Increased attractiveness of honeybee hive product volatiles to adult small hive beetle, Aethina tumida, resulting from small hive beetle larval infestation

The small hive beetle, Aethina tumida Murray (Coleoptera: Nitidulidae), is a recent but significant pest of honeybee Apis mellifera L. (Hymenoptera: Apidae) hives in various regions throughout the world, including Eastern Australia. The larval stage of this beetle damages hives when they feed on brood, pollen, and honeycomb, leaving behind fermented wastes. In cases of extreme damage, hives collapse and are turned to an odorous mass of larvae in fermenting hive products. The yeast Kodamaea ohmeri (Etchells & Bell) Yamada et al. (Ascomycota) has been consistently isolated from the fermenting material as well as each life stage of this beetle. Various studies have noted that the small hive beetle is attracted to volatiles from hive products and those of the yeast K. ohmeri, although earlier studies have not used naturally occurring hive products as their source of fermentation. This study investigated changes through time in the attractiveness of natural honeybee hive products to the small hive beetle as the hive products were altered by the action of beetle larvae and fermentation by K. ohmeri. We used gas chromatography-mass spectrometry and choice-test behavioural assays to investigate these changes using products sampled from three apiaries. Attractiveness of the fermenting hive products (‘slime’) increased as fermentation progressed, and volatile profiles became more complex. Fermenting hive products remained extremely attractive for more than 30 days, significantly longer than previous reports. These results have strong implications for the development of an external attractant trap to assist in the management of this invasive pest.

Commercial-scale Alternaria brown spot resistance screening as the first step in breeding new mandarins for Australia

Rapid screening tests and an appreciation of the simple genetic control of Alternaria brown spot (ABS) susceptibility have existed for many years, and yet the application of this knowledge to commercial-scale breeding programs has been limited. Detached leaf assays were first demonstrated more than 40 years ago and reliable data suggesting a single gene determining susceptibility has been emerging for at least 20 years. However it is only recently that the requirement for genetic resistance in new hybrids has become a priority, following increased disease prevalence in Australian mandarin production areas previously considered too dry for the pathogen. Almost all of the high-fruit-quality parents developed so far by the Queensland-based breeding program are susceptible to ABS necessitating the screening of their progeny to avoid commercialisation of susceptible hybrids. This is done effectively and efficiently by spraying 3-6 month old hybrid seedlings with a spore suspension derived from a toxin-producing field isolate of Alternaria alternate, then incubating these seedlings in a cool room at 25°C and high humidity for 5 days. Susceptible seedlings show clear disease symptoms and are discarded. Analysis of observed and expected segregation ratios loosely support the hypothesis for a single dominant gene for susceptibility, but do not rule out the possibility of alternative genetic models. After implementing the routine screening for ABS resistance for three seasons we now have more than 20,000 hybrids growing in field progeny blocks that have been screened for resistance to the ABS disease.

Methods to improve gene signal: Application to cDNA microarrays

Microarrays are high throughput biological assays that allow the screening of thousands of genes for their expression. The main idea behind microarrays is to compute for each gene a unique signal that is directly proportional to the quantity of mRNA that was hybridized on the chip. A large number of steps and errors associated with each step make the generated expression signal noisy. As a result, microarray data need to be carefully pre-processed before their analysis can be assumed to lead to reliable and biologically relevant conclusions. This thesis focuses on developing methods for improving gene signal and further utilizing this improved signal for higher level analysis. To achieve this, first, approaches for designing microarray experiments using various optimality criteria, considering both biological and technical replicates, are described. A carefully designed experiment leads to signal with low noise, as the effect of unwanted variations is minimized and the precision of the estimates of the parameters of interest are maximized. Second, a system for improving the gene signal by using three scans at varying scanner sensitivities is developed. A novel Bayesian latent intensity model is then applied on these three sets of expression values, corresponding to the three scans, to estimate the suitably calibrated true signal of genes. Third, a novel image segmentation approach that segregates the fluorescent signal from the undesired noise is developed using an additional dye, SYBR green RNA II. This technique helped in identifying signal only with respect to the hybridized DNA, and signal corresponding to dust, scratch, spilling of dye, and other noises, are avoided. Fourth, an integrated statistical model is developed, where signal correction, systematic array effects, dye effects, and differential expression, are modelled jointly as opposed to a sequential application of several methods of analysis. The methods described in here have been tested only for cDNA microarrays, but can also, with some modifications, be applied to other high-throughput technologies. Keywords: High-throughput technology, microarray, cDNA, multiple scans, Bayesian hierarchical models, image analysis, experimental design, MCMC, WinBUGS.

