Evaluation of group electronegativities and hardness (softness) of group 14 elements and containing functional groups through density functional theory and correlation with NMR spectra data

Quantum Chemical calculations for group 14 elements of Periodic Table (C, Si, Ge, Sn, Pb) and their functional groups have been carried out using Density Functional Theory (DFT) based reactivity descriptors such as group electronegativities, hardness and softness. DFT calculations were performed for a large series of tetracoordinated Sn compounds of the CH3SnRR'X type, where X is a halogen and R and R' are alkyl, halogenated alkyl, alkoxy, or alkyl thio groups. The results were interpreted in terms of calculated electronegativity and hardness of the SnRR'X groups, applying a methodology previously developed by Geerlings and coworkers (J. Phys. Chem. 1993, 97, 1826). These calculations allowed to see the regularities concerning the influence of the nature of organic groups RR' and inorganic group X on electronegativities and hardness of the SnRR'X groups; in this case, it was found a very good correlation between the electronegativity of the fragment and experimental 119Sn chemical shifts, a property that sensitively reflects the change in the valence electronic structure of molecules. This work was complemented with the study of some compounds of the EX and ER types, where E= C, Si, Ge, Sn and R= CH3, H, which was performed to study the influence that the central atom has on the electronegativity and hardness of molecules, or whether these properties are mainly affected for the type of ligand bound to the central atom. All these calculations were performed using the B3PW91 functional together with the 6-311++G** basis set level for H, C, Si, Ge, F, Cl and Br atoms and the 3-21G for Sn and I atoms.

Functional characterization of proteins required for mitotic progression and the spindle assembly checkpoint

During mitotic cell division, the genetic material packed into chromosomes is divided equally between two daughter cells. Before the separation of the two copies of a chromosome (sister chromatids), each chromosome has to be properly connected with microtubules of the mitotic spindle apparatus and aligned to the centre of the cell. The spindle assembly checkpoint (SAC) monitors connections between microtubules and chromosomes as well as tension applied across the centromere. Microtubules connect to a chromosome via kinetochores, which are proteinaceous organelles assembled onto the centromeric region of the sister chromatids. Improper kinetochore-microtubule attachments activate the SAC and block chromosome segregation until errors are corrected and all chromosomes are connected to the mitotic spindle in a bipolar manner. The purpose of this surveillance mechanism is to prevent loss or gain of chromosomes in daughter cells that according to current understanding contributes to cancer formation. Numerous proteins participate in the regulation of mitotic progression. In this thesis, the mitotic tasks of three kinetochore proteins, Shugoshin 1 (Sgo1), INCENP, and p38 MAP kinase (p38 MAPK), were investigated. Sgo1 is a protector of centromeric cohesion. It is also described in the tension-sensing mechanism of the SAC and in the regulation of kinetochore-microtubule connections. Our results revealed a central role for Sgo1 in a novel branch of kinetochore assembly. INCENP constitutes part of the chromosomal passenger complex (CPC). The other members of the core complex are the Aurora B kinase, Survivin and Borealin. CPC is an important regulatory element of cell division having several roles at various stages of mitosis. Our results indicated that INCENP and Aurora B are highly dynamic proteins at the mitotic centromeres and suggested a new role for CPC in regulation of chromosome movements and spindle structure during late mitosis. The p38 MAPK has been implicated in G1 and G2 checkpoints during the cell cycle. However, its role in mitotic progression and control of SAC signaling has been controversial. In this thesis, we discovered a novel function for p38γ MAPK in chromosome orientation and spindle structure as well as in promotion of viability of mitotic cells.

