Gene expression analysis in ‘Candidatus Phytoplasma mali’-resistant and -susceptible Malus genotypes

Apple proliferation (AP) disease is the most important graft-transmissible and vector-borne disease of apple in Europe. ‘Candidatus Phytoplasma mali’ (Ca. P. mali) is the causal agent of AP. Apple (Malus x domestica) and other Malus species are the only known woody hosts. In European apple orchards, the cultivars are mainly grafted on one rootstock, M. x domestica cv. M9. M9 like all other M. x domestica cultivars is susceptible to ‘Ca. P. mali’. Resistance to AP was found in the wild genotype Malus sieboldii (MS) and in MS-derived hybrids but they were characterised by poor agronomic value. The breeding of a new rootstock carrying the resistant and the agronomic traits was the major aim of a project of which this work is a part. The objective was to shed light into the unknown resistance mechanism. The plant-phytoplasma interaction was studied by analysing differences between the ‘Ca. P. mali’-resistant and -susceptible genotypes related to constitutively expressed genes or to induced genes during infection. The cDNA-Amplified Fragment Length Polymorphism (cDNA-AFLP) technique was employed in both approaches. Differences related to constitutively expressed genes were identified between two ‘Ca. P. mali’-resistant hybrid genotypes (4551 and H0909) and the ‘Ca. P. mali’-susceptible M9. 232 cDNA-AFLP bands present in the two resistant genotypes but absent in the susceptible one were isolated but several different products associated to each band were found. Therefore, two different macroarray hybridisation experiments were performed with the cDNA-AFLP fragments yielding 40 sequences encoding for genes of unknown function or a wide array of functions including plant defence. In the second approach, individuation and analysis of the induced genes was carried out exploiting an in vitro system in which healthy and ‘Ca. P. mali’-infected micropropagated plants were maintained under controlled conditions. Infection trials using in vitro grafting of ‘Ca. P. mali’ showed that the resistance phenotype could be reproduced in this system. In addition, ex vitro plants were generated as an independent control of the genes differentially expressed in the in vitro plants. The cDNA-AFLP analysis in in vitro plants yielded 63 bands characterised by over-expression in the infected state of both the H0909 and MS genotypes. The major part (37 %) of the associated sequences showed homology with products of unknown function. The other genes were involved in plant defence, energy transport/oxidative stress response, protein metabolism and cellular growth. Real-time qPCR analysis was employed to validate the differential expression of the genes individuated in the cDNA-AFLP analysis. Since no internal controls were available for the study of the gene expression in Malus, an analysis on housekeeping genes was performed. The most stably expressed genes were the elongation factor-1 α (EF1) and the eukaryotic translation initiation factor 4-A (eIF4A). Twelve out of 20 genes investigated through qPCR were significantly differentially expressed in at least one genotype either in in vitro plants or in ex vitro plants. Overall, about 20% of the genes confirmed their cDNA-AFLP expression pattern in M. sieboldii or H0909. On the contrary, 30 % of the genes showed down-regulation or were not differentially expressed. For the remaining 50 % of the genes a contrasting behaviour was observed. The qPCR data could be interpreted as follows: the phytoplasma infection unbalance photosynthetic activity and photorespiration down-regulating genes involved in photosynthesis and in the electron transfer chain. As result, and in contrast to M. x domestica genotypes, an up-regulation of genes of the general response against pathogens was found in MS. These genes involved the pathway of H2O2 and the production of secondary metabolites leading to the hypothesis that a response based on the accumulation of H2O2 in MS would be at the base of its resistance. This resembles a phenomenon known as “recovery” where the spontaneous remission of the symptoms is observed in old susceptible plants but occurring in a stochastic way while the resistance in MS is an inducible but stable feature. As additional product of this work three cDNA-AFLP-derived markers were developed which showed independent distribution among the seedlings of two breeding progenies and were associated to a genomic region characteristic of MS. These markers will contribute to the development of molecular markers for the resistance as well as to map the resistance on the Malus genome.

Statistical analysis of the error associated with the simplification of the stratigraphy in geotechnical models

The uncertainties in the determination of the stratigraphic profile of natural soils is one of the main problems in geotechnics, in particular for landslide characterization and modeling. The study deals with a new approach in geotechnical modeling which relays on a stochastic generation of different soil layers distributions, following a boolean logic – the method has been thus called BoSG (Boolean Stochastic Generation). In this way, it is possible to randomize the presence of a specific material interdigitated in a uniform matrix. In the building of a geotechnical model it is generally common to discard some stratigraphic data in order to simplify the model itself, assuming that the significance of the results of the modeling procedure would not be affected. With the proposed technique it is possible to quantify the error associated with this simplification. Moreover, it could be used to determine the most significant zones where eventual further investigations and surveys would be more effective to build the geotechnical model of the slope. The commercial software FLAC was used for the 2D and 3D geotechnical model. The distribution of the materials was randomized through a specifically coded MatLab program that automatically generates text files, each of them representing a specific soil configuration. Besides, a routine was designed to automate the computation of FLAC with the different data files in order to maximize the sample number. The methodology is applied with reference to a simplified slope in 2D, a simplified slope in 3D and an actual landslide, namely the Mortisa mudslide (Cortina d’Ampezzo, BL, Italy). However, it could be extended to numerous different cases, especially for hydrogeological analysis and landslide stability assessment, in different geological and geomorphological contexts.

