Conformational Regulation of alpha2beta1 Integrin in Ligand Binding

Integrins are heterodimeric cell adhesion receptors involved in cell-cell and cell-extracellular matrix (ECM) interactions. They transmit bidirectional signals across the cell membrane. This results in a wide range of biological events from cell differentiation to apoptosis. alpha2beta1 integrin is an abundant collagen receptor expressed on the surface of several cell types. In addition to ECM ligands, alpha2beta1 integrins are bound by echovirus 1 (EV1) which uses alpha2beta1 as a receptor to initiate its life cycle in the infected cell. The aim of this thesis project was to provide further insight into the mechanisms of alpha2beta1 integrin ligand recognition and receptor activation. Collagen fibrils are the principal tensile elements of the ECM. Yet, the interaction of alpha2beta1 integrin with the fibrillar form of collagen I has received relatively little attention. This research focused on the ability of alpha2beta1 integrin to act as a receptor for type I collagen fibrils. Also the molecular requirements of the EV1 interaction with alpha2beta1 were studied. Conventionally, ligand binding has been suggested to require integrin activation and the binding may further trigger integrin signalling. Another main objective of this study was to elucidate both the inside-out and outside-in signalling mechanisms of alpha2beta1 integrin in adherent cells. The results indicated that alpha2beta1 integrin is the principal integrin-type collagen receptor for type I collagen fibrils, and alpha2beta1 may participate in the regulation of pericellular collagen fibrillogenesis. Furthermore, alpha2beta1 integrin inside-out activation appeared to be synergistically regulated by integrin clustering and conformational activation. The triggering of alpha2beta1 integrin outside-in signalling, however, was shown to require both conformational changes and clustering. In contrast to ECM ligands, EV1 appeared to take advantage of the bent, inactive form of alpha2beta1 integrin in initiating its life cycle in the cell. This research together with other recent studies, has shed light on the molecular mechanisms of integrin activation. It is becoming evident that large ligands are able to bind to the bent form of integrin, which has been previously considered to be physiologically inactive. Consequently, our understanding of the conformational modulation of integrins upon activation is changing.

Carbohydrate intake in children - associations with dietary intakes, growth, serum lipids,and dental health. The STRIP Project

This study is part of the STRIP study, which is a long-term, randomized controlled trial, designed to decrease the exposure of children in the intervention group (n=540) to known risk factors of atherosclerosis. The main focus of the intervention was the quality of dietary fat. The control group (n=522) did not receive any individualized counselling. Food consumption was evaluated with food records, and blood samples were drawn and growth was measured regularly for all participating children from 13 months to 9 years. A subsample of 66 children participated in a dental health survey. The number of studies on children’s carbohydrate intake, especially fibre intake, is insufficient. The current international recommendations for fibre intake in children are based on average assumptions and data extrapolated from intakes in adults and intake recommendations for adults. Finnish nutrition recommendations lack strict recommendations for dietary fibre in children. Due to fibre’s high bulk volume, excessive dietary fibre is considered to decrease energy density and hence it may have an adverse effect on growth. If fats are reduced from the diet, the low-fat diet may become high in sucrose. Therefore, especially in the STRIP study, it is important to determine the use of fibre and sucrose in children and possible associations with growth and nutrition as well as dental health. The results of the present study indicate that a high fibre intake does not displace energy or disturb growth in children and that children with high fibre intake have better quality of diet than those with low fibre intake. Additionally, dietary fibre intake associated inversely with serum cholesterol concentration. Other carbohydrates also affected serum lipid levels as well, since total carbohydrates, sucrose, and fructose increased serum triglyceride concentration. Total carbohydrate intake reduced HDL cholesterol concentration only in children with apoE3 or apoE4 phenotype. Over the period from the 1970s to the 1990s the dental health of children in Finland has substantially improved despite an increase in sucrose intake. The improvement was thought to be due to improved dental hygiene and the use of fluorine. However, during the past twenty years improvement in dental health has stopped. The present study showed that high long-term sugar intake increases risk of caries in children. High intake of sugar had also negative effects on the diet of children, because it worsens dietary quality by displacing essential nutrients. Furthermore, the quality of dietary fat was worse in children with high sucrose intake. In this study the children’s high sucrose intake was not associated with overweight, but interestingly, it associated inversely with growth.