The role of selection in functional explanations

In this essay I will argue that natural selection is more important to functional explanations than what has been thought in some of the literature in philosophy of biology. I start by giving a brief overview of the two paradigms cases of functional explanations: etiological functions and causal-role functions. i then consider one particular attempt to conciliate both perspectives given by David Buller (1998). Buller's trial to conciliate both etiological functions and causal-role functions results in what he calls a weak etiological theory. I argue that Buller has not succeeded in his construal of the weak etiological theory: he underestimates the role that selective processes have in functional explanations and so his theory may not be classified as an etiologial theory. As an alternative, I consider the account of etiological functions given by Ruth Millikan (1984) and I argue that Millikan's theory is more comprehensive to assess contentious case in biology like exaptations. Finally, I conclude by analyzing where the adoption of Millikan's theory leave us. I argue, contrary to Millikan and others, that once we assume the importance of natural selection in functional explanations, there is no strong reason to resist a linguistic reform of the word function and hence that the attempts to conciliate both etiological functions and causal-role functions are misplaced.

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.

Functional Study of Oncogenic Transcription Factor ERG and its Signaling in Prostate Cancer

TMPRSS2–ERG is the most frequent type of genomic rearrangement present in prostate tumors, in which the 5- prime region of the TMPRSS2 gene is fused to the ERG oncogene. TMPRSS2, containing androgen response elements (AREs), is regulated by androgens in the prostate. The truncated TMPRSS2-ERG fusion transcript is overexpressed in half of the prostate cancer patients. The formation of TMPRSS2-ERG transcript is an early event in prostate carcinogenesis and previous in vivo and in vitro studies have shown ectopic ERG expression to be associated with increased cell invasion. However, the molecular function of ERG and its role in cell signaling is poorly understood. In this study, genomic rearrangement of ERG with TMPRSS2 was studied by using comparative genomic hybridization (CGH) in prostate cancer samples. The biological processes associated with the ERG oncogene expression in prostate epithelial cells were studied, and the results were compared with findings observed in clinical prostate tumor samples. The gene expression data indicated that increased WNT signaling and loss of cell adhesion were a characteristic of TMPRSS2- ERG fusion positive prostate tumor samples. Up- regulation of WNT pathway genes were present in ERG positive prostate tumors, with frizzled receptor 4 (FZD4) presenting with the highest association with ERG overexpression, as verified by quantitative reverse transcription-PCR, immunostaining, and immunoblotting in TMPRSS2-ERG positive VCaP prostate cancer cells. Furthermore, ERG and FZD4 silencing increased cell adhesion by inducing active β1-integrin and E-cadherin expression in VCaP cells. Furthermore, we found a novel inhibitor, 4-(chloromethyl) benzoyl chloride which inhibited the WNT signaling and induced similar phenotypic effects as observed after ERG or FZD4 down regulation in VCaP cells. In conclusion, this work deepens our understanding on the complex oncogenic mechanisms of ERG in prostate cancer that may help in developing drugs against TMPRSS2-ERG positive tumors.

Conventional and novel cardiovascular risk factors in young Finns and their associations with structural and functional vascular changes of subclinical atherosclerosis. The Cardiovascular Risk in Young Finns Study

Background: Atherosclerosis begins in early life progressing from asymptomatic to symptomatic as we age. Although substantial progress has been made in identifying the determinants of atherosclerosis in middle to older age adults at increased cardiovascular risk, there is lack of data examining determinants and prediction of atherosclerosis in young adults. Aims: The current study was designed to investigate levels of cardiovascular risk factors in young adults, subclinical measures of atherosclerosis, and prediction of subclinical arterial changes with conventional risk factor measures and novel metabolic profiling of serum samples. Subjects and Methods: This thesis utilised data from the follow-ups performed in 2001 and 2007 in the Cardiovascular Risk in Young Finns study, a Finnish population-based prospective cohort study that examined 2,204 subjects who were aged 30-45 years in 2007. Subclinical atherosclerosis was studied using noninvasive ultrasound measurements of carotid intima-media thickness (IMT), carotid arterial distensibility (CDist) and brachial flow-mediated dilation (FMD). Measurements included conventional risk factors and metabolic profiling using highthroughput nuclear magnetic resonance (NMR) methods that provided data on 42 lipid markers and 16 circulating metabolites. Results: Trends in lipids were favourable between 2001 and 2007, whereas waist circumference, fasting glucose, and blood pressure levels increased. To study the stability of noninvasive ultrasound markers, 6-year tracking (the likelihood to maintain the original fractile over time) in 6 years was examined. IMT tracked more strongly than CDist and FMD. Cardiovascular risk scores (Framingham, SCORE, Finrisk, Reynolds and PROCAM) predicted subclinical atherosclerosis equally. Lipoprotein subclass testing did not improve the prediction of subclinical atherosclerosis over and above conventional risk factors. However, circulating metabolites improved risk stratification. Tyrosine and docosahexaenoic acid were found to be novel biomarkers of high IMT. Conclusions: Prediction of cardiovascular risk in young Finnish adults can be performed with any of the existing risk scores. The addition of metabonomics to risk stratification improves prediction of subclinical changes and enables more accurate targeting of prevention at an early stage.

