Moonlighting Proteins of Lactobacillus crispatus : Extracellular Localization, Cell Wall Anchoring and Interactions with the Host

Moonlighting functions have been described for several proteins previously thought to localize exclusively in the cytoplasm of bacterial or eukaryotic cells. Moonlighting proteins usually perform conserved functions, e. g. in glycolysis or as chaperonins, and their traditional and moonlighting function(s) usually localize to different cell compartments. The most characterized moonlighting proteins in Grampositive bacteria are the glycolytic enzymes enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which function in bacteria-host interactions, e. g. as adhesins or plasminogen receptors. Research on bacterial moonlighting proteins has focused on Gram-positive bacterial pathogens, where many of their functions have been associated with bacterial virulence. In this thesis work I show that also species of the genus Lactobacillus have moonlighting proteins that carry out functions earlier associated with bacterial virulence only. I identified enolase, GAPDH, glutamine synthetase (GS), and glucose-6-phosphate isomerase (GPI) as moonlighting proteins of Lactobacillus crispatus strain ST1 and demonstrated that they are associated with cell surface and easily released from the cell surface into incubation buffer. I also showed that these lactobacillar proteins moonlight either as adhesins with affinity for basement membrane and extracellular matrix proteins or as plasminogen receptors. The mechanisms of surface translocation and anchoring of bacterial moonlighting proteins have remained enigmatic. In this work, the surface localization of enolase, GAPDH, GS and GPI was shown to depend on environmental factors. The members of the genus Lactobacillus are fermentative organisms that lower the ambient pH by producing lactic acid. At acidic pH enolase, GAPDH, GS and GPI were associated with the cell surface, whereas at neutral pH they were released into the buffer. The release did not involve de novo protein synthesis. I showed that purified recombinant His6-enolase, His6-GAPDH, His6-GS and His6-GPI reassociate with cell wall and bind in vitro to lipoteichoic acids at acidic pH. The in-vitro binding of these proteins localizes to cell division septa and cell poles. I also show that the release of moonlighting proteins is enhanced in the presence of cathelicidin LL- 37, which is an antimicrobial peptide and a central part of the innate immunity defence. I found that the LL-37-induced detachment of moonlighting proteins from cell surface is associated with cell wall permeabilization by LL-37. The results in this thesis work are compatible with the hypothesis that the moonlighting proteins of L. crispatus associate to the cell wall via electrostatic or ionic interactions and that they are released into surroundings in stress conditions. Their surface translocation is, at least in part, a result from their release from dead or permeabilized cells and subsequent reassociation onto the cell wall. The results of this thesis show that lactobacillar cells rapidly change their surface architecture in response to environmental factors and that these changes influence bacterial interactions with the host.

The Significance of Wild Plants in the Evolutionary Ecology of Three Major Viruses Infecting Cultivated Sweetpotato in Uganda

