Genotypic variation in seedling root architectural traits and implications for drought adaptation in wheat (Triticum aestivum L.)

Root system characteristics are of fundamental importance to soil exploration and below-ground resource acquisition. Root architectural traits determine the in situ space-filling properties of a root system or root architecture. The growth angle of root axes is a principal component of root system architecture that has been strongly associated with acquisition efficiency in many crop species. The aims of this study were to examine the extent of genotypic variability for the growth angle and number of seminal roots in 27 current Australian and 3 CIMMYT wheat (Triticum aestivum L.) genotypes, and to quantify using fractal analysis the root system architecture of a subset of wheat genotypes contrasting in drought tolerance and seminal root characteristics. The growth angle and number of seminal roots showed significant genotypic variation among the wheat genotypes with values ranging from 36 to 56 (degrees) and 3 to 5 (plant-1), respectively. Cluster analysis of wheat genotypes based on similarity in their seminal root characteristics resulted in four groups. The group composition reflected to some extent the genetic background and environmental adaptation of genotypes. Wheat cultivars grown widely in the Mediterranean environments of southern and western Australia generally had wider growth angle and lower number of seminal axes. In contrast, cultivars with superior performance on deep clay soils in the northern cropping region, such as SeriM82, Baxter, Babax, and Dharwar Dry exhibited a narrower angle of seminal axes. The wheat genotypes also showed significant variation in fractal dimension (D). The D values calculated for the individual segments of each root system suggested that, compared to the standard cultivar Hartog, the drought-tolerant genotypes adapted to the northern region tended to distribute relatively more roots in the soil volume directly underneath the plant. These findings suggest that wheat root system architecture is closely linked to the angle of seminal root axes at the seedling stage. The implications of genotypic variation in the seminal root characteristics and fractal dimension for specific adaptation to drought environment types are discussed with emphasis on the possible exploitation of root architectural traits in breeding for improved wheat cultivars for water-limited environments.

An analysis of assessment outcomes from eight years' operation of the Australian border weed risk assessment system

The majority of Australian weeds are exotic plant species that were intentionally introduced for a variety of horticultural and agricultural purposes. A border weed risk assessment system (WRA) was implemented in 1997 in order to reduce the high economic costs and massive environmental damage associated with introducing serious weeds. We review the behaviour of this system with regard to eight years of data collected from the assessment of species proposed for importation or held within genetic resource centres in Australia. From a taxonomic perspective, species from the Chenopodiaceae and Poaceae were most likely to be rejected and those from the Arecaceae and Flacourtiaceae were most likely to be accepted. Dendrogram analysis and classification and regression tree (TREE) models were also used to analyse the data. The latter revealed that a small subset of the 35 variables assessed was highly associated with the outcome of the original assessment. The TREE model examining all of the data contained just five variables: unintentional human dispersal, congeneric weed, weed elsewhere, tolerates or benefits from mutilation, cultivation or fire, and reproduction by vegetative propagation. It gave the same outcome as the full WRA model for 71% of species. Weed elsewhere was not the first splitting variable in this model, indicating that the WRA has a capacity for capturing species that have no history of weediness. A reduced TREE model (in which human-mediated variables had been removed) contained four variables: broad climate suitability, reproduction in less or than equal to 1 year, self-fertilisation, and tolerates and benefits from mutilation, cultivation or fire. It yielded the same outcome as the full WRA model for 65% of species. Data inconsistencies and the relative importance of questions are discussed, with some recommendations made for improving the use of the system.

Genetics of heifer performance in 'wet' and 'dry' seasons and their relationships with steer performance in two tropical beef genotypes.

