Generation of Flat and Curved Membranes by Inverse-BAR Domain Proteins

Biological membranes are tightly linked to the evolution of life, because they provide a way to concentrate molecules into partially closed compartments. The dynamic shaping of cellular membranes is essential for many physiological processes, including cell morphogenesis, motility, cytokinesis, endocytosis, and secretion. It is therefore essential to understand the structure of the membrane and recognize the players that directly sculpt the membrane and enable it to adopt different shapes. The actin cytoskeleton provides the force to push eukaryotic plasma membrane in order to form different protrusions or/and invaginations. It has now became evident that actin directly co-operates with many membrane sculptors, including BAR domain proteins, in these important events. However, the molecular mechanisms behind BAR domain function and the differences between the members of this large protein family remain largely unresolved. In this thesis, the structure and functions of the I-BAR domain family members IRSp53 and MIM were thoroughly analyzed. By using several methods such as electron microscopy and systematic mutagenesis, we showed that these I-BAR domain proteins bind to PI(4,5)P2-rich membranes, generate negative membrane curvature and are involved in the formation of plasma membrane protrusions in cells e.g. filopodia. Importantly, we characterized a novel member of the BAR-domain superfamily which we named Pinkbar. We revealed that Pinkbar is specifically expressed in kidney and epithelial cells, and it localizes to Rab13-positive vesicles in intestinal epithelial cells. Remarkably, we learned that the I-BAR domain of Pinkbar does not generate membrane curvature but instead stabilizes planar membranes. Based on structural, mutagenesis and biochemical work we present a model for the mechanism of the novel membrane deforming activity of Pinkbar. Collectively, this work describes the mechanism by which I-BAR domain proteins deform membranes and provides new information about the biological roles of these proteins. Intriguingly, this work also gives evidence that significant functional plasticity exists within the I-BAR domain family. I-BAR proteins can either generate negative membrane curvature or stabilize planar membrane sheets, depending on the specific structural properties of their I-BAR domains. The results presented in this thesis expand our knowledge on membrane sculpting mechanisms and shows for the first time how flat membranes can be generated in cells.

Modernity, postmodernity and identity of jazz in Chile : The cases of the generation of 1990 and Ángel Parra

This research is about jazz in Chile in relation to modernity and identity. Final chapters focus and detach latest jazz musician s generation in 1990 decade and composer guitarist Angel Parra. An historic and sociological approach is developed, which will be useful for modernity and identity analysis, and so on post modernity and globalization. Modernity has been studied in texts of Adorno, Baudrillard, Brünner, García Canclini, Habermas and Jameson. Identity has been studied in texts of Aharonián, Cordúa, Garretón, Gissi, Larraín and others. Chapter 3 is about Latin-American musicology and jazz investigations, in relation to approach developed in chapter 2. Chapters 4 and 5 are about history of jazz in Chile until beginning of XXI century. Chapter 6 focuses in Ángel Parra Orrego. Conclusions of this investigation detach the modernist mechanical that has conducted jazz development in Chile, which in Ángel Parra´s case has been overcame by a post modernist behaviour. This behaviour has solved in a creative way, subjects like modernity and identity in jazz practice in a Latin-American country.

Expression Analysis of Host-Induced Genes and Proteins of Potato Pathogen Pectobacterium atrosepticum

