Methods of genetic diversity creation and functional display for directed evolution experiments

Protein engineering aims to improve the properties of enzymes and affinity reagents by genetic changes. Typical engineered properties are affinity, specificity, stability, expression, and solubility. Because proteins are complex biomolecules, the effects of specific genetic changes are seldom predictable. Consequently, a popular strategy in protein engineering is to create a library of genetic variants of the target molecule, and render the population in a selection process to sort the variants by the desired property. This technique, called directed evolution, is a central tool for trimming protein-based products used in a wide range of applications from laundry detergents to anti-cancer drugs. New methods are continuously needed to generate larger gene repertoires and compatible selection platforms to shorten the development timeline for new biochemicals. In the first study of this thesis, primer extension mutagenesis was revisited to establish higher quality gene variant libraries in Escherichia coli cells. In the second study, recombination was explored as a method to expand the number of screenable enzyme variants. A selection platform was developed to improve antigen binding fragment (Fab) display on filamentous phages in the third article and, in the fourth study, novel design concepts were tested by two differentially randomized recombinant antibody libraries. Finally, in the last study, the performance of the same antibody repertoire was compared in phage display selections as a genetic fusion to different phage capsid proteins and in different antibody formats, Fab vs. single chain variable fragment (ScFv), in order to find out the most suitable display platform for the library at hand. As a result of the studies, a novel gene library construction method, termed selective rolling circle amplification (sRCA), was developed. The method increases mutagenesis frequency close to 100% in the final library and the number of transformants over 100-fold compared to traditional primer extension mutagenesis. In the second study, Cre/loxP recombination was found to be an appropriate tool to resolve the DNA concatemer resulting from error-prone RCA (epRCA) mutagenesis into monomeric circular DNA units for higher efficiency transformation into E. coli. Library selections against antigens of various size in the fourth study demonstrated that diversity placed closer to the antigen binding site of antibodies supports generation of antibodies against haptens and peptides, whereas diversity at more peripheral locations is better suited for targeting proteins. The conclusion from a comparison of the display formats was that truncated capsid protein three (p3Δ) of filamentous phage was superior to the full-length p3 and protein nine (p9) in obtaining a high number of uniquely specific clones. Especially for digoxigenin, a difficult hapten target, the antibody repertoire as ScFv-p3Δ provided the clones with the highest affinity for binding. This thesis on the construction, design, and selection of gene variant libraries contributes to the practical know-how in directed evolution and contains useful information for scientists in the field to support their undertakings.

Neurocognitive changes in early-stage Parkinson's disease : functional and structural brain correlates of memory and verbal functions