The genetics of heifer performance in tropical 'wet' and 'dry' seasons, and relationships with steer performance, were studied in Brahman (BRAH) and Tropical Composite (TCOMP) (50% Bos indicus, African Sanga or other tropically adapted Bos taurus; 50% non-tropically adapted Bos taurus) cattle of northern Australia. Data were from 2159 heifers (1027 BRAH, 1132 TCOMP), representing 54 BRAH and 51 TCOMP sires. Heifers were assessed after post-weaning 'wet' (ENDWET) and 'dry' (ENDDRY) seasons. Steers were assessed post-weaning, at feedlot entry, over a 70-day feed test, and after similar to 120-day finishing. Measures studied in both heifers and steers were liveweight (LWT), scanned rump fat, rib fat and M. longissimus area (SEMA), body condition score (CS), hip height (HH), serum insulin-like growth factor-I concentration (IGF-I), and average daily gains (ADG). Additional steer measures were scanned intra-muscular fat%, flight time, and daily (DFI) and residual feed intake (RFI). Uni- and bivariate analyses were conducted for combined genotypes and for individual genotypes. Genotype means were predicted for a subset of data involving 34 BRAH and 26 TCOMP sires. A meta-analysis of genetic correlation estimates examined how these were related to the difference between measurement environments for specific traits. There were genotype differences at the level of means, variances and genetic correlations. BRAH heifers were significantly (P < 0.05) faster-growing in the 'wet' season, slower-growing in the 'dry' season, lighter at ENDDRY, and taller and fatter with greater CS and IGF-I at both ENDWET and ENDDRY. Heritabilities were generally in the 20 to 60% range for both genotypes. Phenotypic and genetic variances, and genetic correlations, were commonly lower for BRAH. Differences were often explained by the long period of tropical adaptation of B. indicus. Genetic correlations were high between corresponding measures at ENDWET and ENDDRY, positive between fat and muscle measures in TCOMP but negative in BRAH (mean of 13 estimates 0.50 and -0.19, respectively), and approximately zero between steer feedlot ADG and heifer ADG in BRAH. Numerous genetic correlations between heifers and steers differed substantially from unity, especially in BRAH, suggesting there may be scope to select differently in the sexes where that would aid the differing roles of heifers and steers in production. Genetic correlations declined as measurement environments became more different, the rates of decline (environment sensitivity) sometimes differing with genotype. Similar measures (LWT, HH and ADG; IGF-I at ENDWET in TCOMP) were genetically correlated with steer DFI in heifers as in steers. Heifer SEMA was genetically correlated with steer feedlot RFI in BRAH (0.75 +/- 0.27 at ENDWET, 0.66 +/- 0.24 at ENDDRY). Selection to reduce steer RFI would reduce SEMA in BRAH heifers but otherwise have only small effects on heifers before their first joining.

An (O)over-tilde(mn) Gomory-Hu Tree Construction Algorithm for Unweighted Graphs

We present a fast algorithm for computing a Gomory-Hu tree or cut tree for an unweighted undirected graph G = (V, E). The expected running time of our algorithm is (O) over tilde (mc) where vertical bar E vertical bar = m and c is the maximum u-v edge connectivity, where u, v is an element of V. When the input graph is also simple (i.e., it has no parallel edges), then the u-v edge connectivity for each pair of vertices u and v is at most n - 1; so the expected run-ning time of our algorithm for simple unweighted graphs is (O) over tilde (mn). All the algorithms currently known for constructing a Gomory-Hu tree [8, 9] use n - 1 minimum s-t cut (i.e., max flow) subroutines. This in conjunction with the current fastest (O) over tilde (n(20/9)) max flow algorithm due to Karger and Levine[11] yields the current best running time of (O) over tilde (n(20/9)n) for Gomory-Hu tree construction on simple unweighted graphs with m edges and n vertices. Thus we present the first (O) over tilde (mn) algorithm for constructing a Gomory-Hu tree for simple unweighted graphs. We do not use a max flow subroutine here; we present an efficient tree packing algorithm for computing Steiner edge connectivity and use this algorithm as our main subroutine. The advantage in using a tree packing algorithm for constructing a Gomory-Hu tree is that the work done in computing a minimum Steiner cut for a Steiner set S subset of V can be reused for computing a minimum Steiner cut for certain Steiner sets S' subset of S.