Pectobacterium atrosepticum on Gram-negatiivinen bakteeri, joka aiheuttaa perunan tyvi- ja märkämätää. P. atrosepticum bakteerin optimilämpötila on melko alhainen ja se on yleinen lauhkeilla alueilla. Tyvimätä leviää pääasiassa siemenperunan välityksellä ja siksi se on ongelma erityisesti siemenperunan tuotannossa. P. atrosepticum kannan SCRI1043 genomi on julkaistu ja sitä tutkitaan malliorganismina märkä- ja tyvimädän taudinaiheuttamisen ymmärtämiseksi. Tämä opportunistinen taudinaiheuttaja voi elää isäntäkasvissa kuukausia piilevänä, aiheuttamatta näkyviä oireita. Suotuisissa olosuhteissa bakteerit alkavat jakautua ja tuottaa kasvin kudoksia hajottavia entsyymejä. Mädäntyvä kasvimassa tarjoaa ravinteita bakteerien kasvuun ja mahdollistaa isäntäkasvin asuttamisen. Soluseiniä hajottavien entsyymien merkitys taudinaiheuttamisessa on hyvin tunnettu, mutta oireettomasta jaksosta ja taudin alkuvaiheista tiedätään vain vähän. Bakteerin genomi sisältää monia toksiineja, adhesiineja, hemolysiineja ja muita proteiineja, joilla saattaa olla merkitys taudinaiheuttamisessa. Tässä työssä käytettiin proteomiikkaa ja mikrosiruanalysiä P. atrosepticum bakteerin erittyvien proteiinien ja geeniekspression tutkimiseen. Proteiinit, jotka eritetään ulos bakteerista, toimivat todennäköisesti taudinaiheuttamisessa, koska ne ovat suorassa kontaktissa isäntäkasvin kanssa. Analyysit suoritettiin olosuhteissa, jotka muistuttavat kasvin soluvälitilaa: matala pH, vähän ravinteita ja matala lämpötila. Isäntäkasvin läsnäolon vaikutusta proteiinien tuottoon ja geeniekspressioon tutkittiin lisäämällä perunauutetta kasvatusalustaan. Tutkimuksessa tunnistettiin P. atrosepticum bakteerin monia jo tunnettuja ja mahdollisesti taudinaiheuttamiseen liittyviä proteiineja. Perunauute lisäsi hiljattain tunnistetun, proteiinien eritysreittiä (tyyppi VI sekreetio, T6SS) koodaavien geenien ilmentymistä. Lisäksi bakteerin havaittiin erittävän useita T6SS:n liittyviä proteiineja kasvualustaan, johon oli lisätty perunauutetta. T6SS:n merkitys bakteereille on vielä epäselvä ja sen vaikutuksesta taudinaiheuttamiseen on julkaistu ristiriitaisia tuloksia. Märkä- ja tyvimädän ymmärtäminen molekulaarisella tasolla luo pohjan tautien kontrollointiin tähtäävään soveltavaan tutkimukseen. Tämä tutkimus lisää tietoa kasvi-patogeeni- interaktiosta ja sitä voidaan tulevaisuudessa käyttää hyväksi esimerkiksi diagnostiikassa, resistenttien perunalajikkeiden jalostuksessa tai viljely- ja varastointiolosuhteiden parantamisessa.

The effects of human induced disturbances on the sediment geochemistry of Baltic Sea embayments with a focus on eutrophication history

Historical sediment nutrient concentrations and heavy-metal distributions were studied in five embayments in the Gulf of Finland and an adjacent lake. The main objective of the study was to examine the response of these water bodies to temporal changes in human activities. Sediment cores were collected from the sites and dated using 210Pb and 137Cs. The cores were analyzed for total carbon (TC), total nitrogen (TN), total phosphorus (TP), organic phosphorus (OP), inorganic phosphorus (IP), biogenic silica (BSi), loss on ignition (LOI), grain size, Cu, Zn, Al, Fe, Mn, K, Ca, Mg and Na. Principal component analysis (PCA) was used to summarize the trends in the geochemical variables and to compare trends between the different sites. The links between the catchment land use and sediment geochemical data were studied using a multivariate technique of redundancy analysis (RDA). Human activities produce marked geochemical variations in coastal sediments. These variations and signals are often challenging to interpret due to various sedimentological and post-depositional factors affecting the sediment profiles. In general, the sites studied here show significant upcore increases in sedimentation rates, TP and TN concentrations. Also Cu, which is considered to be a good indicator of anthropogenic influence, showed clear increases from 1850 towards the top part of the cores. Based on the RDA-analysis, in the least disturbed embayments with high forest cover, the sediments are dominated by lithogenic indicators Fe, K, Al and Mg. In embayments close to urban settlement, the sediments have high Cu concentrations and a high sediment Fe/Mn ratio. This study suggests that sediment accumulation rates vary significantly from site to site and that the overall sedimentation can be linked to the geomorphology and basin bathymetry, which appear to be the major factors governing sedimentation rates; i.e. a high sediment accumulation rate is not characteristic either to urban or to rural sites. The geochemical trends are strongly site specific and depend on the local geochemical background, basin characteristics and anthropogenic metal and nutrient loading. Of the studied geochemical indicators, OP shows the least monotonic trends in all studied sites. When compared to other available data, OP seems to be the most reliable geochemical indicator describing the trophic development of the study sites, whereas Cu and Zn appear to be good indicators for anthropogenic influence. As sedimentation environments, estuarine and marine sites are more complex than lacustrine basins with multiple sources of sediment input and more energetic conditions in the former. The crucial differences between lacustrine and estuarine/coastal sedimentation environments are mostly related to Fe. P sedimentation is largely governed by Fe redox-reactions in estuarine environments. In freshwaters, presence of Fe is clearly linked to the sedimentation of other lithogenic metals, and therefore P sedimentation and preservation has a more direct linkage to organic matter sedimentation.