DPS-Like Peroxide Resistance Protein: Structural and Functional Studies on a Versatile Nanocontainer

Oxidative stress is a constant threat to almost all organisms. It damages a number of biomolecules and leads to the disruption of many crucial cellular functions. It is caused by reactive oxygen species (ROS), such as hydrogen peroxide (H2O₂), superoxide (•O₂ -), and hydroxyl radical (•OH). The most harmful of these compounds is •OH, which is only formed in cells in the presence of redox-cycling transition metals, such as iron and copper. Bacteria have developed a number of mechanisms to cope with ROS. One of the most widespread means employed by bacteria is the DNA-binding proteins from starved cells (Dps). Dps proteins protect the cells by binding and oxidizing Fe2+, thus greatly reducing the production of •OH. The oxidized iron is stored inside the protein as an iron core. In addition, Dps proteins bind directly to DNA forming a protective coating that shields DNA from harmful agents. Moreover, Dps proteins have been found to elicit other protective functions in cells and to participate in bacterial virulence. Dps proteins are of special importance to Streptococci owing to the lack of catalase in this genus of bacteria.This study was focused on structural and functional characterization of streptococcal Dpslike peroxide resistance (Dpr) proteins. Initially, crystal structures of Streptococcus pyogenes Dpr were determined. The data confirmed the presence of a di-metal ferroxidase center (FOC) in Dpr proteins and revealed the presence of a novel N-terminal helix as well as a surface metal-binding site. The crystal structures of Streptococcus suis Dpr complexed with transition metals demonstrated the metal specificity of the FOC. Solution binding studies also indicated the presence of a di-metal FOC. These results suggested a possible role for Dpr in the detoxification of various metals. Iron was found to mineralize inside the protein as ferrihydrite based on X-ray absorption spectroscopy data. The iron core was found to exhibit clear superparamagnetic behaviour using magnetic and Mössbauer measurements. The results from this study are expected to further increase our understanding on the binding, oxidation, and mineralization of iron and other metals in Dpr proteins. In particular, the structural and magnetic properties of the iron core can form a basis for potential new applications in nanotechnology. From the streptococcal viewpoint, the results would help in understanding better the complicated picture of bacterial pathogenesis. Dpr proteins may also provide a novel target for drug design due to their tight involvement in bacterial virulence.

Fractionation of Hen Egg and Oat Lipids with supercritical Fluids Chemical and functional Properties of Fractions