Impacts of grazing management options on pasture and animal productivity in a Heteropogon contortus (black speargrass) pasture in central Queensland. 3. Diet composition in autumn

The diet selected in autumn by steers fistulated at the oesophageous was studied in a subset of treatments in an extensive grazing study conducted in a Heteropogon contortus pasture in central Queensland between 1988 and 2001. These treatments were a factorial array of three stocking rates (4, 3 and 2 ha/steer) and three pasture types (native pasture, legume-oversown native pasture and animal diet supplement/spring-burning native pasture). Seasonal rainfall throughout this study was below the long-term mean and mean annual pasture utilisation ranged from 30 to 61%. Steers consistently selected H. contortus with levels decreasing from 47 to 18% of the diet as stocking rate increased from 4 ha/steer to 2 ha/steer. Stylosanthes scabra cv. Seca was always selected in legume-oversown pastures with diet composition varying from 35 to 66% despite its plant density increasing from 7 to 65 plants/m(2) and pasture composition from 20 to 50%. Steers also selected a diet containing Chrysopogon fallax, forbs and sedges in higher proportions than they were present in the pasture. Greater availability of the intermediate grasses Chloris divaricata and Eragrostis spp. was associated with increased stocking rates. Bothriochloa bladhii was seldom selected in the diet, especially when other palatable species were present in the pasture, despite B. bladhii often being the major contributor to total pasture yield. It was concluded that a stocking rate of 4 ha/steer will maintain the availability of H. contortus in the pasture.

Impacts of grazing management options on pasture and animal productivity in a Heteropogon contortus (black speargrass) pasture in central Queensland. 2. Population dynamics of Heteropogon contortus and Stylosanthes scabra cv. Seca.

The dynamics of Heteropogon contortus and Stylosanthes scabra cv. Seca populations were studied in a subset of treatments in an extensive grazing study conducted in central Queensland between 1988 and 2001. These treatments were 4 stocking rates in native pasture and 2 of these stocking rates in legume oversown and supplement/spring burning treatments. For the 1999-2000 summer, population data for H. contortus in 5 of these native pasture and supplement/burning treatments were compared with those for an additional burnt treatment. Seasonal rainfall throughout this study was below the long-term mean and mean annual pasture utilisation ranged from 24 to 61%. Increasing stocking rate from 5 to 2 ha/steer in native pasture reduced H. contortus plant density. Increasing stocking rate reduced seedling recruitment as a result of its effect on soil seedbanks. Seedling recruitment was the major determinant of change in plant density, although some individual H. contortus plants did survive throughout the study. Burning in spring 1999, particularly at light stocking rate, promoted seedling recruitment above that in both unburnt native and legume oversown pasture and resulted in increased H. contortus plant density. In the legume oversown treatments, S. scabra cv. Seca density increased rapidly from 15 plants/m2 in 1988 to 140 plants/m2 in 2001 following a lag phase between 1988 and 1993. This increased S. scabra density was associated with an eventual decline in H. contortus plant density through reduced seedling recruitment. It was concluded that H. contortus population density is sustainable at stocking rates of 4 and 5 ha/steer (30% pasture utilisation) and that spring burning at light stocking rate can promote H. contortus populations. Increasing densities of S. scabra need to be managed to prevent its dominance.

To reactivate or not to reactivate : Control of KSHV lytic replication is essential for apoptosis in response to p53 restoration

Kaposi's sarcoma herpesvirus (KSHV) is an oncogenic human virus and the causative agent of three human malignancies: Kaposi's sarcoma (KS), Multicentric Castleman's Disease (MCD), and primary effusion lymphoma (PEL). In tumors, KSHV establishes latent infection during which it produces no infectious particles. Latently infected cells can enter the lytic replication cycle, and upon provision of appropriate cellular signals, produce progeny virus. PEL, commonly described in patients with AIDS, represents a diffuse large-cell non-Hodgkin's lymphoma, with median survival time less than six months after diagnosis. As tumor suppressor gene TP53 mutations occur rarely in PEL, the aim of this thesis was to investigate whether non-genotoxic activation of the p53 pathway can eradicate malignant PEL cells. This thesis demonstrates that Nutlin-3, a small-molecule inhibitor of the p53-MDM2 interaction, efficiently restored p53 function in PEL cells, leading to cell cycle arrest and massive apoptosis. Furthermore, we found that KSHV infection activated DNA damage signaling, rendering the cells more sensitive to p53-dependent cell death. We also showed in vivo the therapeutic potential of p53 restoration that led to regression of subcutaneous and intraperitoneal PEL tumor xenografts without adversely affecting normal cells. Importantly, we demonstrated that in a small subset of intraperitoneal PEL tumors, spontaneous induction of viral reactivation dramatically impaired Nutlin-3-induced p53-mediated apoptosis. Accordingly, we found that elevated KSHV lytic transcripts correlated with PEL tumor burden in animals and that inhibition of viral reactivation in vitro restored cytotoxic activity of a small-molecule inhibitor of the p53-MDM2 interaction. Latency provides a unique opportunity for KSHV to escape host immune surveillance and to establish persistent infections. However, to maintain viral reservoirs and spread to other hosts, KSHV must be reactivated from latency and enter into the lytic growth phase. We showed that phosphorylation of nucleolar phosphoprotein nucleophosmin (NPM) by viral cyclin-CDK6 is critical for establishment and maintenance of the KSHV latency. In short, this study provides evidence that the switch between latent phase and lytic replication is a critical step that determines the outcome of viral infection and the pathogenesis of KSHV-induced malignancies. Our data may thus contribute to development of novel targeted therapies for intervention and treatment of KSHV-associated cancers.