Glutamatergic Modulation of Cue-Induced Drug-Seeking Behavior in the Rat

The characteristics of drug addiction include compulsive drug use despite negative consequences and re-occurring relapses, returns to drug use after a period of abstinence. Therefore, relapse prevention is one of the major challenges for the treatment of drug addiction. There are three main factors capable of inducing craving for drugs and triggering relapse long after cessation of drug use and dissipation of physical withdrawal signs: stress, re-exposure to the drug, and environmental stimuli (cues) that have been previously associated with drug use. The neurotransmitters dopamine and glutamate have been implicated in the modulation of drug-seeking behavior. The aim of this project was to examine the role of glutamatergic neurotransmission in relapse triggered by conditioned drug-associated stimuli. The focus was on clarifying whether relapse to drug seeking can be attenuated by blockade of glutamate receptors. In addition, as the nucleus accumbens has been proposed to participate in the modulation of drug-seeking behavior, the effects of glutamate receptor blockade in this brain structure on cue-induced relapse were investigated. The studies employed animals models in which rats were trained to press a lever in a test cage to obtain alcohol or intravenous cocaine. Drug availability was paired with distinct olfactory, auditory, or visual stimuli. This phase was followed by extinction training, during which lever presses did not result in the presentation of the drug or the drug-associated stimuli. Extinction training led to a gradual decrease in the number of lever presses during test sessions. Relapse was triggered by presenting the rats with the drug-associated stimuli in the absence of alcohol or cocaine. The drug-associated stimuli were alone capable of inducing resumption of lever pressing and maintaining this behavior during repeated testing. The number of lever presses during a session represented the intensity of drug-seeking and relapse behavior. The results suggest that glutamatergic neurotransmission is involved in the modulation of drug-seeking behavior. Both alcohol and cocaine relapse were attenuated by systemic pretreatment with glutamate receptor antagonists. However, differences were found in the ability of ionotropic AMPA/kainate and NMDA receptor antagonists to regulate drug-seeking behavior. The AMPA/kainate antagonists CNQX and NBQX, and L-701,324, an antagonist with affinity for the glycine site of the NMDA receptor, attenuated cue-induced drug seeking, whereas the competitive NMDA antagonist CGP39551 and the NMDA channel blocker MK-801 were without effect. MPEP, an antagonist at metabotropic mGlu5 glutamate receptors, also decreased drug seeking, but its administration was found to lead to conditioned suppression of behavior during subsequent treatment sessions, suggesting that MPEP may have undesirable side effects. The mGluR2/3 agonist LY379268 and the mGluR8 agonist (S)-3,4-DCPG decreased both cue-induced relapse to alcohol drinking and alcohol consumption. Control experiments showed however that administration of the agonists was accompanied by motor suppression limiting their usefulness. Administration of the AMPA/kainate antagonist CNQX, the NMDA antagonist D-AP5, and the mGluR5 antagonist MPEP into the nucleus accumbens resulted also in a decrease in drug-seeking behavior, suggesting that the nucleus accumbens is at least one of the anatomical sites regulating drug seeking and mediating the effects of glutamate receptor antagonists on this behavior.