Hen eggs and oats (Avena Sativa) are important materials for the food industry. Today, instead of merely satisfying the feeling of hunger, consumers are asking for healthier, biologically active and environmentally friendly products. The growing awareness of consumers’ increasing demands presents a great challenge to the food industry to develop more sustainable products and utilise modern and effective techniques. The modification of yolk fatty acid composition by means of feed supplements is well understood. Egg yolk phospholipids are polar lipids and are used in several applications including food, cosmetics, pharmaceuticals, and special nutrients. Egg yolk phospholipids are excellent emulsifiers, typically sold as mixtures of phospholipids, triacylglycerols, and cholesterol. However, highly purified and characterised phospholipids are needed in several sophisticated applications. Industrial fractionation of phospholipids is usually based on organic solvents. With these fractionation techniques, some harmful residues of organic solvents may cause problems in further processing. The objective of the present study was to investigate the methods to improve the functional properties of eggs, to develop techniques to isolate the fractions responsible for the specific functional properties of egg yolk lipids, and to apply the developed techniques to plant-based materials, too. Fractionation techniques based on supercritical fluids were utilised for the separation of the lipid fractions of eggs and oats. The chemical and functional characterisation of the fractions were performed, and the produced oat polar lipid fractions were tested as protective barrier in encapsulation processes. Modifying the fatty acid compositions of egg yolks with different types of oil supplements in feed had no affect on their functional or sensory properties. Based on the results of functional and sensory analysis, it is evident that eggs with modified fatty acid compositions are usable in several industrial applications. These applications include liquid egg yolk products used in mayonnaise and salad dressings. Egg yolk powders were utilised in different kinds of fractionation processes. The precipitation method developed in this study resembles the supercritical anti-solvent method, which is typically used in the pharmaceutical industry. With pilot scale supercritical fluid processes, non-polar lipids and polar lipids were successfully separated from commercially produced egg yolk powder and oat flakes. The egg and oat-based polar lipid fractions showed high purities, and the corresponding delipidated fractions produced using supercritical techniques offer interesting starting materials for the further production of bioactive compounds. The oat polar lipid fraction contained especially digalactosyadiacylglycerol, which was shown to have valuable functional properties in the encapsulation of probiotics.

Anatomical and functional characteristics of the pelvic floor in nulliparous women submitted to three-dimensional endovaginal ultrasonography: case control study and evaluation of interobserver agreement

PURPOSE: To determine anatomical and functional pelvic floor measurements performed with three-dimensional (3-D) endovaginal ultrasonography in asymptomatic nulliparous women without dysfunctions detected in previous dynamic 3-D anorectal ultrasonography (echo defecography) and to demonstrate the interobserver reliability of these measurements. METHODS: Asymptomatic nulliparous volunteers were submitted to echo defecography to identify dynamic dysfunctions, including anatomical (rectocele, intussusceptions, entero/sigmoidocele and perineal descent) and functional changes (non-relaxation or paradoxical contraction of the puborectalis muscle) in the posterior compartment and assessed with regard to the biometric index of levator hiatus, pubovisceral muscle thickness, urethral length, anorectal angle, anorectal junction position and bladder neck position with the 3-D endovaginal ultrasonography. All measurements were compared at rest and during the Valsalva maneuver, and perineal and bladder neck descent was determined. The level of interobserver agreement was evaluated for all measurements. RESULTS: A total of 34 volunteers were assessed by echo defecography and by 3-D endovaginal ultrasonography. Out of these, 20 subjects met the inclusion criteria. The 14 excluded subjects were found to have posterior dynamic dysfunctions. During the Valsalva maneuver, the hiatal area was significantly larger, the urethra was significantly shorter and the anorectal angle was greater. Measurements at rest and during the Valsalva maneuver differed significantly with regard to anorectal junction and bladder neck position. The mean values for normal perineal descent and bladder neck descent were 0.6 cm and 0.5 cm above the symphysis pubis, respectively. The intraclass correlation coefficient ranged from 0.62-0.93. CONCLUSIONS: Functional biometric indexes, normal perineal descent and bladder neck descent values were determined for young asymptomatic nulliparous women with the 3-D endovaginal ultrasonography. The method was found to be reliable to measure pelvic floor structures at rest and during Valsalva, and might therefore be suitable for identifying dysfunctions in symptomatic patients.