Alpha2-adrenoceptors: Structure and ligand binding properties at the molecular level

Alpha₂-Adrenoceptors: structure and ligand binding properties at the molecular level The mouse is the most frequently used animal model in biomedical research, but the use of zebrafish as a model organism to mimic human diseases is on the increase. Therefore it is considered important to understand their pharmacological differences from humans also at the molecular level. The zebrafish Alpha_2-adrenoceptors were expressed in mammalian cells and the binding affinities of 20 diverse ligands were determined and compared to the corresponding human receptors. The pharmacological properties of the human and zebrafish Alpha_2--adrenoceptors were found to be quite well conserved. Receptor models based on the crystal structures of bovine rhodopsin and the human Beta₂-adrenoceptor revealed that most structural differences between the paralogous and orthologous Alpha_2--adrenoceptors were located within the second extracellular loop (XL2). Reciprocal mutations were generated in the mouse and human Alpha_2--adrenoceptors. Ligand binding experiments revealed that substitutions in XL2 reversed the binding profiles of the human and mouse Alpha_2--adrenoceptors for yohimbine, rauwolscine and RS-79948-197, evidence for a role for XL2 in the determination of species-specific ligand binding. Previous mutagenesis studies had not been able to explain the subtype preference of several large Alpha_2--adrenoceptor antagonists. We prepared chimaeric Alpha_2--adrenoceptors where the first transmembrane (TM1) domain was exchanged between the three human Alpha_2--adrenoceptor subtypes. The binding affinities of spiperone, spiroxatrine and chlorpromazine were observed to be significantly improved by TM1 substitutions of the Alpha_2a--adrenoceptor. Docking simulations indicated that indirect effects, such as allosteric modulation, are more likely to be involved in this phenomenon rather than specific side-chain interactions between ligands and receptors.

Functional studies on bacterial nucleotide-regulated inorganic pyrophosphatases

CBS domains are ~60 amino acid tandemly repeated regulatory modules forming a widely distributed domain superfamily. Found in thousands of proteins from all kingdoms of life, CBS domains have adopted a variety of functions during evolution, one of which is regulation of enzyme activity through binding of adenylate-containing compounds in a hydrophobic cavity. Mutations in human CBS domain-containing proteins cause hereditary diseases. Inorganic pyrophosphatases (PPases) are ubiquitous enzymes, which pull pyrophosphate (PPi) producing reactions forward by hydrolyzing PPi into phosphate. Of the two nonhomologous soluble PPases, dimeric family II PPases, belonging to the DHH family of phosphoesterases, require a transition metal and magnesium for maximal activity. A quarter of the almost 500 family II PPases, found in bacteria and archaea, contain a 120-250 amino acid N-terminal insertion, comprised of two CBS domains separated in sequence by a DRTGG domain. These enzymes are thus named CBS-PPases. The function of the DRTGG domain in proteins is unknown. The aim of this PhD thesis was to elucidate the structural and functional differences of CBS-PPases in comparison to family II PPases lacking the regulatory insert. To this end, we expressed, purified and characterized the CBS-PPases from Clostridium perfringens (cpCBS-PPase) and Moorella thermoacetica (mtCBS-PPase), the latter lacking a DRTGG domain. Both enzymes are homodimers in solution and display maximal activity against PPi in the presence of Co2+ and Mg2+. Uniquely, the DRTGG domain was found to enable tripolyphosphate hydrolysis at rates similar to that of PPi. Additionally, we found that AMP and ADP inhibit, while ATP and AP4A activate CBSPPases, thus enabling regulation in response to changes in cellular energy status. We then observed substrate- and nucleotide-induced conformational transitions in mtCBS-PPase and found that the enzyme exists in two differentially active conformations, interconverted through substrate binding and resulting in a 2.5-fold enzyme activation. AMP binding was shown to produce an alternate conformation, which is reached through a different pathway than the substrate-induced conformation. We solved the structure of the regulatory insert from cpCBS-PPase in complex with AMP and AP4A and proposed that conformational changes in the loops connecting the catalytic and regulatory domains enable activity regulation. We examined the effects of mutations in the CBS domains of mtCBS-PPase on catalytic activity, as well as, nucleotide binding and inhibition.