Molecular evidence of Ebola Reston virus infection in Philippine bats

Background: In 2008-09, evidence of Reston ebolavirus (RESTV) infection was found in domestic pigs and pig workers in the Philippines. With species of bats having been shown to be the cryptic reservoir of filoviruses elsewhere, the Philippine government, in conjunction with the Food and Agriculture Organization of the United Nations, assembled a multi-disciplinary and multi-institutional team to investigate Philippine bats as the possible reservoir of RESTV. Methods: The team undertook surveillance of bat populations at multiple locations during 2010 using both serology and molecular assays. Results: A total of 464 bats from 21 species were sampled. We found both molecular and serologic evidence of RESTV infection in multiple bat species. RNA was detected with quantitative PCR (qPCR) in oropharyngeal swabs taken from Miniopterus schreibersii, with three samples yielding a product on conventional hemi-nested PCR whose sequences differed from a Philippine pig isolate by a single nucleotide. Uncorroborated qPCR detections may indicate RESTV nucleic acid in several additional bat species (M. australis, C. brachyotis and Ch. plicata). We also detected anti-RESTV antibodies in three bats (Acerodon jubatus) using both Western blot and ELISA. Conclusions: The findings suggest that ebolavirus infection is taxonomically widespread in Philippine bats, but the evident low prevalence and low viral load warrants expanded surveillance to elaborate the findings, and more broadly, to determine the taxonomic and geographic occurrence of ebolaviruses in bats in the region. © 2015 Jayme et al.

Characterization of Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease, reveals a unique mode of DNA-binding, helical distortion and cleavage, compared to a canonical LAGLIDADG homing endonuclease

Mycobacterium leprae, which has undergone reductive evolution leaving behind a minimal set of essential genes, has retained intervening sequences in four of its genes implicating a vital role for them in the survival of the leprosy bacillus. A single in-frame intervening sequence has been found embedded within its recA gene. Comparison of M. leprae recA intervening sequence with the known intervening sequences indicated that it has the consensus amino acid sequence necessary for being a LAGLIDADG-type homing endonuclease. In light of massive gene decay and function loss in the leprosy bacillus, we sought to investigate whether its recA intervening sequence encodes a catalytically active homing endonuclease. Here we show that the purified M. leprae RecA intein (PI-MleI) binds to cognate DNA and displays endonuclease activity in the presence of alternative divalent cations, Mg2+ or Mn2+. A combination of approaches including four complementary footprinting assays such as DNase I, Cu/phenanthroline, methylation protection and KMnO4, enhancement of 2-aminopurine fluorescence and mapping of the cleavage site revealed that PI-MleI binds to cognate DNA flanking its insertion site, induces helical distortion at the cleavage site and generates two staggered double-strand breaks. Taken together, these results implicate that PI-MleI possess a modular structure with separate domains for DNA target recognition and cleavage, each with distinct sequence preferences. From a biological standpoint, it is tempting to speculate that our findings have implications for understanding the evolution of LAGLIDADG family of homing endonucleases

Stable expression of silencing-suppressor protein enhances the performance and longevity of an engineered metabolic pathway