Functional activity of neutrophilic polymorphonuclear leukocytes in the first five days postpartum

PURPOSE: To assess the chemotactic activity and phagocytic response of neutrophilic polymorphonuclear leukocytes among women in the first five days postpartum.METHODS: A prospective, cross-sectional clinical-laboratory study was conducted. Data of 31 postpartum women during the first five days after vaginal delivery were compared with those of 24 healthy non-pregnant non-postpartum women matched for age. The inclusion criteria were postpartum, clinically and obstetrically healthy women; vaginal delivery, singleton pregnancy carried to term; non-hypertensive, hyperglycemic, allergic, malnourished or with autoimmune or neoplastic diseases; not having received vaccines or blood products in the last three months. The Control Group was chosen according to the same inclusion criteria but involving non-pregnant non-postpartum women. The chemotactic activity of neutrophilic polymorphonuclear leukocytes was assessed by determining the distance from directed migration to bacterial lipopolysaccharide, in three Boyden chamber assays. The phagocytic response was identified by assessing the Zymosan particles' ingestion in three assays carried out in Leighton tubes. The Student's t-test was used in the statistical analysis, adopting a 5% level of significance.RESULTS: The chemotactic activity of neutrophilic polymorphonuclear leukocytes from postpartum women in the presence of homologous (73.2±6.9) and autologous (78.6±13.9) sera showed a significant increase compared to the values observed in the Control Group (64.1±4.1 and 66.6±5.4). Both chemotactic response and phagocytosis ingestion phase of neutrophilic polymorphonuclear leukocytes were significantly increased (p<0.05) in postpartum women compared to healthy non-pregnant and non-postpartum women.CONCLUSION: There was an increase in the chemotactic activity and phagocytic response of neutrophilic polymorphonuclear leukocytes during the first five days after vaginal delivery in women.

Clinical evaluation of enalapril maleate and furosemide usage in dogs with degenerative myxomatous mitral valve, CHF functional class Ib

Degenerative myxomatous mitral valve (DMMV) is a heart disease of high incidence in small animal clinical medicine, affecting mainly older dogs and small breeds. Thus, a scientific investigation was performed in order to evaluate the clinical use of the medicines furosemide and enalapril maleate in dogs with this disease in CHF functional class Ib before and after the treatment was established. For this purpose 16 dogs with the given valve disease were used, separated into two groups: the first received furosemide (n=8) and the second received enalapril maleate (n=8) throughout 56 days. The dogs were evaluated in four stages (T0, T14, T28 and T56 day) in relation to clinical signs, hematological, biochemical and serum assessment, which included serum angiotensin converting enzyme (ACE) and aldosterone, as well as radiography, electrocardiography, Doppler-echocardiography and blood pressure. The results regarding the clinical, hematological and serum chemistry evaluations revealed no significant changes in both groups, but significant reductions in the values of ACE and aldosterone in the group receiving enalapril maleate were verified. The radiographic examination revealed reductions of VHS values and variable Pms wave of the electrocardiogram in both groups, but no changes in blood pressure values were identified. The echocardiogram showed a significant decrease of the variables LVDd/s in the studied groups and the FS% in animals that received only enalapril. Therefore, analysis of results showed that monotherapy based on enalapril maleate showed better efficiency of symptoms control in patients with CHF functional class Ib.

Character transformations and their functional significance as a key to the evolution of hystricognath Rodentia

Hystricognathi represent a monophyletic taxon within Rodentia. Since phylogenetically analyzed morphological systems are essential for revealing evolutionary processes, this study identifies evolutionary character transformations on the stem lineage of Hystricognathi as derived from the author's own work and the literature. Data so far indicate that evolutionary transformations in the rostral head region, the loss of tactile ability in the outer nasal skin and the mobile arrangement of the associated cartilage, were allied with a switch from omnivorous to herbivorous and fiber-rich nutrition. Additional character transformations in the skull assist in digesting such food. Structures associated with reproduction and placentation show a remarkable pro portion of derived character conditions: the chorioallantoic placenta has a ring-shaped organization and growth structure which optimizes the capacity for passive diffusion, a subplacenta occurred as a specialized region responsible for placental invasion and the inverted yolk sac facilitates substance exchange with the main placenta. Finally, precocial newborns evolved as a derived condition within Rodentia. All things considered, a mode of reproduction is indicated, which does not demand excessive additional energy intake by the mother and is in accordance with her low energetic diet. Hystricognathi possess major character transformations that represent prerequisites for their successful radiation at the time when more open ecosystems and grasslands evolved during Earth history. The analysis resulted in the reconstruction of a life-near picture of the hystricognath stem species pattern with high explanatory power in terms of changes in space and time and their interdependence with biodiversity.