The studies presented in this thesis contribute to the understanding of evolutionary ecology of three major viruses threatening cultivated sweetpotato (Ipomoea batatas Lam) in East Africa: Sweet potato feathery mottle virus (SPFMV; genus Potyvirus; Potyviridae), Sweet potato chlorotic stunt virus (SPCSV; genus Crinivirus; Closteroviridae) and Sweet potato mild mottle virus (SPMMV; genus Ipomovirus; Potyviridae). The viruses were serologically detected and the positive results confirmed by RT-PCR and sequencing. SPFMV was detected in 24 wild plant species of family Convolvulacea (genera Ipomoea, Lepistemon and Hewittia), of which 19 species were new natural hosts for SPFMV. SPMMV and SPCSV were detected in wild plants belonging to 21 and 12 species (genera Ipomoea, Lepistemon and Hewittia), respectively, all of which were previously unknown to be natural hosts of these viruses. SPFMV was the most abundant virus being detected in 17% of the plants, while SPMMV and SPCSV were detected in 9.8% and 5.4% of the assessed plants, respectively. Wild plants in Uganda were infected with the East African (EA), common (C), and the ordinary (O) strains, or co-infected with the EA and the C strain of SPFMV. The viruses and virus-like diseases were more frequent in the eastern agro-ecological zone than the western and central zones, which contrasted with known incidences of these viruses in sweetpotato crops, except for northern zone where incidences were lowest in wild plants as in sweetpotato. The NIb/CP junction in SPMMV was determined experimentally which facilitated CP-based phylogenetic and evolutionary analyses of SPMMV. Isolates of all the three viruses from wild plants were genetically similar to those found in cultivated sweetpotatoes in East Africa. There was no evidence of host-driven population genetic structures suggesting frequent transmission of these viruses between their wild and cultivated hosts. The p22 RNA silencing suppressor-encoding sequence was absent in a few SPCSV isolates, but regardless of this, SPCSV isolates incited sweet potato virus disease (SPVD) in sweetpotato plants co-infected with SPFMV, indicating that p22 is redundant for synergism between SCSV and SPFMV. Molecular evolutionary analysis revealed that isolates of strain EA of SPFMV that is largely restricted geographically in East Africa experience frequent recombination in comparison to isolates of strain C that is globally distributed. Moreover, non-homologous recombination events between strains EA and C were rare, despite frequent co-infections of these strains in wild plants, suggesting purifying selection against non-homologous recombinants between these strains or that such recombinants are mostly not infectious. Recombination was detected also in the 5 - and 3 -proximal regions of the SPMMV genome providing the first evidence of recombination in genus Ipomovirus, but no recombination events were detected in the characterized genomic regions of SPCSV. Strong purifying selection was implicated on evolution of majority of amino acids of the proteins encoded by the analyzed genomic regions of SPFMV, SPMMV and SPCSV. However, positive selection was predicted on 17 amino acids distributed over the whole the coat protein (CP) in the globally distributed strain C, as compared to only 4 amino acids in the multifunctional CP N-terminus (CP-NT) of strain EA largely restricted geographically to East Africa. A few amino acid sites in the N-terminus of SPMMV P1, the p7 protein and RNA silencing suppressor proteins p22 and RNase3 of SPCSV were also submitted to positive selection. Positively selected amino acids may constitute ligand-binding domains that determine interactions with plant host and/or insect vector factors. The P1 proteinase of SPMMV (genus Ipomovirus) seems to respond to needs of adaptation, which was not observed with the helper component proteinase (HC-Pro) of SPMMV, although the HC-Pro is responsible for many important molecular interactions in genus Potyvirus. Because the centre of origin of cultivated sweetpotato is in the Americas from where the crop was dispersed to other continents in recent history (except for the Australasia and South Pacific region), it would be expected that identical viruses and their strains occur worldwide, presuming virus dispersal with the host. Apparently, this seems not to be the case with SPMMV, the strain EA of SPFMV and the strain EA of SPCSV that are largely geographically confined in East Africa where they are predominant and occur both in natural and agro-ecosystems. The geographical distribution of plant viruses is constrained more by virus-vector relations than by virus-host interactions, which in accordance of the wide range of natural host species and the geographical confinement to East Africa suggest that these viruses existed in East African wild plants before the introduction of sweetpotato. Subsequently, these studies provide compelling evidence that East Africa constitutes a cradle of SPFMV strain EA, SPCSV strain EA, and SPMMV. Therefore, sweet potato virus disease (SPVD) in East Africa may be one of the examples of damaging virus diseases resulting from exchange of viruses between introduced crops and indigenous wild plant species. Keywords: Convolvulaceae, East Africa, epidemiology, evolution, genetic variability, Ipomoea, recombination, SPCSV, SPFMV, SPMMV, selection pressure, sweetpotato, wild plant species Author s Address: Arthur K. Tugume, Department of Agricultural Sciences, Faculty of Agriculture and Forestry, University of Helsinki, Latokartanonkaari 7, P.O Box 27, FIN-00014, Helsinki, Finland. Email: tugume.arthur@helsinki.fi Author s Present Address: Arthur K. Tugume, Department of Botany, Faculty of Science, Makerere University, P.O. Box 7062, Kampala, Uganda. Email: aktugume@botany.mak.ac.ug, tugumeka@yahoo.com