LKB1 tumor suppression in Peutz-Jeghers Syndrome

Kasvainten, ajatellaan syntyvän yksittäisen solun perimän mutaatioista, jonka seurauksena tuon solun kasvu häiriintyy. Ruoansulatuskanavan polyyppien syntyä käytetään usein mallina siitä, miten nämä epiteelisoluun kerääntyvät mutaatiot aiheuttavat asteittain pahenevan kasvuhäiriön. Peutz–Jeghersin oireyhtymä (PJS) on perinnöllinen polypoosisyndrooma, jossa oireita aiheuttavat erityisesti maha-suolikanavan hamartomatoottiset polyypit. Noin puolella PJS potilaista havaitaan mutaatioita LKB1 kasvunrajoite geenissä. Hiirille joilta toinen Lkb1 alleeli on poistettu (Lkb1+/-) kehittyy PJS-tyypin maha-suolikanavan polyyppeja, joissa on epiteelin liikakasvun lisäksi merkittävä sileälihaskomponentti, aivan kuten PJS polyypeissa. Kuten myös muissa ruoansulatuskanavan polypooseissa, sekä PJS että hiirten polyypeissa Cyclo-oxygenaasi-2:n (COX-2) määrä on usein kohonnut. PJS-polyyppien kehittymisen molekulaarinen mekanismi on kuitenkin selvittämättä. Koska vain osa PJS potilaista kantaa LKB1 mutaatioita, mutaatiot jossakin toisessa lokuksessa saattaisivat selittää osan PJS tapauksista. Jotta PJS:n geneettinen tausta selviäisi, seulottiin kolmen LKB1:n kanssa interaktoivan proteiinin (BRG1, STRADα ja MO25α) geenit PJS potilaista joilla ei ole havaittu LKB1 mutaatioita. Yhdessäkään tutkituista geeneistä ei havaittu tautia aiheuttavia mutaatioita. Näiden kolmen geenin pois sulkeminen, ja uusien menetelmien ansiosta kasvanut havaittujen Lkb1 mutaatioden määrä viittaavat LKB1:n olevan useimpien PJS tapausten taustalla. COX-2:n estäjien käyttö on tehokkaasti vähentänyt polyyppien määrää familiaarisessa adenomatoottisessa polypoosissa. Tästä johtuen COX-2:n eston tehokkuutta tutkittiin PJS polypoosissa. PJS-tyypin polypoosin havaittin pienenevän merkittävästi Lkb1+/- hiirissä, joilta oli lisäksi poistettu toinen tai molemmat COX-2:n alleeleista. Lisäksi farmakologinen COX-2:n esto Celecoxib:lla vähensi polypoosia tehokkaasti. Näin ollen COX-2:n eston tehokkuutta tutkittiin seuraavaksi PJS potilaissa. Kuuden kuukauden Celecoxib hoidon jälkeen polypoosin havaittiin vähentyneen merkittävästi osalla potilaista (2/6). Nämä tulokset osoittavat COX-2:n roolin PJS-polyyppien kehityksessä, ja viittaavat COX-2:n eston vähentävän polypoosia. Kasvunrajoitegeenin klassisen määritelmän mukaan kasvaimen kehitys vaatii perinnöllisen mutaation lisäksi geenin toisenkin alleelin mutaation, mutta PJS-polyyppien häiriintyneestä epiteelistä ei kuitenkaan systemaattisesti löydy toista LKB1:n mutaatiota. Havainto johti tutkimukseen, jossa selvitettiin voisiko LKB1:n kasvun rajoitus välittyäkin epäsuorasti tukikudokseksi ajatelluista sileälihassoluista. Tätä tutkittiin kehittämällä poistogeeninen hiirimalli jossa Lkb1 on mutatoitunut vain sileälihassoluissa. Näille hiirille kehittyi polyyppeja, jotka ovat kaikin tavoin PJS-polyyppien kaltaisia. Lkb1:n menettäneiden solujen havaittiin tuottavan vähemmän transformoivaa kasvutekijä beetaa (TGFß), joka aiheutti solujen välisen viestinnän heikentymisen ja mahdollisesti viereisten epiteelisolujen liikakasvun. Vastaava häiriö havaittiin myös PJS-potilaiden polyypeissa, mikä viittaa siihen, että potilaillakin sileälihassolujen häiriö on polyyppien taustalla. Havainto suuntaa täten hoitokohteiden etsintää ja osoittaa että LKB1 toimii kasvunrajoittajana epätyypillisellä tavalla pitäen naapurisolujen kasvun kurissa.