Resultaten påvisade små, men mätbara försämringar i minnes- och verbal förmåga hos personer som haft Parkinsons sjukdom under tre år. Jämfört med en kontrollgrupp uppvisade Parkinsonpatienter avvikande responser i hjärnans elektriska aktivitet under en korttidsminnesuppgift, och de presterade även sämre i olika typer av andra minnesuppgifter. Försämring i en specifik typ av minnesuppgift korrelerade med förminskad volym i höger hjärnhalva. Samband hittades också mellan sämre verbal förmåga och förminskad volym i djupa hjärnstrukturer. Förminskad hjärnvolym har tidigare påvisats hos dementa patienter i senare sjukdomsstadier. Forskningsresultaten bidrar med ny kunskap om kognitiva symptom och deras neurala bakgrund vid Parkinsons sjukdom. De tyder också på att tidig kognitiv funktionsnedsättning kan identifieras, vilket kan bidra till utvecklingen av sjukdomens behandling. Parkinsons sjukdom är den näst vanligaste neurogeriatriska sjukdomen efter Alzheimers sjukdom. Symptomen uppstår som följd av förminskad produktion av hjärnans transmittorämne dopamin. Parkinsons sjukdom har traditionellt betraktats som en progressiv motorisk sjukdom. Ny forskning tyder på att multipla hjärnsystem skadas, vilket resulterar i att även tankeprocesser påverkas. 75-80% uppskattas insjukna i demens 10-15 år efter diagnos, men det kognitiva sjukdomsförloppet och orsaken till demenssymptomen är tillsvidare okänd. I Finland uppskattas ca 10-12 000 personer ha Parkinsons sjukdom, varav ca 3 000 uppskattas ha demens. ----------------------------------------------------------------------------------------------------------------------------------------------------- Tutkimuksessa todettiin lieviä muutoksia muisti- ja kielellisissä toiminnoissa alle kolme vuotta sairastaneilla Parkinson-potilailla. Potilailla havaittiin poikkeavia aivosähkötoiminnan vasteita lyhytkestoista muistia mittaavan tehtävän aikana. Potilaat suoriutuivat myös verrokkiryhmää heikommin useissa muistitehtävissä. Heikentynyt tahaton muisti liittyi pienempään aivokuoren harmaan aineen paikalliseen tilavuuteen. Heikompi kielellinen suoriutuminen liittyi pienempään harmaan aineen tilavuuteen aivojen syvissä rakenteissa. Pienentyneitä aivorakenteiden tilavuuksia on aiemmin todettu dementoituneilla Parkinson-potilailla sairauden myöhemmissä vaiheissa. Tutkimustulokset tuovat uutta tietoa Parkinsonin taudin kognitiivisista oireista ja niiden aivoperäisestä taustasta. Tulosten perusteella on mahdollista tunnistaa jo varhaisia kognitiivisia muutoksia, mikä voi mahdollistaa tehokkaamman hoidon kohdentamisen. Parkinsonin tauti on Alzheimerin taudin jälkeen toiseksi yleisin neurogeriatrinen sairaus. Oirekuva liittyy aivojen dopaminergisen järjestelmän rappeutumiseen. Perinteisesti liikehäiriösairaudeksi luokiteltu sairaus vaurioittaa lukuisia muita aivojärjestelmiä aiheuttaen muutoksia myös mm. ajattelutoiminnoissa. Pitkään sairastaneista 75–80 prosentilla esiintyy dementiaoireita, mutta oireiden syy ja kehityskaari tunnetaan toistaiseksi huonosti. Suomessa on arviolta 10–12 000 Parkinson-potilasta, joista noin 3 000 arvioidaan kärsivän dementiaoireista.

Characterisation of Functional Brown Adipose Tissue In Adult Humans

The white adipose tissue mainly serves the purpose of energy storage, while brown adipose tissue (BAT) has the capacity to generate heat under cold conditions in mammals and in human infants. BAT is controlled by the central nervous system, and BAT function is accompanied by increased energy expenditure. However, it was not previously certain whether adult humans also have functional BAT. The aim of this doctoral work was to identify functional BAT in adult humans and to characterise its glucose uptake and blood flow under cold and insulin stimulation conditions in lean and in obese humans, by using positron emission tomography. Further, the impact of weight loss on BAT glucose uptake was assessed. Cerebral glucose uptake was also studied in relation to BAT function and cold exposure. The results showed that healthy adult humans have functional BAT, as assessed by the intense cold-induced glucose uptake and by biopsies. BAT was also found to be a highly insulinsensitive tissue in lean humans, but the effects of insulin and cold exposure were attenuated in obese humans, although the glucose uptake capacity of cold-activated BAT might be increased by weight loss. Blood flow in the BAT of lean humans was associated with whole-body energy expenditure. The presence of cold-activated BAT was related to lower body mass index and higher insulin sensitivity. Finally, BAT activation was linked to the activity of the cerebellum, the thalamus and certain neocortical regions. The cold-induced cerebral glucose uptake was also lower in obese than in lean adult humans.