Cellular Membranes as a Playground for Semliki Forest Virus Replication Complex

All positive-strand RNA viruses utilize cellular membranes for the assembly of their replication complexes, which results in extensive membrane modification in infected host cells. These alterations act as structural and functional scaffolds for RNA replication, providing protection for the viral double-stranded RNA against host defences. It is known that different positive-strand RNA viruses alter different cellular membranes. However, the origin of the targeted membranes, the mechanisms that direct replication proteins to specific membranes and the steps in the formation of the membrane bound replication complex are not completely understood. Alphaviruses (including Semliki Forest virus, SFV), members of family Togaviridae, replicate their RNA in association with membranes derived from the endosomal and lysosomal compartment, inducing membrane invaginations called spherules. Spherule structures have been shown to be the specific sites for RNA synthesis. Four replication proteins, nsP1-nsP4, are translated as a polyprotein (P1234) which is processed autocatalytically and gives rise to a membrane-bound replication complex. Membrane binding is mediated via nsP1 which possesses an amphipathic α-helix (binding peptide) in the central region of the protein. The aim of this thesis was to characterize the association of the SFV replication complex with cellular membranes and the modification of the membranes during virus infection. Therefore, it was necessary to set up the system for determining which viral components are needed for inducing the spherules. In addition, the targeting of the replication complex, the formation site of the spherules and their intracellular trafficking were studied in detail. The results of current work demonstrate that mutations in the binding peptide region of nsP1 are lethal for virus replication and change the localization of the polyprotein precursor P123. The replication complex is first targeted to the plasma membrane where membrane invaginations, spherules, are induced. Using a specific regulated endocytosis event the spherules are internalized from the plasma membrane in neutral carrier vesicles and transported via an actin-and microtubule-dependent manner to the pericentriolar area. Homotypic fusions and fusions with pre-existing acidic organelles lead to the maturation of previously described cytopathic vacuoles with hundreds of spherules on their limiting membranes. This work provides new insights into the membrane binding mechanism of SFV replication complex and its role in the virus life cycle. Development of plasmid-driven system for studying the formation of the replication complex described in this thesis allows various applications to address different steps in SFV life cycle and virus-host interactions in the future. This trans-replication system could be applied for many different viruses. In addition, the current work brings up new aspects of membranes and cellular components involved in SFV replication leading to further understanding in the formation and dynamics of the membrane-associated replication complex.

Surface Proteins of Lactobacillus crispatus: Adhesive Properties and Cell Wall Anchoring

Bacterial surface-associated proteins are important in communication with the environment and bacteria-host interactions. In this thesis work, surface molecules of Lactobacillus crispatus important in host interaction were studied. The L. crispatus strains of the study were known from previous studies to be efficient in adhesion to intestinal tract and ECM. L. crispatus JCM 5810 possess an adhesive surface layer (S-layer) protein, whose functions and domain structure was characterized. We cloned two S-layer protein genes (cbsA; collagen-binding S-layer protein A and silent cbsB) and identified the protein region in CbsA important for adhesion to host tissues, for polymerization into a periodic layer as well as for attachment to the bacterial cell surface. The analysis was done by extensive mutation analysis and by testing His6-tagged fusion proteins from recombinant Escherichia coli as well as by expressing truncated CbsA peptides on the surface of Lactobacillus casei. The N-terminal region (31-274) of CbsA showed efficient and specific binding to collagens, laminin and extracellular matrix on tissue sections of chicken intestine. The N-terminal region also contained the information for formation of periodic S-layer polymer. This region is bordered at both ends by a conserved short region rich in valines, whose substitution to leucines drastically affected the periodic polymer structure. The mutated CbsA proteins that failed to form a periodic polymer, did not bind collagens, which indicates that the polymerized structure of CbsA is needed for collagen-binding ability. The C-terminal region, which is highly identical in S-layer proteins of L. crispatus, Lactobacillus acidophilus and Lactobacillus helveticus, was shown to anchor the protein to the bacterial cell wall. The C-terminal CbsA peptide specifically bound to bacterial teichoic acid and lipoteichoic acids. In conclusion, the N-terminal domain of the S-layer protein of L. crispatus is important for polymerization and adhesion to host tissues, whereas the C-terminal domain anchors the protein to bacterial cell-wall teichoic acids. Lactobacilli are fermentative organisms that effectively lower the surrounding pH. While this study was in progress, plasminogen-binding proteins enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were identified in the extracellular proteome of L. crispatus ST1. In this work, the cell-wall association of enolase and GAPDH were shown to rely on pH-reversible binding to the cell-wall lipoteichoic acids. Enolase from L. crispatus was functionally compared with enolase from L. johnsonii as well as from pathogenic streptococci (Streptococcus pneumoniae, Streptococcus pyogenes) and Staphylococcus aureus. His6-enolases from commensal lactobacilli bound human plasminogen and enhanced its activation by human plasminogen activators similarly to, or even better than, the enolases from pathogens. Similarly, the His6-enolases from lactobacilli exhibited adhesive characteristics previously assigned to pathogens. The results call for more detailed analyses of the role of the host plasminogen system in bacterial pathogenesis and commensalism as well of the biological role and potential health risk of the extracellular proteome in lactobacilli.