Stochastic foundations of dynamic trade and labor market models

This dissertation consists of three self-contained papers that are related to two main topics. In particular, the first and third studies focus on labor market modeling, whereas the second essay presents a dynamic international trade setup.rnrnIn Chapter "Expenses on Labor Market Reforms during Transitional Dynamics", we investigate the arising costs of a potential labor market reform from a government point of view. To analyze various effects of unemployment benefits system changes, this chapter develops a dynamic model with heterogeneous employed and unemployed workers.rn rnIn Chapter "Endogenous Markup Distributions", we study how markup distributions adjust when a closed economy opens up. In order to perform this analysis, we first present a closed-economy general-equilibrium industry dynamics model, where firms enter and exit markets, and then extend our analysis to the open-economy case.rn rnIn Chapter "Unemployment in the OECD - Pure Chance or Institutions?", we examine effects of aggregate shocks on the distribution of the unemployment rates in OECD member countries.rn rnIn all three chapters we model systems that behave randomly and operate on stochastic processes. We therefore exploit stochastic calculus that establishes clear methodological links between the chapters.

Uncovering the dynamics of cardiac systems using stochastic pacing and frequency domain analyses

Alternans of cardiac action potential duration (APD) is a well-known arrhythmogenic mechanism which results from dynamical instabilities. The propensity to alternans is classically investigated by examining APD restitution and by deriving APD restitution slopes as predictive markers. However, experiments have shown that such markers are not always accurate for the prediction of alternans. Using a mathematical ventricular cell model known to exhibit unstable dynamics of both membrane potential and Ca2+ cycling, we demonstrate that an accurate marker can be obtained by pacing at cycle lengths (CLs) varying randomly around a basic CL (BCL) and by evaluating the transfer function between the time series of CLs and APDs using an autoregressive-moving-average (ARMA) model. The first pole of this transfer function corresponds to the eigenvalue (λalt) of the dominant eigenmode of the cardiac system, which predicts that alternans occurs when λalt≤−1. For different BCLs, control values of λalt were obtained using eigenmode analysis and compared to the first pole of the transfer function estimated using ARMA model fitting in simulations of random pacing protocols. In all versions of the cell model, this pole provided an accurate estimation of λalt. Furthermore, during slow ramp decreases of BCL or simulated drug application, this approach predicted the onset of alternans by extrapolating the time course of the estimated λalt. In conclusion, stochastic pacing and ARMA model identification represents a novel approach to predict alternans without making any assumptions about its ionic mechanisms. It should therefore be applicable experimentally for any type of myocardial cell.

Markers Models in Survival Analysis and Applications to Issues Associated with AIDS

Jewell and Kalbfleisch (1992) consider the use of marker processes for applications related to estimation of the survival distribution of time to failure. Marker processes were assumed to be stochastic processes that, at a given point in time, provide information about the current hazard and consequently on the remaining time to failure. Particular attention was paid to calculations based on a simple additive model for the relationship between the hazard function at time t and the history of the marker process up until time t. Specific applications to the analysis of AIDS data included the use of markers as surrogate responses for onset of AIDS with censored data and as predictors of the time elapsed since infection in prevalent individuals. Here we review recent work on the use of marker data to tackle these kinds of problems with AIDS data. The Poisson marker process with an additive model, introduced in Jewell and Kalbfleisch (1992) may be a useful "test" example for comparison of various procedures.

On Time Series Analysis of Public Health and Biomedical Data

A time series is a sequence of observations made over time. Examples in public health include daily ozone concentrations, weekly admissions to an emergency department or annual expenditures on health care in the United States. Time series models are used to describe the dependence of the response at each time on predictor variables including covariates and possibly previous values in the series. Time series methods are necessary to account for the correlation among repeated responses over time. This paper gives an overview of time series ideas and methods used in public health research.