Chemical derivatization of galactoglucomannan for functional materials

In the framework of the biorefinery concept researchers aspire to optimize the utilization of plant materials, such as agricultural wastes and wood. For most of the known processes, the first steps in the valorisation of biomass are the extraction and purification of the individual components. The obtained raw products by means of a controlled separation can consecutively be modified to result in biofuels or biogas for energy production, but also in value-added products such as additives and important building blocks for the chemical and material industries. Considerable efforts are undertaken in order to substitute the use of oil-based starting materials or at least minimize their processing for the production of everyday goods. Wood is one of the raw materials, which have gained large attention in the last decades and its composition has been studied in detail. Nowadays, the extraction of water-soluble hemicelluloses from wood is well known and so for example xylan can be obtained from hardwoods and O-acetyl galactoglucomannans (GGMs) from softwoods. The aim of this work was to develop water-soluble amphiphilic materials of GGM and to assess their potential use as additives. Furthermore, GGM was also applied as a crosslinker in the synthesis of functional hydrogels for the removal of toxic metals and metalloid ions from aqueous solutions. The distinguished products were obtained by several chemical approaches and analysed by nuclear magnetic resonance spectroscopy (NMR), Fourier transform infrared spectroscopy (FTIR), size exclusion chromatography (SEC), thermal gravimetric analysis (TGA), scanning electron microscope SEM, among others. Bio-based surfactants were produced by applying GGM and different fatty acids as starting materials. On one hand, GGM-grafted-fatty acids were prepared by esterification and on the other hand, well-defined GGM-block-fatty acid derivatives were obtained by linking amino-functional fatty acids to the reducing end of GGM. The reaction conditions for the syntheses were optimized and the resultant amphiphilic GGM derivatives were evaluated concerning their ability to reduce the surface tension of water as surfactants. Furthermore, the block-structured derivatives were tested in respect to their applicability as additives for the surface modification of cellulosic materials. Besides the GGM surfactants with a bio-based hydrophilic and a bio-based hydrophobic part, also GGM block-structured derivatives with a synthetic hydrophobic tail, consisting of a polydimethylsiloxane chain, were prepared and assessed for the hydrophobization of surface of nanofibrillated cellulose films. In order to generate GGM block-structured derivatives containing a synthetic tail with distinguished physical and chemical properties, as well as a tailored chain length, a controlled polymerization method was used. Therefore, firstly an initiator group was introduced at the reducing end of the GGM and consecutively single electron transfer-living radical polymerization (SET-LRP) was performed by applying three different monomers in individual reactions. For the accomplishment of the synthesis and the analysis of the products, challenges related to the solubility of the reactants had to be overcome. Overall, a synthesis route for the production of GGM block-copolymers bearing different synthetic polymer chains was developed and several derivatives were obtained. Moreover, GGM with different molar masses were, after modification, used as a crosslinker in the synthesis of functional hydrogels. Hereby, a cationic monomer was used during the free radical polymerization and the resultant hydrogels were successfully tested for the removal of chromium and arsenic ions from aqueous solutions. The hydrogel synthesis was tailored and materials with distinguished physical properties, such as the swelling rate, were obtained after purification. The results generated in this work underline the potential of bio-based products and the urge to continue carrying out research in order to be able to use more green chemicals for the manufacturing of biorenewable and biodegradable daily products.

A 37-kb restriction map of the human immunoglobulin lambda variable locus, VB cluster, harboring four functional genes and two non-coding Vl sequences

The human immunoglobulin lambda variable locus (IGLV) is mapped at chromosome 22 band q11.1-q11.2. The 30 functional germline v-lambda genes sequenced untill now have been subgrouped into 10 families (Vl1 to Vl10). The number of Vl genes has been estimated at approximately 70. This locus is formed by three gene clusters (VA, VB and VC) that encompass the variable coding genes (V) responsible for the synthesis of lambda-type Ig light chains, and the Jl-Cl cluster with the joining segments and the constant genes. Recently the entire variable lambda gene locus was mapped by contig methodology and its one- megabase DNA totally sequenced. All the known functional V-lambda genes and pseudogenes were located. We screened a human genomic DNA cosmid library and isolated a clone with an insert of 37 kb (cosmid 8.3) encompassing four functional genes (IGLV7S1, IGLV1S1, IGLV1S2 and IGLV5a), a pseudogene (VlA) and a vestigial sequence (vg1) to study in detail the positions of the restriction sites surrounding the Vl genes. We generated a high resolution restriction map, locating 31 restriction sites in 37 kb of the VB cluster, a region rich in functional Vl genes. This mapping information opens the perspective for further RFLP studies and sequencing

Functional evidence that the central renin-angiotensin system plays a role in the pressor response induced by central injection of carbachol