Meta-analysis of 375,000 individuals identifies 38 susceptibility loci for migraine

Migraine is a debilitating neurological disorder affecting around 1 in 7 people worldwide, but its molecular mechanisms remain poorly understood. Some debate exists over whether migraine is a disease of vascular dysfunction, or a result of neuronal dysfunction with secondary vascular changes. Genome-wide association (GWA) studies have thus far identified 13 independent loci associated with migraine. To identify new susceptibility loci, we performed the largest genetic study of migraine to date, comprising 59,674 cases and 316,078 controls from 22 GWA studies. We identified 45 independent single nucleotide polymorphisms (SNPs) significantly associated with migraine risk (P < 5 x 10-8) that map to 38 distinct genomic loci, including 28 loci not previously reported and the first locus identified on chromosome X. Furthermore, a subset analysis for migraine without aura (MO) identified seven of the same loci as from the full sample, whereas no loci reached genome-wide significance in the migraine with aura (MA) subset. In subsequent computational analyzes, the identified loci showed enrichment for genes expressed in vascular and smooth muscle tissues, consistent with a predominant theory of migraine that highlights vascular etiologies.

No evidence for genetic association with the let-7 microRNA-binding site or other common KRAS variants in risk of endometriosis

STUDY QUESTION Is there a contribution of the minor allele at the KRAS single nucleotide polymorphism (SNP) rs61764370 in the let-7 microRNA-binding site to endometriosis risk? SUMMARY ANSWER We found no evidence for association between endometriosis risk and rs61764370 or any other SNPs in KRAS. WHAT IS KNOWN ALREADY The rs61764370 SNP in the 3' untranslated region of the KRAS gene is predicted to disrupt a complementary binding site (LCS6) for the let-7 microRNA, and was recently reported to be at a high frequency (31%) in 132 women of varying ancestry with endometriosis compared with frequencies in a database of population controls (up to 7.6% depending on ancestry), suggesting a strong effect of this KRAS SNP in the aetiology of endometriosis. STUDY DESIGN, SIZE AND DURATION This was a case-control study with a total of 11 206 subjects. The study was performed between February 2012 and July 2012. PARTICIPANTS/MATERIALS, SETTINGAND METHODS We first investigated a possible association between common markers in KRAS and endometriosis risk from our genome-wide association (GWA) data in 3194 surgically confirmed endometriosis cases and 7060 controls of European ancestry. Although rs61764370 was not genotyped on the GWA arrays, five SNPs typed in the study were highly correlated with this variant. The rs61764370 and two SNPs highly correlated with rs61764370 were then genotyped in 933 endometriosis cases and 952 controls using the Sequenom MassARRAY platform. MAIN RESULTS AND THE ROLE OF CHANCE There was no evidence for an association between rs61764370 and endometriosis risk P = 0.411 and odds ratio = 1.10 (95% confidence intervals: 0.88-1.36). We also found no evidence for an association between the highly correlated SNP rs17387019 and endometriosis. Their minor allele frequencies in cases and controls were of 0.087-0.091 similar to the population frequency reported previously for this variant in controls. Analyses of endometriosis cases with revised American Fertility Society stage III/IV disease also showed no evidence for an association between these SNPs and endometriosis risk. LIMITATIONS AND REASONS FOR CAUTION The GWA and genotyped data sets were not independent since individuals and cases from some families overlap. Controls in our GWA study were not screened for endometriosis. WIDER IMPLICATIONS OF THE FINDINGS The key SNP, rs61764370, was genotyped in a subset of samples. Our results do not support the suggestion that carrying the minor allele at rs61764370 contributes to a significant number of endometriosis cases and rs61764370 is, therefore, unlikely to be a useful marker of endometriosis risk. STUDY FUNDING/COMPETING INTEREST(S) The research was funded by grants from the Australian National Health and Medical Research Council and Wellcome Trust. None of the authors has competing interests for the study.