Transgenic engineering of plants is important in both basic and applied research. However, the expression of a transgene can dwindle over time as the plant's small (s)RNA-guided silencing pathways shut it down. The silencing pathways have evolved as antiviral defence mechanisms, and viruses have co-evolved viral silencing-suppressor proteins (VSPs) to block them. Therefore, VSPs have been routinely used alongside desired transgene constructs to enhance their expression in transient assays. However, constitutive, stable expression of a VSP in a plant usually causes pronounced developmental abnormalities, as their actions interfere with endogenous microRNA-regulated processes, and has largely precluded the use of VSPs as an aid to stable transgene expression. In an attempt to avoid the deleterious effects but obtain the enhancing effect, a number of different VSPs were expressed exclusively in the seeds of Arabidopsis thaliana alongside a three-step transgenic pathway for the synthesis of arachidonic acid (AA), an ω-6 long chain polyunsaturated fatty acid. Results from independent transgenic events, maintained for four generations, showed that the VSP-AA-transformed plants were developmentally normal, apart from minor phenotypes at the cotyledon stage, and could produce 40% more AA than plants transformed with the AA transgene cassette alone. Intriguingly, a geminivirus VSP, V2, was constitutively expressed without causing developmental defects, as it acts on the siRNA amplification step that is not part of the miRNA pathway, and gave strong transgene enhancement. These results demonstrate that VSP expression can be used to protect and enhance stable transgene performance and has significant biotechnological application.

High-throughput Phenotyping of Wheat Seminal Root Traits in a Breeding Context

Water availability is a major limiting factor for wheat (Triticum aestivum L.) in rain-fed agricultural systems worldwide. Root architecture has important functional implications for the timing and extent of soil water extraction, yet selection for root traits in wheat breeding programs has been largely limited due to the lack of suitable phenotyping methods. The aim of this research was to develop a low-cost high-throughput phenotyping method to facilitate selection for desirable root traits. We developed a method to assess ‘seminal root angle’ and ‘seminal root number’ in seedlings – two proxy traits associated to root architecture of mature wheat plants (1). The method involves measuring the angle between the first pair of seminal roots and the number of roots of wheat seedlings grown in transparent pots (Figure 1). Images captured at 5 to 10 days after sowing are analyzed to calculate seminal root angle and number. Performing this technique under “speed breeding” conditions (plants grown at a density of 600 plants / m2, under controlled temperature and constant light) allows the selection based on the desired root traits of up to 5 consecutive generations within 12 months. Alternatively, when focusing only on germplasm screening, up to 52 successive phenotypic assays can be conducted within 12 months. This approach has been shown to be highly reproducible, it requires little resource (time, space, and labour) and can be used to rapidly enrich breeding populations with desirable alleles for narrow root angle and a high number of seminal roots to indirectly target the selection of deeper root system with higher branching at depth. Such root characteristics are highly desirable in wheat to cope with the climate model projections, especially in summer rainfall dominant regions including some Australian, Indian, South American and African cropping regions, where winter crops mainly rely on deep stored water.

Rapid transfection assay for screening mutagens and carcinogens

Testing for mutagenicity and carcinogenicity has become an integral part of the toxicological evaluation of drugs and chemicals. Standard carcinogenicity tests in vivo require both large numbers of animals and prolonged experiments. To circumvent these problems, several rapid tests have been developed for preliminary screening of mutagens and carcinogens in vitro. Ames and his associates, the first to develop a mutation test, used mutant strains of Salmonella typhimurium [1]. Mutation tests with Escherichia coli, Bacillus subtilis, Neurospora crassa and Saccharomyces cerevisiae, and DNA-repair tests with E. coli and B. subtilis, have been developed. Cytogenetic assays, in vivo as well as in vitro, in both plant and animal systems, are also used to detect potential mutagens and carcinogens. Transfection is inhibited by base mutation, cleavage of DNA, loss of cohesive ends, interaction with histones, spermidine, nalidixic acid, etc. [3]. The efficiency of transfection is affected by temperature, DNA structure and the condition of the competence of the recipient cells [3]. Transfection assays with phages MS: RNA and ~i, x 174-DNA have been reported [15]. A fast and easy transfection assay using colitis bacteriophage DNA is reported in this communication.

Studies on the relative biopotencies and intestinal absorption of different apo-beta-carotenoids in rats and chickens.

