. For purification and analysing the labelled end-product, an effective radioanalytical method combined with an optimal radioactivity detection technique is of great importance. In this study, a fluorine-18 labelling synthesis method for two tracers was developed and optimized, and the usefulness of these tracers for possible prospective human studies was evaluated. N-(3-[18F]fluoropropyl)-2β-carbomethoxy-3β-(4-fluorophenyl)nortropane ([18F]β-CFT-FP) is a candidate PET tracer for the dopamine transporter (DAT), and 1H-1-(3-[18F]fluoro-2-hydroxypropyl)-2-nitroimidazole ([18F]FMISO) is a well-known hypoxia marker for hypoxic but viable cells in tumours. The methodological aim of this thesis was to evaluate the status of thin-layer chromatography (TLC) combined with proper radioactivity detection measurement systems as a radioanalytical method. Three different detection methods of radioactivity were compared: radioactivity scanning, film autoradiography, and digital photostimulated luminescence (PSL) autoradiography. The fluorine-18 labelling synthesis for [18F]β-CFT-FP was developed and carbon-11 labelled [11C]β-CFT-FP was used to study the specificity of β-CFT-FP for the DAT sites in human post-mortem brain slices. These in vitro studies showed that β-CFT-FP binds to the caudate-putamen, an area rich of DAT. The synthesis of fluorine-18 labelled [18F]FMISO was optimized, and the tracer was prepared using an automated system with good and reproducible yields. In preclinical studies, the action of the radiation sensitizer estramustine phosphate on the radiation treatment and uptake of [18F]FMISO was evaluated, with results of great importance for later human studies. The methodological part of this thesis showed that radioTLC is the method of choice when combined with an appropriate radioactivity detection technique. Digital PSL autoradiography proved to be the most appropriate when compared to the radioactivity scanning and film autoradiography methods. The very high sensitivity, good resolution, and wide dynamic range of digital PSL autoradiography are its advantages in detection of β-emitting radiolabelled substances.
Resumo:
Austria and Finland are persistently referred to as the “success stories” of post-1945 European history. Notwithstanding their different points of departure, in the course of the Cold War both countries portrayed themselves as small and neutral border-states in the world dictated by superpower politics. By the 1970s, both countries frequently ranked at the top end in various international classifications regarding economic development and well-being in society. This trend continues today. The study takes under scrutiny the concept of consensus which figures centrally in the two national narratives of post-1945 success. Given that the two domestic contexts as such only share few direct links with one another and are more obviously different than similar in terms of their geographical location, historical experiences and politico-cultural traditions, the analogies and variations in the anatomies of the post-1945 “cultures of consensus” provide an interesting topic for a historical comparative and cross-national examination. The main research question concerns the identification and analysis of the conceptual and procedural convergence points of the concepts of the state and consensus. The thesis is divided into six main chapters. After the introduction, the second chapter presents the theoretical framework in more detail by focusing on the key concepts of the study – the state and consensus. Chapter two also introduces the comparative historical and cross-national research angles. Chapter three grounds the key concepts of the state and consensus in the historical contexts of Austria and Finland by discussing the state, the nation and democracy in a longer term comparative perspective. The fourth and fifth chapter present case studies on the two policy fields, the “pillars”, upon which the post-1945 Austrian and Finnish cultures of consensus are argued to have rested. Chapter four deals with neo-corporatist features in the economic policy making and chapter five discusses the building up of domestic consensus regarding the key concepts of neutrality policies in the 1950s and 1960s. The study concludes that it was not consensus as such but the strikingly intense preoccupation with the theme of domestic consensus that cross-cut, in a curiously analogous manner, the policy-making processes studied. The main challenge for the post-1945 architects of Austrian and Finnish cultures of consensus was to find strategies and concepts for consensus-building which would be compatible with the principles of democracy. Discussed at the level of procedures, the most important finding of the study concerns the triangular mechanism of coordination, consultation and cooperation that set into motion and facilitated a new type of search for consensus in both post-war societies. In this triangle, the agency of the state was central, though in varying ways. The new conceptions concerning a small state’s position in the Cold War world also prompted cross-nationally perceivable willingness to reconsider inherited concepts and procedures of the state and the nation. At the same time, the ways of understanding the role of the state and its relation to society remained profoundly different in Austria and Finland and this basic difference was in many ways reflected in the concepts and procedures deployed in the search for consensus and management of domestic conflicts. For more detailed information, please consult the author.