Identification of two novel human neuronal ceroid lipofuscinosis genes

The neuronal ceroid lipofuscinoses (NCLs) are a group of mostly autosomal recessively inherited neurodegenerative disorders. The aim of this thesis was to characterize the molecular genetic bases of these, previously genetically undetermined, NCL forms. Congenital NCL is the most aggressive form of NCLs. Previously, a mutation in the cathepsin D (CTSD) gene was shown to cause congenital NCL in sheep. Based on the close resemblance of the phenotypes between congenital NCLs in sheep and human, CTSD was considered as a potential candidate gene in humans as well. When screened for mutations by sequencing, a homozygous nucleotide duplication creating a premature stop codon was identified in CTSD in one family with congenital NCL. While in vitro the overexpressed truncated mutant protein was stable although inactive, the absence of CTSD staining in brain tissue samples of patients indicated degradation of the mutant CTSD in vivo. A lack of CTSD staining was detected also in another, unrelated family with congenital NCL. These results imply that CTSD deficiency underlies congenital NCL. While initially Turkish vLINCL was considered a distinct genetic entity (CLN7), mutations in the CLN8 gene were later reported to account for the disease in a subset of Turkish patients with vLINCL. To further dissect the genetic basis of the disease, all known NCL genes were screened for homozygosity by haplotype analysis of microsatellite markers and/or sequenced in 13 mainly consanguineous, Turkish vLINCL families. Two novel, family-specific homozygous mutations were identified in the CLN6 gene. In the remaining families, all known NCL loci were excluded. To identify novel gene(s) underlying vLINCL, a genomewide single nucleotide polymorphism scan, homozygosity mapping, and positional candidate gene sequencing were performed in ten of these families. On chromosome 4q28.1-q28.2, a novel major facilitator superfamily domain containing 8 (MFSD8) gene with six family-specific homozygous mutations in vLINCL patients was identified. MFSD8 transcript was shown to be ubiquitously expressed with a complex pattern of alternative splicing. Our results suggest that MFSD8 is a novel lysosomal integral membrane protein which, as a member of the major facilitator superfamily, is predicted to function as a transporter. Identification of MFSD8 emphasizes the genetic heterogeneity of Turkish vLINCL. In families where no MFSD8 mutations were detected, additional NCL-causing genes remain to be identified. The identification of CTSD and MFSD8 increases the number of known human NCL-causing genes to eight, and is an important step towards the complete understanding of the genetic spectrum underlying NCLs. In addition, it is a starting point for dissecting the molecular mechanisms behind the associated NCLs and contributes to the challenging task of understanding the molecular pathology underlying the group of NCL disorders.

Implementation of CSP-S for description of distributed algorithms

The implementation of CSP-S (a subset of CSP)—a high level language for distributed programming—is presented in this paper. The language CSP-S features a parallel command, communication by message passing and the use of guarded command. The implementation consists of a compiler translating the CSP-S constructs into intermediate language. The execution is carried out by a scheduler which creates an illusion of concurrency. Using the CSP-S language constructs, distributed algorithms are written, executed and tested with the compiler designed.