We investigated the effects of losartan, an AT1-receptor blocker, and ramipril, a converting enzyme inhibitor, on the pressor response induced by angiotensin II (ANG II) and carbachol (a cholinergic receptor agonist). Male Holtzman rats (250-300 g) with a stainless steel cannula implanted into the lateral ventricle (LV) were used. The injection of losartan (50 nmol/1 µl) into the LV blocked the pressor response induced by ANG II (12 ng/1 µl) and carbachol (2 nmol/1 µl). After injection of ANG II and carbachol into the LV, mean arterial pressure (MAP) increased to 31 ± 1 and 28 ± 2 mmHg, respectively. Previous injection of losartan abolished the increase in MAP induced by ANG II and carbachol into the LV (2 ± 1 and 5 ± 2 mmHg, respectively). The injection of ramipril (12 ng/1 µl) prior to carbachol blocked the pressor effect of carbachol to 7 ± 3 mmHg. These results suggest an interaction between central cholinergic pathways and the angiotensinergic system in the regulation of arterial blood pressure

Transgenic animal models for the functional analysis of vasoactive peptides

The interplay of vasoactive peptide systems is an essential determinant of blood pressure regulation in mammals. While the endothelin and the renin-angiotensin systems raise blood pressure by inducing vasoconstriction and sodium retention, the kallikrein-kinin and the natriuretic-peptide systems reduce arterial pressure by eliciting vasodilatation and natriuresis. Transgenic technology has proven to be very useful for the functional analysis of vasoactive peptide systems. As an outstanding example, transgenic rats overexpressing the mouse Ren-2 renin gene in several tissues become extremely hypertensive. Several other transgenic rat and mouse strains with genetic modifications of components of the renin-angiotensin system have been developed in the past decade. Moreover, in recent years gene-targeting technology was employed to produce mouse strains lacking these proteins. The established animal models as well as the main insights gained by their analysis are summarized in this review.

Functional analysis of newly discovered growth control genes: experimental approaches

A large number of DNA sequences corresponding to human and animal transcripts have been filed in data banks, as cDNAs or ESTs (expression sequence tags). However, the actual function of their corresponding gene products is still largely unknown. Several of these genes may play a role in regulation of important biological processes such as cell division, differentiation, malignant transformation and oncogenesis. Elucidation of gene function is based on 2 main approaches, namely, overexpression and expression interference, which respectively mimick or suppress a given phenotype. The currently available tools and experimental approaches to gene functional analysis and the most recent advances in mass cDNA screening by functional analysis are discussed.

Functional characterization and localization of AQP3 in the human colon

Water channels or aquaporins (AQPs) have been identified in a large variety of tissues. Nevertheless, their role in the human gastrointestinal tract, where their action is essential for the reabsorption and secretion of water and electrolytes, is still unclear. The purpose of the present study was to investigate the structure and function of water channels expressed in the human colon. A cDNA fragment of about 420 bp with a 98% identity to human AQP3 was amplified from human stomach, small intestine and colon by reverse transcription polymerase chain reaction (RT-PCR) and a transcript of 2.2 kb was expressed more abundantly in colon than in jejunum, ileum and stomach as indicated by Northern blots. Expression of mRNA from the colon of adults and children but not from other gastrointestinal regions in Xenopus oocytes enhanced the osmotic water permeability, and the urea and glycerol transport in a manner sensitive to an antisense AQP3 oligonucleotide, indicating the presence of functional AQP3. Immunocytochemistry and immunofluorescence studies in human colon revealed that the AQP3 protein is restricted to the villus epithelial cells. The immunostaining within these cells was more intense in the apical than in the basolateral membranes. The presence of AQP3 in villus epithelial cells suggests that AQP3 is implicated in water absorption across human colonic surface cells.

Activity and functional interaction of alternative oxidase and uncoupling protein in mitochondria from tomato fruit

Cyanide-resistant alternative oxidase (AOX) is not limited to plant mitochondria and is widespread among several types of protists. The uncoupling protein (UCP) is much more widespread than previously believed, not only in tissues of higher animals but also in plants and in an amoeboid protozoan. The redox energy-dissipating pathway (AOX) and the proton electrochemical gradient energy-dissipating pathway (UCP) lead to the same final effect, i.e., a decrease in ATP synthesis and an increase in heat production. Studies with green tomato fruit mitochondria show that both proteins are present simultaneously in the membrane. This raises the question of a specific physiological role for each energy-dissipating system and of a possible functional connection between them (shared regulation). Linoleic acid, an abundant free fatty acid in plants which activates UCP, strongly inhibits cyanide-resistant respiration mediated by AOX. Moreover, studies of the evolution of AOX and UCP protein expression and of their activities during post-harvest ripening of tomato fruit show that AOX and plant UCP work sequentially: AOX activity decreases in early post-growing stages and UCP activity is decreased in late ripening stages. Electron partitioning between the alternative oxidase and the cytochrome pathway as well as H+ gradient partitioning between ATP synthase and UCP can be evaluated by the ADP/O method. This method facilitates description of the kinetics of energy-dissipating pathways and of ATP synthase when state 3 respiration is decreased by limitation of oxidizable substrate.