Resumo:
This work is concerned with presenting a modified theoretical approach to the study of centre-periphery relations in the Russian Federation. In the widely accepted scientific discourse, the Russian federal system under the Yeltsin Administration (1991-2000) was asymmetrical; largely owing to the varying amount of structural autonomy distributed among the federation s 89 constituent units. While providing an improved understanding as to which political and socio-economic structures contributed to federal asymmetry, it is felt that associated large N-studies have underemphasised the role played by actor agency in re-shaping Russian federal institutions. It is the main task of this thesis to reintroduce /re-emphasise the importance of actor agency as a major contributing element of institutional change in the Russian federal system. By focusing on the strategic agency of regional elites simultaneously within regional and federal contexts, the thesis adopts the position that political, ethnic and socio-economic structural factors alone cannot fully determine the extent to which regional leaders were successful in their pursuit of economic and political pay-offs from the institutionally weakened federal centre. Furthermore, this work hypothesises that under conditions of federal institutional uncertainty, it is the ability of regional leaders to simultaneously interpret various mutable structural conditions then translate them into plausible strategies which accounts for the regions ability to extract variable amounts of economic and political pay-offs from the Russian federal system. The thesis finds that while the hypothesis is accurate in its theoretical assumptions, several key conclusions provide paths for further inquiry posed by the initial research question. First, without reliable information or stable institutions to guide their actions, both regional and federal elites were forced into ad-hoc decision-making in order to maintain their core strategic focus: political survival. Second, instead of attributing asymmetry to either actor agency or structural factors exclusively, the empirical data shows that both agency and structures interact symbiotically in the strategic formulation process, thus accounting for the sub-optimal nature of several of the actions taken in the adopted cases. Third, as actor agency and structural factors mutate over time, so, too do the perceived payoffs from elite competition. In the case of the Russian federal system, the stronger the federal centre became, the less likely it was that regional leaders could extract the high degree of economic and political pay-offs that they clamoured for earlier in the Yeltsin period. Finally, traditional approaches to the study of federal systems which focus on institutions as measures of federalism are not fully applicable in the Russian case precisely because the institutions themselves were a secondary point of contention between competing elites. Institutional equilibriums between the regions and Moscow were struck only when highly personalised elite preferences were satisfied. Therefore the Russian federal system is the product of short-term, institutional solutions suited to elite survival strategies developed under conditions of economic, political and social uncertainty.
Resumo:
Eukaryotic cells are characterized by having a subset of internal membrane compartments, each one with a specifi c identity, structure and function. Proteins destined to be targeted to the exterior of the cell need to enter and progress through the secretory pathway. Transport of secretory proteins from the endoplasmic reticulum (ER) to the Golgi takes place by the selective packaging of proteins into COPII-coated vesicles at the ER membrane. Taking advantage of the extensive genetic tools available for S. cerevisiae we found that Hsp150, a yeast secretory glycoprotein, selectively exited the ER in the absence of any of the three Sec24p family members. Sec24p has been thought to be an essential component of the COPII coat and thus indispensable for exocytic membrane traffic. Next we analyzed the ability of Hsp150 to be secreted in mutants, where post-Golgi transport is temperature sensitive. We found that Hsp150 could be selectively secreted under conditions where the exocyst component Sec15p is defective. Analysis of the secretory vesicles revealed that Hsp150 was packaged into a subset of known secretory vesicles as well as in a novel pool of secretory vesicles at the level of the Golgi. Secretion of Hsp150 in the absence of Sec15p function was dependent of Mso1p, a protein capable of interacting with vesicles intended to fuse with the plasma membrane, with the SNARE machinery and with Sec1p. This work demonstrated that Hsp150 is capable of using alternative secretory pathways in ER-to-Golgi and Golgi-to-plasma membrane traffi c. The sorting signals, used at both stages of the secretory pathway, for secretion of Hsp150 were different, revealing the highly dynamic nature and spatial organization of the secretory pathway. Foreign proteins usually misfold in the yeast ER. We used Hsp150 as a carrier to assist folding and transport of heterologous proteins though the secretory pathway to the culture medium in both S. cerevisiae and P. pastoris. Using this technique we expressed Hsp150Δ-HRP and developed a staining procedure, which allowed the visualization of the organelles of the secretory pathway of S. cerevisiae.