A conditionally linearized Monte Carlo filter in non-linear structural dynamics

State and parameter estimations of non-linear dynamical systems, based on incomplete and noisy measurements, are considered using Monte Carlo simulations. Given the measurements. the proposed method obtains the marginalized posterior distribution of an appropriately chosen (ideally small) subset of the state vector using a particle filter. Samples (particles) of the marginalized states are then used to construct a family of conditionally linearized system of equations and thus obtain the posterior distribution of the states using a bank of Kalman filters. Discrete process equations for the marginalized states are derived through truncated Ito-Taylor expansions. Increased analyticity and reduced dispersion of weights computed over a smaller sample space of marginalized states are the key features of the filter that help achieve smaller sample variance of the estimates. Numerical illustrations are provided for state/parameter estimations of a Duffing oscillator and a 3-DOF non-linear oscillator. Performance of the filter in parameter estimation is also assessed using measurements obtained through experiments on simple models in the laboratory. Despite an added computational cost, the results verify that the proposed filter generally produces estimates with lower sample variance over the standard sequential importance sampling (SIS) filter.

Development of regulatory T cells in the human thymus

The role of the immune system is to protect an organism against pathogens while maintaining tolerance against self. T cells are an essential component of the immune system and they develop in the thymus. The AIRE (autoimmune regulator) gene product plays an important role in T cell development, as it promotes expression of peripheral tissue antigens in the thymus. Developing T cells, thymocytes, which recognize self-antigens with high affinity are deleted. However, this deletion process is not perfect and not all autoreactive T cells are destroyed. When the distinction between self and non-self fails, tolerance breaks and the immune system attacks the host s own tissues. This results in autoimmunity. Regulatory T cells contribute to the maintenance of self-tolerance. They can actively suppress the function of autoreactive cells. Several populations of cells with regulatory properties have been described, but the best characterized population is the natural regulatory T cells (Treg cells), which develop in the thymus and express the transcription factor FOXP3. The thymic development of Treg cells in humans is the subject of this thesis. Thymocytes at different developmental stages were analyzed using flow cytometry. The CD4-CD8- double-negative (DN) thymocytes are the earliest T cell precursors in the T cell lineage. My results show that the Treg cell marker FOXP3 is up-regulated already in a subset of these DN thymocytes. FOXP3+ cells were also found among the more mature CD4+CD8+ double-positive (DP) cells and among the CD4+ and CD8+ single-positive (SP) thymocytes. The different developmental stages of the FOXP3+ thymocytes were isolated and their gene expression examined by quantitative PCR. T cell receptor (TCR) repertoire analysis was used to compare these different thymocyte populations. My data show that in humans commitment to the Treg cell lineage is an early event and suggest that the development of Treg cells follows a linear developmental pathway, FOXP3+ DN precursors evolving through the DP stage to become mature CD4+ Treg cells. Most T cells have only one kind of TCR on their cell surface, but a small fraction of cells expresses two different TCRs. My results show that the expression of two different TCRs is enriched among Treg cells. Furthermore, both receptors were capable of transmitting signals when bound by a ligand. By extrapolating flow cytometric data, it was estimated that the majority of peripheral blood Treg cells are indeed dual-specific. The high frequency of dual-specific cells among human Treg cells suggests that dual-specificity has a role in directing these cells to the Treg cell lineage. It is known that both genetic predisposition and environmental factors influence the development of autoimmunity. It is also known that the dysfunction or absence of Treg cells leads to the development of autoimmune manifestations. APECED (autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy) is a rare monogenic autoimmune disease, caused by mutations in the AIRE gene. In the absence of AIRE gene product, deletion of self-specific T cells is presumably disturbed and autoreactive T cells escape to the periphery. I examined whether Treg cells are also affected in APECED. I found that the frequency of FOXP3+ Treg cells and the level of FOXP3 expression were significantly lower in APECED patients than in controls. Additionally, when studied in cell cultures, the suppressive capacity of the patients' Treg cells was impaired. Additionally, repertoire analysis showed that the TCR repertoire of Treg cells was altered. These results suggest that AIRE contributes to the development of Treg cells in humans and the selection of Treg cells is impaired in APECED patients. In conclusion, my thesis elucidates the developmental pathway of Treg cells in humans. The differentiation of Tregs begins early during thymic development and both the cells dual-specificity and AIRE probably affect the final commitment of Treg cells.