Assessment of neuroleptic-induced movement disorders in a naturalistic schizophrenia population

The prevalence and assessment of neuroleptic-induced movement disorders (NIMDs) in a naturalistic schizophrenia population that uses conventional neuroleptics were studied. We recruited 99 chronic schizophrenic institutionalized adult patients from a state nursing home in central Estonia. The total prevalence of NIMDs according to the diagnostic criteria of the Diagnostic and Statistical Manual of Mental Disorders, 4th edition (DSM-IV) was 61.6%, and 22.2% had more than one NIMD. We explored the reliability and validity of different instruments for measuring these disorders. First, we compared DSM-IV with the established observer rating scales of Barnes Akathisia Rating Scale (BARS), Simpson-Angus Scale (SAS) (for neuroleptic-induced parkinsonism, NIP) and Abnormal Involuntary Movement Scale (AIMS) (for tardive dyskinesia), all three of which have been used for diagnosing NIMD. We found a good overlap of cases for neuroleptic-induced akathisia (NIA) and tardive dyskinesia (TD) but somewhat poorer overlap for NIP, for which we suggest raising the commonly used threshold value of 0.3 to 0.65. Second, we compared the established observer rating scales with an objective motor measurement, namely controlled rest lower limb activity measured by actometry. Actometry supported the validity of BARS and SAS, but it could not be used alone in this naturalistic population with several co-existing NIMDs. It could not differentiate the disorders from each other. Quantitative actometry may be useful in measuring changes in NIA and NIP severity, in situations where the diagnosis has been made using another method. Third, after the relative failure of quantitative actometry to show diagnostic power in a naturalistic population, we explored descriptive ways of analysing actometric data, and demonstrated diagnostic power pooled NIA and pseudoakathisia (PsA) in our population. A subjective question concerning movement problems was able to discriminate NIA patients from all other subjects. Answers to this question were not selective for other NIMDs. Chronic schizophrenia populations are common worldwide, NIMD affected two-thirds of our study population. Prevention, diagnosis and treatment of NIMDs warrant more attention, especially in countries where typical antipsychotics are frequently used. Our study supported the validity and reliability of DSM-IV diagnostic criteria for NIMD in comparison with established rating scales and actometry. SAS can be used with minor modifications for screening purposes. Controlled rest lower limb actometry was not diagnostically specific in our naturalistic population with several co-morbid NIMDs, but it may be sensitive in measuring changes in NIMDs.

Molecular mechanisms of hypertension-induced heart failure : Experimental studies with special emphasis on local renin-angiotensin system, cardiac metabolism and levosimendan