Host-parasitoid relationship in different Cotesia melitaearum and Melitaea cinxia populations around the Baltic Sea

The relationship between hosts and parasites is one of the most studied interactions between living organisms, and it is both universal and common in nature. Parasitoids are special type of parasites whose offspring develop attached to or within a single host organism that it ultimately consumes and kills. Hosts are arthropods and most parasitoids belong to the insect order Hymenoptera. For almost two decades metapopulation research on the Glanville fritillary butterfly (Melitaea cinxia) has been conducted in the Åland Islands, Finland. The studies have been concerned with the population dynamics, evolution, genetics, behavior, natural history and life history characteristics of M. cinxia, as well as with species interacting with the butterfly. The parasitoids of M. cinxia have been under long term studies and much has been learned about specific host-parasitoid interactions during the past decade. The research for this Master s thesis was done in the Åland Islands during summer 2010. I conducted a reciprocal transplant style experiment in order to compare the performance of host butterflies (M. cinxia) under attack by different parasitoid wasps (C. melitaearum). I used hosts and parasitoids from five origins around the Baltic Sea: Öland, Uppland, Åland, Saaremaa and Pikku-Tytärsaari. The host-parasitoid relationship was studied in terms of host susceptibility and parasitoid virulence, addressing specifically the possible effects of inbreeding and local adaptation of both parasitoids and their hosts. I compared various factors such as host defence ratio, parasitoid development rate, cocoon production rate etc. I also conducted a small scale C. melitaearum egg development experiment and C. melitaearum external morphology comparison between different parasitoid populations. The results show that host resistance and parasitoid virulence differ between both host and parasitoid populations. For example, Öland hosts were most susceptible to parasitoids and especially vulnerable to Pikku-Tytärsaari wasps. Pikku-Tytärsaari wasps were most successful in terms of parasitoids virulence and efficiency except in Saaremaa hosts, where the wasp did not succeed. Saaremaa hosts were resistant except towards Åland parasitoids. I did not find any simple pattern concerning host resistance and parasitoid virulence between inbred and outbred populations. Also, the effect of local adaptation was not detected, perhaps because metapopulation processes disturb local adaptation of the studied populations. Morphological comparisons showed differences between studied wasp populations and sexual dimorphism was obvious with females being bigger that males. There were also interesting differences among populations in male and female wing shapes. The results raise many further questions. Especially interesting were Pikku-Tytärsaari wasps that did well in terms of efficiency and virulence. Pikku-Tytärsaari is a small, isolated island in the Gulf of Finland and both the host and parasitoids are extremely inbred. For the host and parasitoid to persist in the island, the host has to have some mechanisms to escape the parasitoid. Further research will be done on the subject to discover the mechanisms of Pikku-Tytärsaari host s ability to escape parasitism. Also, genetic analyses will be conducted in the near future to determine the relatedness of used C. melitaearum populations.

Rhizoctonia -Scots pine interactions: detection, impact on seedling performance and host defence gene response