Episodes in carbohydrate chemistry : from β-linked mannosides to glycoconjugates

Carbohydrates are one of the most abundant classes of biomolecules on earth. In the initial stages of research on carbohydrates much effort was focused on investigation and determination of the structural aspects and complex nature of individual monosaccharides. Later on, development of protective group strategies and methods for oligosaccharide synthesis became the main topics of research. Today, the methodologies developed early on are being utilized in the production of carbohydrates for biological screening events. This multidisciplinary approach has generated the new discipline of glycobiology which focuses on research related to the appearance and biological significance of carbohydrates. In more detail, studies in glycobiology have revealed the essential roles of carbohydrates in cell-cell interactions, biological recognition events, protein folding, cell growth and tumor cell metastasis. As a result of these studies, carbohydrate derived diagnostic and therapeutic agents are likely to be of growing interest in the future. In this doctoral thesis, a journey through the fundamentals of carbohydrate synthesis is presented. The research conducted on this journey was neither limited to the study of any particular phenomena nor to the addressing of a single synthetic challenge. Instead, the focus was deliberately shifted from time to time in order to broaden the scope of the thesis, to continue the learning process and to explore new areas of carbohydrate research. Throughout the work, several previously reported synthetic protocols, especially procedures related to glycosylation reactions and protective group manipulations, were evaluated, modified and utilized or rejected. The synthetic molecules targeted within this thesis were either required for biological evaluations or utilized to study phenomena occuring in larger molecules. In addition, much effort was invested in the complete structural characterization of the synthesized compounds by a combination of NMR spectroscopic techniques and spectral simulations with the PERCH-software. This thesis provides the basics of working with carbohydrate chemistry. In more detail, synthetic strategies and experimental procedures for many different reactions and guidelines for the NMR-spectroscopic characterization of oligosaccharides and glycoconjugates are provided. Therefore, the thesis should prove valuable to researchers starting their own journeys in the ever expanding field of carbohydrate chemistry.

Identification and characterisation of a novel adhesin of Streptococcus suis and its use as a target of adhesion inhibition and bacterial detection

Streptococcus suis is an important pig pathogen but it is also zoonotic, i.e. capable of causing diseases in humans. Human S. suis infections are quite uncommon but potentially life-threatening and the pathogen is an emerging public health concern. This Gram-positive bacterium possesses a galabiose-specific (Galalpha1−4Gal) adhesion activity, which has been studied for over 20 years. P-fimbriated Escherichia coli−bacteria also possess a similar adhesin activity targeting the same disaccharide. The galabiose-specific adhesin of S. suis was identified by an affinity proteomics method. No function of the protein identified was formerly known and it was designated streptococcal adhesin P (SadP). The peptide sequence of SadP contains an LPXTG-motif and the protein was proven to be cell wall−anchored. SadP may be multimeric since in SDS-PAGE gel it formed a protein ladder starting from about 200 kDa. The identification was confirmed by producing knockout strains lacking functional adhesin, which had lost their ability to bind to galabiose. The adhesin gene was cloned in a bacterial expression host and properties of the recombinant adhesin were studied. The galabiose-binding properties of the recombinant protein were found to be consistent with previous results obtained studying whole bacterial cells. A live-bacteria application of surface plasmon resonance was set up, and various carbohydrate inhibitors of the galabiose-specific adhesins were studied with this assay. The potencies of the inhibitors were highly dependent on multivalency. Compared with P-fimbriated E. coli, lower concentrations of galabiose derivatives were needed to inhibit the adhesion of S. suis. Multivalent inhibitors of S. suis adhesion were found to be effective at low nanomolar concentrations. To specifically detect galabiose adhesin−expressing S. suis bacteria, a technique utilising magnetic glycoparticles and an ATP bioluminescence bacterial detection system was also developed. The identification and characterisation of the SadP adhesin give valuable information on the adhesion mechanisms of S. suis, and the results of this study may be helpful for the development of novel inhibitors and specific detection methods of this pathogen.