Hypertension is a major risk factor for stroke, ischaemic heart disease, and the development of heart failure. Hypertension-induced heart failure is usually preceded by the development of left ventricular hypertrophy (LVH), which represents an adaptive and compensatory response to the increased cardiac workload. Biomechanical stress and neurohumoral activation are the most important triggers of pathologic hypertrophy and the transition of cardiac hypertrophy to heart failure. Non-clinical and clinical studies have also revealed derangements of energy metabolism in hypertensive heart failure. The goal of this study was to investigate in experimental models the molecular mechanisms and signalling pathways involved in hypertension-induced heart failure with special emphasis on local renin-angiotensin-aldosterone system (RAAS), cardiac metabolism, and calcium sensitizers, a novel class of inotropic agents used currently in the treatment of acute decompensated heart failure. Two different animal models of hypertensive heart failure were used in the present study, i.e. hypertensive and salt-sensitive Dahl/Rapp rats on a high salt diet (a salt-sensitive model of hypertensive heart failure) and double transgenic rats (dTGR) harboring human renin and human angiotensinogen genes (a transgenic model of hypertensive heart failure with increased local RAAS activity). The influence of angiotensin II (Ang II) on cardiac substrate utilization and cardiac metabolomic profile was investigated by using gas chromatography coupled to time-of-flight mass spectrometry to detect 247 intermediary metabolites. It was found that Ang II could alter cardiac metabolomics both in normotensive and hypertensive rats in an Ang II receptor type 1 (AT1)-dependent manner. A distinct substrate use from fatty acid oxidation towards glycolysis was found in dTGR. Altered cardiac substrate utilization in dTGR was associated with mitochondrial dysfunction. Cardiac expression of the redox-sensitive metabolic sensor sirtuin1 (SIRT1) was increased in dTGR. Resveratrol supplementation prevented cardiovascular mortality and ameliorated Ang II-induced cardiac remodeling in dTGR via blood pressure-dependent pathways and mechanisms linked to increased mitochondrial biogenesis. Resveratrol dose-dependently increased SIRT1 activity in vitro. Oral levosimendan treatment was also found to improve survival and systolic function in dTGR via blood pressure-independent mechanisms, and ameliorate Ang II-induced coronary and cardiomyocyte damage. Finally, using Dahl/Rapp rats it was demonstrated that oral levosimendan as well as the AT1 receptor antagonist valsartan improved survival and prevented cardiac remodeling. The beneficial effects of levosimendan were associated with improved diastolic function without significantly improved systolic changes. These positive effects were potentiated when the drug combination was administered. In conclusion, the present study points to an important role for local RAAS in the pathophysiology of hypertension-induced heart failure as well as its involvement as a regulator of cardiac substrate utilization and mitochondrial function. Our findings suggest a therapeutic role for natural polyphenol resveratrol and calcium sensitizer, levosimendan, and the novel drug combination of valsartan and levosimendan, in prevention of hypertension-induced heart failure. The present study also provides a better understanding of the pathophysiology of hypertension-induced heart failure, and may help identify potential targets for novel therapeutic interventions.

Mother-Infant Antibodies to Pneumococcal Polysaccharides and Proteins : Transfer and Persistence of Maternal Antibodies and Development of Vaccine-Induced and Naturally Acquired Antibodies in Infants

Symptomless nasopharyngeal carriage of Streptococcus pneumoniae (pneumococcus) is very common in young children. Occasionally the carriage proceeds into mild mucosal diseases, such as sinusitis or acute otitis media, or into serious life-threatening diseases, such as pneumonia, sepsis or meningitis. Each year, up to one million children less than five years of age worldwide die of invasive pneumococcal diseases (IPD). Especially in the low-income countries IPD is a leading health problem in infants; 75% of all IPD cases occur before one year of age. This stresses the need of increased protection against pneumococcus in infancy. Anti-pneumococcal antibodies form an important component in the defence against pneumococcal infection. Maternal immunisation and early infant immunisation are two possible ways by which potentially protective antibody concentrations against pneumococci could be achieved in early infancy. The aim of this thesis is to increase the knowledge of antibody mediated protection against pneumococcal disease in infants and young children. We investigated the transfer of maternal anti-pneumococcal antibodies from Filipino mothers to their infants, the persistence of the transferred antibodies in the infants, the immunogenicity of the 23-valent pneumococcal polysaccharide vaccine (PPV) in infants and the response of the children to a second dose of PPV at three years of age. We also investigated the development of antibodies to pneumococcal protein antigens in relation to culture-confirmed pneumococcal carriage in infants. Serum samples were collected from the mothers, the umbilical cords and from the infants at young age as well as at three years of age. The samples were used to determine the antibody concentrations to pneumococcal serotypes 1, 5, 6B, 14, 18C and 19F, as well as to the pneumococcal proteins PspA, PsaA, Ply, PspC, PhtD, PhtDC and LytC by the enzyme immunoassay. The findings of the present study confirm previously obtained results and add to the global knowledge of responses to PPV in young children. Immunising pregnant women with PPV provides the infants with increased concentrations of pneumococcal polysaccharide antibodies. Of the six serotypes examined, serotypes 1 and 5 were immunogenic already in infants. At three years of age, the children responded well to the second dose of PPV suggesting that maternal and early infant immunisations might not induce hyporesponsiveness to polysaccharide antigens after subsequent immunisations. The anti-protein antibody findings provide useful information for the development of pneumococcal protein vaccines. All six proteins studied were immunogenic in infancy and the development of anti-protein antibodies started early in life in relation to pneumococcal carriage.