Rhizoctonia spp. are ubiquitous soil inhabiting fungi that enter into pathogenic or symbiotic associations with plants. In general Rhizoctonia spp. are regarded as plant pathogenic fungi and many cause root rot and other plant diseases which results in considerable economic losses both in agriculture and forestry. Many Rhizoctonia strains enter into symbiotic mycorrhizal associations with orchids and some hypovirulent strains are promising biocontrol candidates in preventing host plant infection by pathogenic Rhizoctonia strains. This work focuses on uni- and binucleate Rhizoctonia (respectively UNR and BNR) strains belonging to the teleomorphic genus Ceratobasidium, but multinucleate Rhizoctonia (MNR) belonging to teleomorphic genus Thanatephorus and ectomycorrhizal fungal species, such as Suillus bovinus, were also included in DNA probe development work. Strain specific probes were developed to target rDNA ITS (internal transcribed spacer) sequences (ITS1, 5.8S and ITS2) and applied in Southern dot blot and liquid hybridization assays. Liquid hybridization was more sensitive and the size of the hybridized PCR products could be detected simultaneously, but the advantage in Southern hybridization was that sample DNA could be used without additional PCR amplification. The impacts of four Finnish BNR Ceratorhiza sp. strains 251, 266, 268 and 269 were investigated on Scot pine (Pinus sylvestris) seedling growth, and the infection biology and infection levels were microscopically examined following tryphan blue staining of infected roots. All BNR strains enhanced early seedling growth and affected the root architecture, while the infection levels remained low. The fungal infection was restricted to the outer cortical regions of long roots and typical monilioid cells detected with strain 268. The interactions of pathogenic UNR Ceratobasidium bicorne strain 1983-111/1N, and endophytic BNR Ceratorhiza sp. strain 268 were studied in single or dual inoculated Scots pine roots. The fungal infection levels and host defence-gene activity of nine transcripts [phenylalanine ammonia lyase (pal1), silbene synthase (STS), chalcone synthase (CHS), short-root specific peroxidase (Psyp1), antimicrobial peptide gene (Sp-AMP), rapidly elicited defence-related gene (PsACRE), germin-like protein (PsGER1), CuZn- superoxide dismutase (SOD), and dehydrin-like protein (dhy-like)] were measured from differentially treated and un-treated control roots by quantitative real time PCR (qRT-PCR). The infection level of pathogenic UNR was restricted in BNR- pre-inoculated Scots pine roots, while UNR was more competitive in simultaneous dual infection. The STS transcript was highly up-regulated in all treated roots, while CHS, pal1, and Psyp1 transcripts were more moderately activated. No significant activity of Sp-AMP, PsACRE, PsGER1, SOD, or dhy-like transcripts were detected compared to control roots. The integrated experiments presented, provide tools to assist in the future detection of these fungi in the environment and to understand the host infection biology and defence, and relationships between these interacting fungi in roots and soils. This study further confirms the complexity of the Rhizoctonia group both phylogenetically and in their infection biology and plant host specificity. The knowledge obtained could be applied in integrated forestry nursery management programmes.

Spatial ecology of food webs : herbivore-parasitoid communities on the pedunculate oak

Herbivorous insects, their host plants and natural enemies form the largest and most species-rich communities on earth. But what forces structure such communities? Do they represent random collections of species, or are they assembled by given rules? To address these questions, food webs offer excellent tools. As a result of their versatile information content, such webs have become the focus of intensive research over the last few decades. In this thesis, I study herbivore-parasitoid food webs from a new perspective: I construct multiple, quantitative food webs in a spatially explicit setting, at two different scales. Focusing on food webs consisting of specialist herbivores and their natural enemies on the pedunculate oak, Quercus robur, I examine consistency in food web structure across space and time, and how landscape context affects this structure. As an important methodological development, I use DNA barcoding to resolve potential cryptic species in the food webs, and to examine their effect on food web structure. I find that DNA barcoding changes our perception of species identity for as many as a third of the individuals, by reducing misidentifications and by resolving several cryptic species. In terms of the variation detected in food web structure, I find surprising consistency in both space and time. From a spatial perspective, landscape context leaves no detectable imprint on food web structure, while species richness declines significantly with decreasing connectivity. From a temporal perspective, food web structure remains predictable from year to year, despite considerable species turnover in local communities. The rate of such turnover varies between guilds and species within guilds. The factors best explaining these observations are abundant and common species, which have a quantitatively dominant imprint on overall structure, and suffer the lowest turnover. By contrast, rare species with little impact on food web structure exhibit the highest turnover rates. These patterns reveal important limitations of modern metrics of quantitative food web structure. While they accurately describe the overall topology of the web and its most significant interactions, they are disproportionately affected by species with given traits, and insensitive to the specific identity of species. As rare species have been shown to be important for food web stability, metrics depicting quantitative food web structure should then not be used as the sole descriptors of communities in a changing world. To detect and resolve the versatile imprint of global environmental change, one should rather use these metrics as one tool among several.