Enhanced muscle strength with carbohydrate supplement two hours before open cholecystectomy: a randomized, double-blind study

Objective: to investigate the effects of preoperative fasting abbreviation with oral supplementation with carbohydrate in the evolution of grip strength in patients undergoing cholecystectomy by laparotomy. Methods : we conducted a clinical, randomizeddouble blind study with adult female patients, aged 18-60 years. Patients were divided into two groups: Control Group, with fasting prescription 6-8h until the time of operation; and Intervention Group, which received prescription of fasting for solids 6-8h before surgery, but ingested an oral supplement containing 12.5% carbohydrate, six (400ml) and two (200ml) hours before theprocedure. The handgrip strength was measured in both hands in both groups, at patient's admission (6h before surgery), the immediate pre-operative time (1h before surgery) and 12-18h postoperatively. Results : we analyzed 27 patients, 14 in the intervention group and 13 in the control group. There was no mortality. The handgrip strength (mean [standard deviation]) was significantly higher in the intervention group in the three periods studied, in at least one hand: preoperatively in the dominant hand (27.8 [2.6] vs 24.1 [3.7] kg; p=0.04), in the immediate preoperative in both hands, and postoperatively in the non-dominant hand (28.5 [3.0] vs 21.3 [5.9] kg; p=0.01). Conclusion : the abbreviation of preoperative fasting to two hours with drink containing carbohydrate improves muscle function in the perioperative period.

Power cycling lifetime estimation of IGBT power modules based on chip temperature modeling

In this doctoral thesis, methods to estimate the expected power cycling life of power semiconductor modules based on chip temperature modeling are developed. Frequency converters operate under dynamic loads in most electric drives. The varying loads cause thermal expansion and contraction, which stresses the internal boundaries between the material layers in the power module. Eventually, the stress wears out the semiconductor modules. The wear-out cannot be detected by traditional temperature or current measurements inside the frequency converter. Therefore, it is important to develop a method to predict the end of the converter lifetime. The thesis concentrates on power-cycling-related failures of insulated gate bipolar transistors. Two types of power modules are discussed: a direct bonded copper (DBC) sandwich structure with and without a baseplate. Most common failure mechanisms are reviewed, and methods to improve the power cycling lifetime of the power modules are presented. Power cycling curves are determined for a module with a lead-free solder by accelerated power cycling tests. A lifetime model is selected and the parameters are updated based on the power cycling test results. According to the measurements, the factor of improvement in the power cycling lifetime of modern IGBT power modules is greater than 10 during the last decade. Also, it is noticed that a 10 C increase in the chip temperature cycle amplitude decreases the lifetime by 40%. A thermal model for the chip temperature estimation is developed. The model is based on power loss estimation of the chip from the output current of the frequency converter. The model is verified with a purpose-built test equipment, which allows simultaneous measurement and simulation of the chip temperature with an arbitrary load waveform. The measurement system is shown to be convenient for studying the thermal behavior of the chip. It is found that the thermal model has a 5 C accuracy in the temperature estimation. The temperature cycles that the power semiconductor chip has experienced are counted by the rainflow algorithm. The counted cycles are compared with the experimentally verified power cycling curves to estimate the life consumption based on the mission profile of the drive. The methods are validated by the lifetime estimation of a power module in a direct-driven wind turbine. The estimated lifetime of the IGBT power module in a direct-driven wind turbine is 15 000 years, if the turbine is located in south-eastern Finland.

Prediction of 3D structure and ligand-binding properties : ABC transporters and flavodiiron proteins from Synechocystis sp. strain PCC 6803