Resumo:
It has been only recently realized that sexual selection does not end at copulation but that post-copulatory processes are often important in determining the fitness of individuals. In this thesis, I experimentally studied both pre- and post-copulatory sexual selection in the least killifish, Heterandria formosa. I found that this species suffers from severe inbreeding depression in male reproductive behaviour, offspring viability and offspring maturation times. Neither sex showed pre-copulatory inbreeding avoidance but when females mated with their brothers, less sperm were retrieved from their reproductive system compared to the situation when females mated with unrelated males. Whether the difference in sperm numbers is due to female or male effect could not be resolved. Based on theory, females should be more eager to avoid inbreeding than males in this species, because females invest more in their offspring than males do. Inbreeding seems to be an important part of this species biology and the severe inbreeding depression has most likely selected for the evolution of the post-copulatory inbreeding avoidance mechanism that I found. In addition, I studied the effects of polyandry on female reproductive success. When females mated with more than one male, they were more likely to get pregnant. However, I also found a cost of polyandry. The offspring of females mated to four males took longer to reach sexual maturity compared to the offspring of monandrous females. This cost may be explained by parent-offspring conflict over maternal resource allocation. In another experiment, in which within-brood relatedness was manipulated, offspring sizes decreased over time when within-brood relatedness was low. This result is partly in accordance with the kinship theory of genomic imprinting. When relatedness decreases, offspring are expected to be less co-operative and demand fewer resources from their mother, which leads to impaired development. In the last chapter of my thesis, I show that H. formosa males do not prefer large females as in other Poeciliidae species. I suggest that males view smaller females as more profitable mates because those are more likely virgin. In conclusion, I found both pre- and post-copulatory sexual selection to be important factors in determining reproductive success in H. formosa.
Resumo:
Plasma phospholipid transfer protein (PLTP) plays a crucial role in high-density lipoprotein (HDL) metabolism and reverse cholesterol transport (RCT). It mediates the generation of pre-beta-HDL particles, enhances the cholesterol efflux from peripheral cells to pre-beta-HDL, and metabolically maintains the plasma HDL levels by facilitating the transfer of post-lipolytic surface remnants of triglyceride-rich lipoproteins to HDL. In addition to the antiatherogenic properties, recent findings indicate that PLTP has also proatherogenic characteristics, and that these opposite characteristics of PLTP are dependent on the site of PLTP expression and action. In human plasma, PLTP exists in a high-activity (HA-PLTP) and a low-activity form (LA-PLTP), which are associated with macromolecular complexes of different size and composition. The aims of this thesis were to isolate the two PLTP forms from human plasma, to characterize the molecular complexes in which the HA- and LA-PLTP reside, and to study the interactions of the PLTP forms with apolipoproteins (apo) and the ability of apolipoproteins to regulate PLTP activity. In addition, we aimed to study the distribution of the two PLTP forms in a Finnish population sample as well as to find possible regulatory factors for PLTP by investigating the influence of lipid and glucose metabolism on the balance between the HA- and LA-PLTP. For these purposes, an enzyme-linked immunosorbent assay (ELISA) capable of determining the serum total PLTP concentration and quantitating the two PLTP forms separately was developed. In this thesis, it was demonstrated that the HA-PLTP isolated from human plasma copurified with apoE, whereas the LA-PLTP formed a complex with apoA-I. The separation of these two PLTP forms was carried out by a dextran