Functional Genomic Approaches for Deciphering Plant-Virus Interactions

Plant-virus interactions are very complex in nature and lead to disease and symptom formation by causing various physiological, metabolic and developmental changes in the host plants. These interactions are mainly the outcomes of viral hijacking of host components to complete their infection cycles and of host defensive responses to restrict the viral infections. Viral genomes contain only a small number of genes often encoding for multifunctional proteins, and all are essential in establishing a viral infection. Thus, it is important to understand the specific roles of individual viral genes and their contribution to the viral life cycles. Among the most important viral proteins are the suppressors of RNA silencing (VSRs). These proteins function to suppress host defenses mediated by RNA silencing and can also serve in other functions, e.g. in viral movement, transactivation of host genes, virus replication and protein processing. Thus these proteins are likely to have a significant impact on host physiology and metabolism. In the present study, I have examined the plant-virus interactions and the effects of three different VSRs on host physiology and gene expression levels by microarray analysis of transgenic plants that express these VSR genes. I also studied the gene expression changes related to the expression of the whole genome of Tobacco mosaic virus (TMV) in transgenic tobacco plants. Expression of the VSR genes in the transgenic tobacco plants causes significant changes in the gene expression profiles. HC-Pro gene derived from the Potyvirus Y (PVY) causes alteration of 748 and 332 transcripts, AC2 gene derived from the African cassava mosaic virus (ACMV) causes alteration of 1118 and 251transcripts, and P25 gene derived from the Potyvirus X (PVX) causes alterations of 1355 and 64 transcripts in leaves and flowers, respectively. All three VSRs cause similar up-regulation in defense, hormonally regulated and different stress-related genes and down-regulation in the photosynthesis and starch metabolism related genes. They also induce alterations that are specific to each viral VSR. The phenotype and transcriptome alterations of the HC-Pro expressing transgenic plants are similar to those observed in some Potyvirus-infected plants. The plants show increased protein degradation, which may be due to the HC-Pro cysteine endopeptidase and thioredoxin activities. The AC2-expressing transgenic plants show a similar phenotype and gene expression pattern as HC-Pro-expressing plants, but also alter pathways related to jasmonic acid, ethylene and retrograde signaling. In the P25 expressing transgenic plants, high numbers of genes (total of 1355) were up-regulated in the leaves, compared to a very low number of down-regulated genes (total of 5). Despite of strong induction of the transcripts, only mild growth reduction and no other distinct phenotype was observed in these plants. As an example of whole virus interactions with its host, I also studied gene expression changes caused by Tobacco mosaic virus (TMV) in tobacco host in three different conditions, i.e. in transgenic plants that are first resistant to the virus, and then become susceptible to it and in wild type plants naturally infected with this virus. The microarray analysis revealed up and down-regulation of 1362 and 1422 transcripts in the TMV resistant young transgenic plants, and up and down-regulation of a total of 1150 and 1200 transcripts, respectively, in the older plants, after the resistance break. Natural TMV infections in wild type plants caused up-regulation of 550 transcripts and down-regulation of 480 transcripts. 124 up-regulated and 29 down-regulated transcripts were commonly altered between young and old TMV transgenic plants, and only 6 up-regulated and none of the down-regulated transcripts were commonly altered in all three plants. During the resistant stage, the strong down-regulation in translation-related transcripts (total of 750 genes) was observed. Additionally, transcripts related to the hormones, protein degradation and defense pathways, cell division and stress were distinctly altered. All these alterations may contribute to the TMV resistance in the young transgenic plants, and the resistance may also be related to RNA silencing, despite of the low viral abundance and lack of viral siRNAs or TMV methylation activity in the plants.

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.