Cyanobacteria are unicellular, non-nitrogen-fixing prokaryotes, which perform photosynthesis similarly as higher plants. The cyanobacterium Synechocystis sp. strain PCC 6803 is used as a model organism in photosynthesis research. My research described herein aims at understanding the function of the photosynthetic machinery and how it responds to changes in the environment. Detailed knowledge of the regulation of photosynthesis in cyanobacteria can be utilized for biotechnological purposes, for example in the harnessing of solar energy for biofuel production. In photosynthesis, iron participates in electron transfer. Here, we focused on iron transport in Synechocystis sp. strain PCC 6803 and particularly on the environmental regulation of the genes encoding the FutA2BC ferric iron transporter, which belongs to the ABC transporter family. A homology model built for the ATP-binding subunit FutC indicates that it has a functional ATPbinding site as well as conserved interactions with the channel-forming subunit FutB in the transporter complex. Polyamines are important for the cell proliferation, differentiation and apoptosis in prokaryotic and eukaryotic cells. In plants, polyamines have special roles in stress response and in plant survival. The polyamine metabolism in cyanobacteria in response to environmental stress is of interest in research on stress tolerance of higher plants. In this thesis, the potd gene encoding an polyamine transporter subunit from Synechocystis sp. strain PCC 6803 was characterized for the first time. A homology model built for PotD protein indicated that it has capability of binding polyamines, with the preference for spermidine. Furthermore, in order to investigate the structural features of the substrate specificity, polyamines were docked into the binding site. Spermidine was positioned very similarly in Synechocystis PotD as in the template structure and had most favorable interactions of the docked polyamines. Based on the homology model, experimental work was conducted, which confirmed the binding preference. Flavodiiron proteins (Flv) are enzymes, which protect the cell against toxicity of oxygen and/or nitric oxide by reduction. In this thesis, we present a novel type of photoprotection mechanism in cyanobacteria by the heterodimer of Flv2/Flv4. The constructed homology model of Flv2/Flv4 suggests a functional heterodimer capable of rapid electron transfer. The unknown protein sll0218, encoded by the flv2-flv4 operon, is assumed to facilitate the interaction of the Flv2/Flv4 heterodimer and energy transfer between the phycobilisome and PSII. Flv2/Flv4 provides an alternative electron transfer pathway and functions as an electron sink in PSII electron transfer.

Effect of exposure to sublethal concentrations of sodium cyanide on the carbohydrate metabolism of the Indian Major Carp Labeo rohita (Hamilton, 1822)

Experiments were designed to study in-vivo effects of sodium cyanide on biochemical endpoints in the freshwater fish Labeo rohita. Fish were exposed to two sublethal concentrations (0.106 and 0.064mg/L) for a period of 15 days. Levels of glycogen, pyruvate, lactate and the enzymatic activities of lactate dehydrogenase (LDH), succinate dehydrogenase (SDH), glucose-6-phosphate dehydrogenase (G6PDH), phosphorylase, alkaline phosphatase (ALP), acid phosphatase (AcP) were assessed in different tissues (liver, muscle and gills). Result indicated a steady decrease in glycogen, pyruvate, SDH, ALP and AcP activity with a concomitant increase in the lactate, phosphorylase, LDH and G6PD activity in all selected tissues. The alterations in all the above biochemical parameters were significantly (p<0.05) time and dose dependent. In all the above parameters, liver pointing out the intensity of cyanide intoxication compare to muscle and gills. Study revealed change in the metabolic energy by means of altered metabolic profile of the fish. Further, these observations indicated that even sublethal concentrations of sodium cyanide might not be fully devoid of deleterious influence on metabolism in L. rohita.

Methodology for Photovoltaic Modules Characterization and Shading Effects Analysis

This work describes the methodology, basic procedures and instrumental employed by the Solar Energy Laboratory at Universidade Federal do Rio Grande do Sul for the determination of current-voltage characteristic curves of photovoltaic modules. According to this methodology, I-V characteristic curves were acquired for several modules under diverse conditions. The main electrical parameters were determined and the temperature and irradiance influence on photovoltaic modules performance was quantified. It was observed that most of the tested modules presented output power values considerably lower than those specified by the manufacturers. The described hardware allows the testing of modules with open-circuit voltage up to 50 V and short-circuit current up to 8 A.