Gap junctions in cells of the immune system: structure, regulation and possible functional roles

Gap junction channels are sites of cytoplasmic communication between contacting cells. In vertebrates, they consist of protein subunits denoted connexins (Cxs) which are encoded by a gene family. According to their Cx composition, gap junction channels show different gating and permeability properties that define which ions and small molecules permeate them. Differences in Cx primary sequences suggest that channels composed of different Cxs are regulated differentially by intracellular pathways under specific physiological conditions. Functional roles of gap junction channels could be defined by the relative importance of permeant substances, resulting in coordination of electrical and/or metabolic cellular responses. Cells of the native and specific immune systems establish transient homo- and heterocellular contacts at various steps of the immune response. Morphological and functional studies reported during the last three decades have revealed that many intercellular contacts between cells in the immune response present gap junctions or "gap junction-like" structures. Partial characterization of the molecular composition of some of these plasma membrane structures and regulatory mechanisms that control them have been published recently. Studies designed to elucidate their physiological roles suggest that they might permit coordination of cellular events which favor the effective and timely response of the immune system.

Structural, functional and immunological studies on a polymeric bacterial protein

The characterization of proteins from Brucella spp, the causative agent of brucellosis, has been the subject of intensive research. We have described an 18-kDa cytoplasmic protein of Brucella abortus and shown the potential usefulness of this protein as an antigen for the serologic diagnosis of brucellosis. The amino acid sequence of the protein showed a low but significant homology with that of lumazine synthases. Lumazine is an intermediate product in bacterial riboflavin biosynthesis. The recombinant form of the 18-kDa protein (expressed in E. coli) folds like the native Brucella protein and has lumazine-synthase enzymatic activity. Three-dimensional analysis by X-ray crystallography of the homolog Bacillus subtilis lumazine synthase has revealed that the enzyme forms an icosahedral capsid. Recombinant lumazine synthase from B. abortus was crystallized, diffracted X rays to 2.7-Å resolution at room temperature, and the structure successfully solved by molecular replacement procedures. The macromolecular assembly of the enzyme differs from that of the enzyme from B. subtilis. The Brucella enzyme remains pentameric (90 kDa) in its crystallographic form. Nonetheless, the active sites of the two enzymes are virtually identical at the structural level, indicating that inhibitors of these enzymes could be viable pharmaceuticals across a broad species range. We describe the structural reasons for the differences in their quaternary arrangement and also discuss the potential use of this protein as a target for the development of acellular vaccines.

Functional, Social and Emotional Values as Determinants of Environmentally Responsible Media Consumption

The primary purpose of this research is to develop an enhanced understanding of how consumption values influence environmentally responsible consumption of print and digital media. Theoretical elaboration considers the associations of functional, social and emotional consumption values, green consumer segmentation and media consumption. Additionally, the purpose is to identify consumer perceptions of print and digital media’s environmental responsibility. Empirical analysis was based on qualitative interviews with a sample of 20 Finnish consumers categorized in two segments: young adults and middle aged consumers. Primary data collection was conducted through individual, semi-structured interviews. To analyze the respondents’ approach on the topic, the interviews disclosed themes of media consumption, perceived environmental friendliness of media, norms of behavior and consumers’ general consumption patterns. The results implicate functional value dominated the consumption decision-making process both in a general level and in media consumption. In addition to functional value, environmental responsibility does provide consumers with both emotional and social values. Analysis on perceived environmental responsibility of media demonstrated consumers generally perceive digital media as an environmentally responsible alternative because it does not create physical paper waste. Nevertheless, the perceptions of environmental responsibility and media consumption patterns lacked a consistent connection. Though, both theory and empirical results indicated an average consumer lacks a comprehensive understanding of digital and print media’s life-cycle and hence their environmental advantages and disadvantages.