1. The biopotencies relative to beta-carotene of several apocarotenoids, such as 8'-, 10'- and 12'-apo-beta-carotenal and methyl 8'-apo-beta-carotenoate, were investigated in rats, on a molar basis, by both curative-growth assay and liver-storage tests. 2. In the curative-growth assays, on a molar basis the biopotencies of 8'-, 10'- and 12'-apo-beta-carotenal and methyl 8'-apo-beta-carotenoate were 72, 78, 72 and 53% respectively, whereas on a weight basis the corresponding values were 93, 111, 111 and 63%, with respect to beta-carotene taken as 100%. In terms of yield of vitamin A, these values were much lower in the liver-storage tests. 3. When 8'-apo-beta-carotenal was fed, the unchanged aldehyde together with small amounts of the corresponding alcohol and larger proportions of the acid rapidly appeared in the tissues of both rats and chickens. The 8'-apocarotenol, 8'-apocarotenoic acid and its methyl ester were absorbed unchanged. The free acid disappeared most rapidly from the tissues, but its methyl ester persisted in the tissues longest. 4. On the basis of these observations it is suggested that most of an apocarotenal is oxidized to the corresponding acid, which, in turn, is mostly degraded to retinoic acid, with small proportions of it being attacked by the dioxygenase system giving retinal.

Antimicrobial efficacy of a novel silver hydrogel dressing compared to two common silver burn wound dressings: Acticoat™ and PolyMem Silver

A novel burn wound hydrogel dressing has been previously developed which is composed of 2-acrylamido-2-methylpropane sulfonic acid sodium salt with silver nanoparticles. This study compared the antimicrobial efficacy of this novel dressing to two commercially available silver dressings; Acticoat™ and PolyMem Silver(®). Three different antimicrobial tests were used: disc diffusion, broth culture, and the Live/Dead(®) Baclight™ bacterial viability assay. Burn wound pathogens (P. aeruginosa, MSSA, A. baumannii and C. albicans) and antibiotic resistant strains (MRSA and VRE) were tested. All three antimicrobial tests indicated that Acticoat™ was the most effective antimicrobial agent, with inhibition zone lengths of 13.9-18.4mm. It reduced the microbial inocula below the limit of detection (10(2)CFU/ml) and reduced viability by 99% within 4h. PolyMem Silver(®) had no zone of inhibition for most tested micro-organisms, and it also showed poor antimicrobial activity in the broth culture and Live/Dead(®) Baclight™ assays. Alarmingly, it appeared to promote the growth of VRE. The silver hydrogel reduced most of the tested microbial inocula below the detection limit and decreased bacterial viability by 94-99% after 24h exposure. These results support the possibility of using this novel silver hydrogel as a burn wound dressing in the future

Effects of 17α-ethinyl estradiol on the mating system of the sand goby (Pomatoschistus minutus)

In aquatic environments, endocrine disrupting chemicals (EDCs) that interfere with the endocrinology of males and females form a threat to the maintenance of populations. EDCs are a diverse group of natural and manmade chemicals that already at very low concentrations (at nanogram levels) can have severe effects on reproduction by individuals, e.g. complete sex reversal, feminisation of males, impaired reproduction even resulting in near extinction of populations. With regard to fish, despite the extensive literature on physiological effects of EDCs, very little is known about potential population-level effects. In this thesis, I examined how 17α-ethinyl estradiol (EE2), a synthetic estrogen used in oral contraceptive pills, affects the reproductive behaviour of a marine fish, the sand goby (Pomatoschistus minutus). The aims were fourfold. First, I investigated how exposure to EE2 affects courtship and parental care of sand goby males. Secondly, I looked at effects on the mating system and sexual selection. In the third study, I observed the effects of exposure in a social context where exposed males had to compete with non-exposed males for resources and mates. Finally, I studied the effects of exposure on male-male competition and male aggressive behaviour. This work revealed that EE2 exposure impairs the ability of males to acquire and defend a nest, as well as diminishes the attractiveness of males to females by decreasing their courtship and aggressive behaviour. These effects are harmful for a male whose reproductive success is determined by the ability to compete for limited resources and to attract mates. Furthermore, this thesis showed that selection on male size was relaxed after EE2 exposure and male size had a smaller effect on mating success. These effects can be of a profound nature as they interfere with sexual selection, and may in the long run lead to the loss of traits maintained through sexual selection. The thesis shows that an exposure to environmentally relevant levels of EE2 clearly reduces the chances of individuals to reproduce successfully. Furthermore, it strongly suggests that several types of biomarkers should be used to detect and assess the effects of EDC exposure because severe behavioural effects can sometimes be seen before effects are detectable at the molecular or morphometric level. Behavioural assays should be considered an important complementary tool for the standard ecotoxicological assays because observed behavioural changes have direct and negative effects on fitness, while the connection between changes in molecular expression and fitness may be less obvious.