Molecular Interactions Chlorin Assemblies and NMR Separations

Photosynthesis is a chemical process in which the energy of the light quanta is transformed into chemical energy. Chlorophyll (Chl) molecules play a key role in photosynthesis; they function in the antennae systems and in the photosynthetic reaction center where the primary charge separation (CS) takes place. Bio-inspired mimicry of the CS is an essential unit in dye-sensitized solar cells. Aim of this study was to design and develop electron donor-acceptor (EDA) pairs from Chls and fullerenes (C60) or carbon nanotubes (CNT). The supramolecular approach was chosen, as long synthetic sequences required by the covalent approach lead to long reaction schemes and low yields. Here, a π-interaction between soluble CNTs and Chl was used in EDA construction. Also, a beta-face selective two-point bound Chl-C60 EDA was introduced. In addition, the photophysical properties of the supramolecular EDA dyads were analyzed. In organic chemistry, nuclear magnetic resonance (NMR) spectroscopy is the most vital analytical technique in use. Multi-dimensional NMR experiments have enabled a structural analysis of complex natural products and proteins. However, in mixture analysis NMR is still facing difficulties. In many cases overlapping signals can t be resolved even with the help of multi-dimensional experiments. In this work, an NMR tool based on simple host-guest chemistry between analytes and macromolecules was developed. Diffusion ordered NMR spectroscopy (DOSY) measures the mobilities of compounds in an NMR sample. In a liquid state NMR sample, each of the analytes has a characteristic diffusion coefficient, which is proportional to the size of the analyte. With normal DOSY experiment, provided that the diffusion coefficients of the analytes differ enough, individual spectra of analytes can be extracted. When similar sized analytes differ chemically, an additive can be introduced into the sample. Since macromolecules in a liquid state NMR sample can be considered practically stationary, even faint supramolecular interaction can change the diffusion coefficient of the analyte sufficiently for a successful resolution in DOSY. In this thesis, polyvinylpyrrolidone and polyethyleneglycol enhanced DOSY NMR techniques, which enable mixture analysis of similar in size but chemically differing natural products, are introduced.

Phylogeny of a taxonomically difficult group and evolution of host location mechanism

The parasitic wasps are one of the largest insect groups and their life histories are remarkably variable. Common to all parasitic wasps is that they kill their hosts, which are usually beetles, butterflies and sometimes spiders. Hosts are often at a larval or pupal stage and live in concealed conditions, such as in plant tissue. Parasitic wasps have two main ways of finding their host. 1) They can detect chemical compounds emitted by damaged plant material or released by larvae living in plant tissue, and 2) detect the larvae by sound vibrations. Even though pupae are immobile and silent, and therefore do not cause vibration, parasitoids have, however, adapted to find passive developmental stages by producing vibration themselves by knocking the substrate with their antennae, and then detecting the echoes with their legs. This echolocation allows a parasitoid to locate its potential hosts that are deeply buried in wood. This study focuses on the relationships of the subfamily Cryptinae (Hymenoptera: Ichneumonidae) and related taxa, and the evolution of host location mechanism. There are no earlier studies of the phylogeny of the Cryptinae, and the position of related taxa are unclear. According to the earlier classification, which is entirely intuitional, the Cryptinae is divided into three tribes: Cryptini, Hemigasterini and Phygadeuontini. Further, these tribes are subdiveded into numerous subtribes. This work, based on molecular characters, shows that the cryptine tribes Cryptini, Phygadeuon¬tini and Hemigasterini come out largely as monophyletic groups, thus agreeing with the earlier classification. The earlier subtribal classification had no support. In addition, it is shown that modified antennal structures are associated with host usage of wood-boring coleopteran hosts. The cryptines have a clear modification series on their antennal tips from a simply tip to a hammer-like structure. The species with strongly modified antennae belong mostly to the tribe Cryptini and they utilise wood-boring beetles as hosts. Also, field observations on insect behaviour support this result.

Interactions of natural products with β-lactoglobulins, members of the lipocalin family