sulfate (DxSO4)-CaCl2 precipitation of plasma samples before the mass determination. A similar immunoreactivity of the two PLTP forms in the ELISA could be reached after a partial sample denaturation by SDS. Among normolipidemic Finnish individuals, the mean PLTP mass was 6.6 +/- 1.5 mg/l and the mean PLTP activity 6.6 +/- 1.7 umol/ml/h. Of the serum PLTP concentration, almost 50% represented HA-PLTP. The results indicate that plasma HDL levels could regulate PLTP concentration, while PLTP activity could be regulated by plasma triglyceride-rich very low-density lipoprotein (VLDL) concentration. Furthermore, new evidence is presented that PLTP could also play a role in glucose metabolism. Finally, both PLTP forms were found to interact with apoA-I, apoA-IV, and apoE. In addition, both apoE and apoA-IV, but not apoA-I, were capable of activating the LA-PLTP. These findings suggest that the distribution of the HA- and LA-PLTP in human plasma is subject to dynamic regulation by apolipoproteins.
Resumo:
The Golgi complex is a central organelle of the secretory pathway, responsible for a range of post-translational modifications, as well as for membrane traffic to the plasma membrane and to the endosomal-lysosomal pathway. In addition, this organelle has roles in cell migration, in the regulation of traffic, and as a mitotic check point. The structure of the Golgi complex is highly dynamic and able to respond to the amount of cargo being transported and the stage of the cell cycle. The Golgi proteome reflects the functions and structure of this organelle, and can be divided into three major groups: the Golgi resident proteins (e.g. modification enzymes), the Golgi matrix proteins (involved in structure and tethering events), and trafficking proteins (e.g. vesicle coat proteins and Rabs). The Golgi proteome has been studied on several occasions, from both rat liver and mammary gland Golgi membranes using proteomic approaches, but still little more than half of the estimated Golgi proteome is known. Nevertheless, methodological improvements and introduction of shotgun proteomics have increased the number of identified proteins, and especially the number of identified transmembrane proteins. Cartilage, even though not a typical tissue in which to study membrane traffic, secretes large amounts of extracellular matrix proteins that are extensively modified, especially by amino acid hydroxylation, glycosylation and sulfation. Furthermore, the cartilage ECM contains several, large oligomeric proteins (such as collagen II) that are difficult to assemble and transport. Indeed, cartilage has been shown to be susceptible to changes both in secretory pathway (e.g. the COPII coat assembly) and in post-translational modifications (e.g. heparan sulfate formation). Dental follicle, and the periodontal ligament (PDL) that it forms, are another type of connective tissue, and they have a role in anchoring teeth to bone. This anchorage is achieved by numerous matrix fibres that connect the bone matrix with the cementum. These tissues have in common the secretion of large matrix molecules. In this study the Golgi proteome was analysed from purified, stacked Golgi membranes isolated from rat liver. The identified, extensive proteome included a protein similar to Ab2-095, or Golgi protein 49kDa (GoPro49), which was shown to localise to the Golgi complex as an EGFP fusion protein. Surprisingly, in situ hybridisation showed the GoPro49 expression to be highly restricted to different mesenchymal tissues, especially in cartilage, and this expression pattern was clearly developmentally regulated. In addition to cartilage, GoPro49 was also expressed in the dental follicle, but was not observed in the mature PDL. Importantly, GoPro49 is the first specific marker for the dental follicle. Endogenous GoPro49 protein co-localised with β-COP in both chondrosarcoma and primary dental follicle cell lines. The COPI staining in these cells was highly dynamic, showing a number of tubules. This may reflect the type of secretory cargo they secrete. Currently GoPro49 is the only Golgi protein with such a restricted expression pattern.