Genetic Basis of Pituitary Adenoma Predisposition

Much of the global cancer research is focused on the most prevalent tumors; yet, less common tumor types warrant investigation, since A rare disorder is not necessarily an unimportant one . The present work discusses a rare tumor type, the benign adenomas of the pituitary gland, and presents the advances which, during the course of this thesis work, contributed to the elucidation of a fraction of their genetic background. Pituitary adenomas are benign neoplasms of the anterior pituitary lobe, accounting for approximately 15% of all intracranial tumors. Pituitary adenoma cells hypersecrete the hormones normally produced by the anterior pituitary tissue, such as growth hormone (GH) and prolactin (PRL). Despite their non-metastasizing nature, these adenomas can cause significant morbidity and have to be adequately treated; otherwise, they can compromise the patient s quality of life, due to conditions provoked by hormonal hypersecretion, such as acromegaly in the case of GH-secreting adenomas, or due to compressive effects to surrounding tissues. The vast majority of pituitary adenomas arise sporadically, whereas a small subset occur as component of familial endocrine-related tumor syndromes, such as Multiple Endocrine Neoplasia type 1 (MEN1) and Carney complex (CNC). MEN1 is caused by germline mutations in the MEN1 tumor suppressor gene (11q13), whereas the majority of CNC cases carry germline mutations in the PRKAR1A gene (17q24). Pituitary adenomas are also encountered in familial settings outside the context of MEN1 and CNC, but unlike in the latter syndromes, their genetic background until recently remained elusive. Evidence in previous literature supported the notion that a tumor suppressor gene on 11q13, residing very close to but still distinct from MEN1, causes genetic susceptibility to pituitary tumors. The aim of the study was to identify the genetic cause of a low penetrance form of Pituitary Adenoma Predisposition (PAP) in families from Northern Finland. The present work describes the methodological approach that led to the identification of aryl hydrocarbon receptor interacting protein (AIP) as the gene causing PAP. Combining chip-based technologies (SNP and gene expression arrays) with traditional gene mapping methods and genealogy data, we showed that germline AIP mutations cause PAP in familial and sporadic settings. PAP patients were diagnosed with mostly adenomas of the GH/PRL-secreting cell lineage. In Finland, two AIP mutations accounted for 16% of all patients diagnosed with GH-secreting adenomas, and for 40% of patients being younger than 35 years of age at diagnosis. AIP is suggested to act as a tumor suppressor gene, a notion supported by the nature of the identified mutations (most are truncating) and the biallelic inactivation of AIP in the tumors studied. AIP has been best characterized as a cytoplasmic interaction partner of aryl hydrocarbon receptor (AHR), also known as dioxin receptor, but it has other partners as well. The mechanisms that underlie AIP-mediated pituitary tumorigenesis are to date largely unknown and warrant further investigation. Because AIP was identified in the genetically homogeneous Finnish population, it was relevant to examine its contribution to PAP in other, more heterogeneous, populations. Analysis of pituitary adenoma patient series of various ethnic origins and differing clinical settings revealed germline AIP mutations in all cohorts studied, albeit with low frequencies (range 0.8-7.4%). Overall, PAP patients were typically diagnosed at a young age (range 8-41 years), mainly with GH-secreting adenomas, without strong family history of endocrine disease. Because many PAP patients did not display family history of pituitary adenomas, detection of the condition appeared challenging. AIP immunohistochemistry was tested as a molecular pre-screening tool on mutation-positive versus mutation-negative tumors, and proved to be a potentially useful predictor of PAP. Mutation screening of a large cohort of colorectal, breast, and prostate tumors did not reveal somatic AIP mutations. These tumors, apart from being the most prevalent among men and women worldwide, have been associated with acromegaly, particularly colorectal neoplasia. In this material, AIP did not appear to contribute to the pathogenesis of these common tumor types and other genes seem likely to play a role in such tumorigenesis. Finally, the contribution of AIP in pediatric onset pituitary adenomas was examined in a unique population-based cohort of sporadic pituitary adenoma patients from Italy. Germline AIP mutations may account for a subset of pediatric onset GH-secreting adenomas (in this study one of seven GH-secreting adenoma cases or 14.3%), and appear to be enriched among young (≤25 years old) patients. In summary, this work reveals a novel tumor susceptibility gene, namely AIP, which causes genetic predisposition to pituitary adenomas, in particular GH-secreting adenomas. Moreover, it provides molecular tools for identification of individuals predisposed for PAP. Further elaborate studies addressing the functional role of AIP in normal and tumor cells will hopefully expand our knowledge on endocrine neoplasia and reveal novel cellular mechanisms of pituitary tumorigenesis, including potential drug targets.