Natural products constitute an important source of new drugs. The bioavailability of the drugs depends on their absorption, distribution, metabolism and elimination. To achieve good bioavailability, the drug must be soluble in water, stable in the gastrointestinal tract and palatable. Binding proteins may improve the solubility of drug compounds, masking unwanted properties, such as bad taste, bitterness or toxicity, transporting or protecting these compounds during processing and storage. The focus of this thesis was to study the interactions, including ligand binding and the effect of pH and temperature, of bovine and reindeer β-lactoglobulin (βLG) with such compounds as retinoids, phenolic compounds as well as with compounds from plant extracts, and to investigate the transport properties of the βLG-ligand complex. To examine the binding interactions of different ligands to βLG, new methods were developed. The fluorescence binding method for the evaluation of ligand binding to βLG was miniaturized from a quartz cell to a 96-well plate. A method of ultrafiltration sampling combined with high-performance liquid chromatography was developed to assess the binding of compounds from extracts. The interactions of phenolic compounds or retinoids and βLG were investigated using the 96-well plate method. The majority of flavones, flavonols, flavanones and isoflavones and all of the retinoids included were shown to bind to bovine and reindeer βLG. Phenolic compounds, contrary to retinol, were not released at acidic pH. Those results suggest that βLG may have more binding sites, probably also on the surface of βLG. An extract from Camellia sinensis (L.) O. Kunze (black tea), Urtica dioica L. (nettle) and Piper nigrum (black pepper) were used to evaluate whether βLG could bind compounds from plant extracts. Piperine from P. nigrum was found to bind tightly and rutin from U. dioica weakly to βLG. No components from C. sinensis bound to βLG in our experiment. The uptake and membrane permeation of bovine and reindeer βLG, free and bound with retinol, palmitic acid and cholesterol, were investigated using Caco-2 cell monolayers. Both bovine and reindeer βLG were able to cross the Caco-2 cell membrane. Free and βLG-bound retinol and palmitic acid were transported equally, whereas cholesterol could not cross the Caco-2 cell monolayer free or bound to βLG. Our results showed that βLG can bind different natural product compounds, but cannot enhance transport of retinol, palmitic acid or cholesterol through Caco-2 cells. Despite this, βLG, as a water-soluble binding protein, may improve the solubility of natural compounds, possibly protecting them from early degradation and transporting some of them through the stomach. Furthermore, it may decrease their bad or bitter taste during oral administration of drugs or in food preparations. βLG can also enhance or decrease the health benefits of herbal teas and food preparations by binding compounds from extracts.

Immunomodulatory effects of probiotic bacteria in healthy adults

Probiooteilla kantakohtaisia vaikutuksia ihmisen immuunijärjestelmään terveillä aikuisilla Probiooteilla on kantakohtaisia tulehduksen välittäjäaineita vähentäviä vaikutuksia ja probioottien yhdistelmien vaikutukset eroavat yksittäisten kantojen vaikutuksista selviää TtM Riina Kekkosen tuoreesta väitöstutkimuksesta. TtM Riina Kekkonen on selvittänyt väitöskirjassaan eri probioottikantojen vaikutuksia immuunivasteeseen valkosolumallissa sekä terveillä aikuisilla lumekontrolloiduissa kliinisissä tutkimuksissa. Aikaisemmin probioottien vaikutuksia on tutkittu lähinnä allergian ja erilaisten vatsavaivojen ehkäisyssä ja hoidossa. Probiootteja sisältäviä tuotteita käyttävät kuluttajat ovat kuitenkin useimmiten terveitä aikuisia, ja probioottien vaikutus terveiden aikuisten immuunijärjestelmään on ollut puutteellisesti selvitettyä. Valkosolumallissa probioottikantojen havaittiin poikkeavan toisistaan niiden kyvyssä aktivoida immuunivasteen välittäjäaineiden, sytokiinien, tuotantoa. Anti-inflammatorisia, eli tulehdusta lievittäviä vaikutuksia nähtiin lähinnä Bifidobacterium ja Propionibacterium sukuihin kuuluvilla kannoilla. Streptococcus ja Leuconostoc sukuihin kuuluvat kannat puolestaan aktivoivat Th1 tyyppistä, soluvälitteistä immuunivastetta. Eri probioottien kombinaatiot eivät saaneet aikaan voimakkaampaa aktivaatiota yksittäisiin kantoihin verrattuna, joka viittaa probioottien keskinäiseen kilpailuun niiden ollessa kontaktissa ihmisen solujen kanssa. Probioottikantojen valinta kliinisiin tutkimuksiin tehtiin niiden anti-inflammatoristen ominaisuuksien perusteella. Parhaita anti-inflammatorisia kantoja olivat B. lactis ssp. animalis Bb12 ja P. freudenreichii ssp. shermanii JS, joiden lisäksi tutkimuksiin valittiin myös L. rhamnosus GG (LGG) hyvin tutkittuna referenssikantana. Solutöiden tulokset eivät olleet täysin verrannollisia kliinisen työn tuloksiin, koska LGG näytti omaavan parhaat anti-inflammatoriset ominaisuudet kliinisissä tutkimuksissa vaikka solutyössä sen aikaansaamat vasteet olivat melko vaimeita. Kolmen viikon kliinisessä tutkimuksessa terveillä aikuisilla LGG alensi mm. tulehdusta kuvaavan C-reaktiivisen proteiinin ja inflammatoristen sytokiinien määrää. Pidemmässä kolmen kuukauden pituisessa kliinisessä tutkimuksessa LGG:llä ei ollut vaikutusta terveiden aikuisten infektiosairastavuuteen, mutta LGG lyhensi vatsavaivojen kestoa. Probioottien vaikutukset immuunijärjestelmään näyttävät olevan kantakohtaisia ja erityisesti Lactobacillus rhamnosus GG:llä havaittiin anti-inflammatorisia vaikutuksia. Valkosolumallia ei tulisi käyttää ainoana probioottikantojen skriinausmenetelmänä niiden immunologisia vaikutuksia selvitettäessä, koska solutöiden tulokset eivät olleet täysin verrannollisia kliinisten tutkimusten tuloksiin. Sen sijaan veren perifeeristen lymfosyyttien eristäminen ja niiden aktivoitumisen selvittäminen lyhytaikaisessa kliinisessä tutkimuksessa voisi toimia suhteellisen helppona skiinausmenetelmänä.