Molecular characterisation of the Vacuolating Autotransporter Toxin in Uropathogenic Escherichia coli

The vacuolating autotransporter (AT) toxin (Vat) contributes to Uropathogenic Escherichia coli (UPEC) fitness during systemic infection. Here we characterised Vat and investigated its regulation in UPEC. We assessed the prevalence of vat in a collection of 45 UPEC urosepsis strains and showed that it was present in 31 (68%) of the isolates. The isolates containing the vat gene corresponded to three major E. coli sequence types (ST12, 73 and 95) and these strains secreted the Vat protein. Further analysis of the vat genomic locus identified a conserved gene located directly downstream of vat that encodes a putative MarR-like transcriptional regulator, which we termed vatX. The vat-vatX genes were present in the UPEC reference strain CFT073 and RT-PCR revealed both genes are co-transcribed. Over-expression of vatX in CFT073 led to a 3-fold increase in vat gene transcription. The vat promoter region contained three putative nucleation sites for the global transcriptional regulator H-NS; thus the hns gene was mutated in CFT073 (to generate CFT073hns). Western blot analysis using a Vat-specific antibody revealed a significant increase in Vat expression in CFT073hns compared to wild-type CFT073. Direct H-NS binding to the vat promoter region was demonstrated using purified H-NS in combination with electrophoresis mobility shift assays. Finally, Vat-specific antibodies were detected in plasma samples from urosepsis patients infected by vat-containing UPEC strains, demonstrating Vat is expressed during infection. Overall, this study has demonstrated that Vat is a highly prevalent and tightly regulated immunogenic SPATE secreted by UPEC during infection.

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.

Pathogenicity, functional significance and clinical phenotype of mismatch repair gene MSH2 variants found in cancer patients

DNA ja siinä sijaitsevat geenit ohjaavat kaikkea solujen toimintaa. DNA-molekyyleihin kuitenkin kertyy mutaatioita sekä ympäristön vaikutuksen, että solujen oman toiminnan tuloksena. Mikäli virheitä ei korjata, saattaa tuloksena olla solun muuttuminen syöpäsoluksi. Soluilla onkin käytössä useita DNA-virheiden korjausmekanismeja, joista yksi on ns. mismatch repair (MMR). MMR vastaa DNA:n kahdentumisessa syntyvien virheiden korjauksesta. Periytyvät mutaatiot geeneissä, jotka vastaavat MMR-proteiinien rakentamisesta, aiheuttavat ongelmia DNA:n korjauksessa ja altistavat kantajansa periytyvälle ei-polypoottiselle paksusuolisyöpäoireyhtymälle (hereditary nonpolyposis colorectal cancer, HNPCC). Yleisimmin mutatoituneet MMR-geenit ovat MLH1 ja MSH2. HNPCC periytyy vallitsevasti, eli jo toiselta vanhemmalta peritty geenivirhe altistaa syövälle. MMR-geenivirheen kantaja sairastuu syöpään elämänsä aikana suurella todennäköisyydellä, ja sairastumisikä on vain noin 40 vuotta. Syövälle altistavan geenivirheen löytäminen mutaation kantajilta on hyvin tärkeää, sillä säännöllinen seuranta mahdollistaa kehittymässä olevan kasvaimen havaitsemisen ja poistamisen jo aikaisessa vaiheessa. Tämän on osoitettu alentavan syöpäkuolleisuutta merkittävästi. Varma tieto altistuksen alkuperästä on tärkeä myös niille syöpäsuvun jäsenille, jotka eivät kanna kyseistä mutaatiota. Syövälle altistavien mutaatioiden ohella MMR-geeneistä löydetään säännöllisesti muutoksia, jotka ovat normaalia henkilöiden välistä geneettistä vaihtelua, eikä niiden oleteta lisäävän syöpäaltistusta. Altistavien mutaatioiden erottaminen näistä neutraaleista variaatioista on vaikeaa, mutta välttämätöntä altistuneiden tehokkaan seurannan varmistamiseksi. Tässä väitöskirjassa tutkittiin 18:a MSH2 -geenin mutaatiota. Mutaatiot oli löydetty perheistä, joissa esiintyi paljon syöpiä, mutta niiden vaikutus DNA:n korjaustehoon ja syöpäaltistukseen oli epäselvä. Työssä tutkittiin kunkin mutaation vaikutusta MSH2-proteiinin normaaliin toimintaan, ja tuloksia verrattiin potilaiden ja sukujen kliinisiin tietoihin. Tutkituista mutaatiosta 12 aiheutti puutteita MMR-korjauksessa. Nämä mutaatiot tulkittiin syövälle altistaviksi. Analyyseissä normaalisti toimineet 4 mutaatiota eivät todennäköisesti ole syynä syövän syntyyn kyseisillä perheillä. Tulkinta jätettiin avoimeksi 2 mutaation kohdalla. Tutkimuksesta hyötyivät suoraan kuvattujen mutaatioiden kantajaperheet, joiden geenivirheen syöpäaltistuksesta saatiin tietoa, mahdollistaen perinnöllisyysneuvonnan ja seurannan kohdentamisen sitä tarvitseville. Työ selvensi myös mekanismeja, joilla mutatoitunut MSH2-proteiini voi menettää toimintakykynsä.