Muscarinic toxins : characterization of adrenoceptor binding and applicability of membrane anchoring via glycosylphosphatidylinositol tail

Adrenoceptors (ARs), G-protein coupled receptors (GPCRs) at the plasma membrane, respond to endogenous catecholamines noradrenaline and adrenaline. These receptors mediate several important physiological functions being especially important in the cardiovascular system and in the regulation of smooth muscle contraction. Impairments in the function of these receptors can thus lead to severe diseases and disorders such as to cardiovascular diseases and benign prostatic hyperplasia. The Eastern green mamba (Dendroaspis angusticeps) venom has been shown to contain toxins that can antagonize the functions of GPCRs. The most well-known are muscarinic toxins (MTs) targeting muscarinic acetylcholine receptors (mAChRs) with high affinity and selectivity. However, some reports have indicated that these toxins might also act on the α1- and α2-ARs which can be divided into various subtypes; the α1-ARs to α1A-, α1B- and α1D-ARs and α2-ARs to α2A-, α2B- and α2C-ARs. In this thesis, the interaction of four common MTs (MT1, MT3, MT7 and MTα) with the adrenoceptors was characterized. It was also evaluated whether these toxins could be anchored to the plasma membrane via glycosylphosphatidylinositol (GPI) tail. Results of this thesis reveal that muscarinic toxins are targeting several α-adrenoceptor subtypes in addition to their previously identified target receptors, mAChRs. MTα was found to interact with high affinity and selectivity with the α2B-AR whereas MT7 confirmed its selectivity for the M1 mAChR. Unlike MTα and MT7, MT1 and MT3 have a broad range of target receptors among the α-ARs. All the MTs characterized were found to behave as non-competitive antagonists of receptor action. The interaction between MTα and the α2B-AR was studied more closely and it was observed that the second extracellular loop of the receptor functions as a structural entity enabling toxin binding. The binding of MTα to the α2B-AR appears to be rather complex and probably involves dimerized receptor. Anchoring MTs to the plasma membrane did not interfere with their pharmacological profile; all the GPI-anchored toxins created retained their ability to block their target receptors. This thesis shows that muscarinic toxins are able to target several subtypes of α-ARs and mAChRs. These toxins offer thus a possibility to create new subtype specific ligands for the α-AR subtypes. Membrane anchored MTs on the other hand could be used to block α-AR and mAChR actions in disease conditions such as in hypertension and in gastrointestinal and urinary bladder disorders in a cell-specific manner and to study the physiological functions of ARs and mAChRs in vivo in model organisms.

Intracellular and intercellular compartmentation of carbohydrate metabolism in leaves of temperate gramineae

This review considers the importance of compartmentation in the regulation of carbohydrate metabolism in leaves. We draw particular attention to the role of the vacuole as a site for storage of soluble sugars based on sucrose, and discuss briefly their characteristic metabolism. We also point out inconsistencies between the observed properties of vacuoles and the behaviour in vitro of the enzymes of fructan biosynthesis that do not support the hypothesis that the vacuole is the site of synthesis as well as of storage. We also consider compartmentation of carbohydrate metabolism between different cell types, using mainly our studies on leaves of temperate C3 gramineae. Here we present evidence of significant differences in carbon metabolism between epidermis, mesophyll, bundle sheath and vasculature based upon both single-cell sampling and immunolocalisation. The implications of these differences for the control of metabolism in leaves are discussed.

Diurnal changes in storage carbohydrate metabolism in cotyledons of the tropical tree Hymenaea courbaril L. (Leguminosae)

The cotyledons of Hymenaea courbaril store large amounts of xyloglucan, a cell wall polysaccharide that is believed to serve as storage for the period of seedling establishment. During storage mobilisation, xyloglucan seems to be degraded by a continuous process that starts right after radicle protrusion and follows up to the establishment of photosynthesis. Here we show evidence that events related to the hydrolases activities and production (α-xylosidase, β-galactosidase, β-glucosidase and xyloglucan endo-β-transglucosilase) as well as auxin, showed changes that follow the diurnal cycle. The period of higher hydrolases activities was between 6pm and 6am, which is out of phase with photosynthesis. Among the enzymes, α-xilosidase seems to be more important than β-glucosidase and β-galactosidase in the xyloglucan disassembling mechanism. Likewise, the sugars related with sucrose metabolism followed the rhythm of the hydrolases, but starch levels were shown to be practically constant. A high level of auxin was observed during the night, what is compatible with the hypothesis that this hormone would be one of the regulators of the whole process. The probable biological meaning of the existence of such a complex control mechanism during storage mobilisation is likely to be related to a remarkably high level of efficiency of carbon usage by the growing seedling of Hymenaea courbaril, allowing the establishment of very vigorous seedlings in the tropical forest.