Molecular Mechanisms of Androgen Receptor Interactions

The androgen receptor (AR) mediates the effects of the male sex-steroid hormones (androgens), testosterone and 5?-dihydrotestosterone. Androgens are critical in the development and maintenance of male sexual characteristics. AR is a member of the steroid receptor ligand-inducible transcription factor family. The steroid receptor family is a subgroup of the nuclear receptor superfamily that also includes receptors for the active forms of vitamin A, vitamin D3, and thyroid hormones. Like all nuclear receptors, AR has a conserved modular structure consisting of a non-conserved amino-terminal domain (NTD), containing the intrinsic activation function 1, a highly conserved DNA-binding domain, and a conserved ligand-binding domain (LBD) that harbors the activation function 2. Each of these domains plays an important role in receptor function and signaling, either via intra- and inter-receptor interactions, interactions with specific DNA sequences, termed hormone response elements, or via functional interactions with domain-specific proteins, termed coregulators (coactivators and corepressors). Upon binding androgens, AR acquires a new conformational state, translocates to the nucleus, binds to androgen response elements, homodimerizes and recruits sequence-specific coregulatory factors and the basal transcription machinery. This set of events is required to activate gene transcription (expression). Gene transcription is a strictly modulated process that governs cell growth, cell homeostasis, cell function and cell death. Disruptions of AR transcriptional activity caused by receptor mutations and/or altered coregulator interactions are linked to a wide spectrum of androgen insensitivity syndromes, and to the pathogenesis of prostate cancer (CaP). The treatment of CaP usually involves androgen depletion therapy (ADT). ADT achieves significant clinical responses during the early stages of the disease. However, under the selective pressure of androgen withdrawal, androgen-dependent CaP can progress to an androgen-independent CaP. Androgen-independent CaP is invariably a more aggressive and untreatable form of the disease. Advancing our understanding of the molecular mechanisms behind the switch in androgen-dependency would improve our success of treating CaP and other AR related illnesses. This study evaluates how clinically identified AR mutations affect the receptor s transcriptional activity. We reveal that a potential molecular abnormality in androgen insensitivity syndrome and CaP patients is caused by disruptions of the important intra-receptor NTD/LBD interaction. We demonstrate that the same AR LBD mutations can also disrupt the recruitment of the p160 coactivator protein GRIP1. Our investigations reveal that 30% of patients with advanced, untreated local CaP have somatic mutations that may lead to increases in AR activity. We report that somatic mutations that activate AR may lead to early relapse in ADT. Our results demonstrate that the types of ADT a CaP patient receives may cause a clustering of mutations to a particular region of the receptor. Furthermore, the mutations that arise before and during ADT do not always result in a receptor that is more active, indicating that coregulator interactions play a pivotal role in the progression of androgen-independent CaP. To improve CaP therapy, it is necessary to identify critical coregulators of AR. We screened a HeLa cell cDNA library and identified small carboxyl-terminal domain phosphatase 2 (SCP2). SCP2 is a protein phosphatase that directly interacts with the AR NTD and represses AR activity. We demonstrated that reducing the endogenous cellular levels of SCP2 causes more AR to load on to the prostate specific antigen (PSA) gene promoter and enhancer regions. Additionally, under the same conditions, more RNA polymerase II was recruited to the PSA promoter region and overall there was an increase in androgen-dependent transcription of the PSA gene, revealing that SCP2 could play a role in the pathogenesis of CaP.