Immune response to insulin and changes in the gut immune system in children with or at risk for type 1 diabetes

Type 1 diabetes (T1D) is considered to be an autoimmune disease. The cause of T1D is the destruction of insulin-producing β-cells in the pancreatic islets. The autoimmune nature of T1D is characterized by the presence of autoreactive T-cells and autoantibodies against β-cell molecules. Insulin is the only β-cell-specific autoantigen associated with T1D but the insulin autoantibodies (IAAs) are difficult to measure with proper sensitivity. T-cell assays for detection of autoreactive T-cells, such as insulin-specific T-cells, have also proven to be difficult to perform. The genetic risk of T1D is associated with the HLA gene region but the environmental factors also play an important role. The most studied environmental risk factors of T1D are enteroviruses and cow's milk which both affect the immune system through the gut. One hypothesis is that the insulin-specific immune response develops against bovine insulin in cow's milk during early infancy and later spreads to include human insulin. The aims of this study were to determine whether the separation of immunoglobulin (Ig)G from plasma would improve the sensitivity of the IAA assay and how insulin treatment affects the cellular immune response to insulin in newly diagnosed patients. Furthermore, the effect of insulin concentration in mother's breast milk on the development of antibodies to dietary insulin in the child was examined. Small intestinal biopsies were also obtained from children with T1D to characterize any immunological changes associated with T1D in the gut. The isolation of the IgG fraction from the plasma of T1D patients negative for plasma IAA led to detectable IAA levels that exceeded those in the control children. Thus the isolation of IgG may improve the sensitivity of the IAA assay. The effect of insulin treatment on insulin-specific T-cells was studied by culturing peripheral blood mononuclear cells with insulin. The insulin stimulation induced increased expression of regulatory T-cell markers, such as Foxp3, in those patients treated with insulin than in patients examined before initiating insulin treatment. This finding suggests that insulin treatment in patients with T1D stimulates regulatory T-cells in vivo and this may partly explain the difficulties in measuring autoantigen-specific T-cell responses in recently diagnosed patients. The stimulation of regulatory T-cells by insulin treatment may also explain the remission period often seen after initiating insulin treatment. In the third study we showed that insulin concentration in mother's breast milk correlates inversely with the levels of bovine insulin-specific antibodies in those infants who were exposed to cow's milk proteins in their diet, suggesting that human insulin in breast milk induces tolerance to dietary bovine insulin. However, in infants who later developed T1D-associated autoantibodies, the insulin concentration in their mother's breast milk was increased. This finding may indicate that in those children prone to β-cell autoimmunity, breast milk insulin does not promote tolerance to insulin. In the small intestinal biopsies the presence of several immunological markers were quantified with the RT-PCR. From these markers the expression of the interleukin (IL)-18 cytokine was significantly increased in the gut in patients with T1D compared with children with celiac disease or control children. The increased IL-18 expression lends further support for the hypothesis that the gut immune system is involved in the pathogenesis of T1D.