Risk of severe asthma episodes predicted from fluctuation analysis of airway function

Asthma is an increasing health problem worldwide, but the long-term temporal pattern of clinical symptoms is not understood and predicting asthma episodes is not generally possible. We analyse the time series of peak expiratory flows, a standard measurement of airway function that has been assessed twice daily in a large asthmatic population during a long-term crossover clinical trial. Here we introduce an approach to predict the risk of worsening airflow obstruction by calculating the conditional probability that, given the current airway condition, a severe obstruction will occur within 30 days. We find that, compared with a placebo, a regular long-acting bronchodilator (salmeterol) that is widely used to improve asthma control decreases the risk of airway obstruction. Unexpectedly, however, a regular short-acting beta2-agonist bronchodilator (albuterol) increases this risk. Furthermore, we find that the time series of peak expiratory flows show long-range correlations that change significantly with disease severity, approaching a random process with increased variability in the most severe cases. Using a nonlinear stochastic model, we show that both the increased variability and the loss of correlations augment the risk of unstable airway function. The characterization of fluctuations in airway function provides a quantitative basis for objective risk prediction of asthma episodes and for evaluating the effectiveness of therapy.

Acute effects of stochastic resonance whole body vibration

AIM: To investigate the acute effects of stochastic resonance whole body vibration (SR-WBV) training to identify possible explanations for preventive effects against musculoskeletal disorders. METHODS: Twenty-three healthy, female students participated in this quasi-experimental pilot study. Acute physiological and psychological effects of SR-WBV training were examined using electromyography of descending trapezius (TD) muscle, heart rate variability (HRV), different skin parameters (temperature, redness and blood flow) and self-report questionnaires. All subjects conducted a sham SR-WBV training at a low intensity (2 Hz with noise level 0) and a verum SR-WBV training at a higher intensity (6 Hz with noise level 4). They were tested before, during and after the training. Conclusions were drawn on the basis of analysis of variance. RESULTS: Twenty-three healthy, female students participated in this study (age = 22.4 ± 2.1 years; body mass index = 21.6 ± 2.2 kg/m2). Muscular activity of the TD and energy expenditure rose during verum SR-WBV compared to baseline and sham SR-WBV (all P < 0.05). Muscular relaxation after verum SR-WBV was higher than at baseline and after sham SR-WBV (all P < 0.05). During verum SR-WBV the levels of HRV were similar to those observed during sham SR-WBV. The same applies for most of the skin characteristics, while microcirculation of the skin of the middle back was higher during verum compared to sham SR-WBV (P < 0.001). Skin redness showed significant changes over the three measurement points only in the middle back area (P = 0.022). There was a significant rise from baseline to verum SR-WBV (0.86 ± 0.25 perfusion units; P = 0.008). The self-reported chronic pain grade indicators of pain, stiffness, well-being, and muscle relaxation showed a mixed pattern across conditions. Muscle and joint stiffness (P = 0.018) and muscular relaxation did significantly change from baseline to different conditions of SR-WBV (P < 0.001). Moreover, muscle relaxation after verum SR-WBV was higher than after sham SR-WBV (P < 0.05). CONCLUSION: Verum SR-WBV stimulated musculoskeletal activity in young healthy individuals while cardiovascular activation was low. Training of musculoskeletal capacity and immediate increase in musculoskeletal relaxation are potential mediators of pain reduction in preventive trials.

Economic efficiency analysis of different strategies to control post-weaning multi-systemic wasting syndrome and porcine circovirus type 2 subclinical infection in 3-weekly batch system farms.

The study assessed the economic efficiency of different strategies for the control of post-weaning multi-systemic wasting syndrome (PMWS) and porcine circovirus type 2 subclinical infection (PCV2SI), which have a major economic impact on the pig farming industry worldwide. The control strategies investigated consisted on the combination of up to 5 different control measures. The control measures considered were: (1) PCV2 vaccination of piglets (vac); (2) ensuring age adjusted diet for growers (diets); (3) reduction of stocking density (stock); (4) improvement of biosecurity measures (bios); and (5) total depopulation and repopulation of the farm for the elimination of other major pathogens (DPRP). A model was developed to simulate 5 years production of a pig farm with a 3-weekly batch system and with 100 sows. A PMWS/PCV2SI disease and economic model, based on PMWS severity scores, was linked to the production model in order to assess disease losses. This PMWS severity scores depends on the combination post-weaning mortality, PMWS morbidity in younger pigs and proportion of PCV2 infected pigs observed on farms. The economic analysis investigated eleven different farm scenarios, depending on the number of risk factors present before the intervention. For each strategy, an investment appraisal assessed the extra costs and benefits of reducing a given PMWS severity score to the average score of a slightly affected farm. The net present value obtained for each strategy was then multiplied by the corresponding probability of success to obtain an expected value. A stochastic simulation was performed to account for uncertainty and variability. For moderately affected farms PCV2 vaccination alone was the most cost-efficient strategy, but for highly affected farms it was either PCV2 vaccination alone or in combination with biosecurity measures, with the marginal profitability between 'vac' and 'vac+bios' being small. Other strategies such as 'diets', 'vac+diets' and 'bios+diets' were frequently identified as the second or third best strategy. The mean expected values of the best strategy for a moderately and a highly affected farm were £14,739 and £57,648 after 5 years, respectively. This is the first study to compare economic efficiency of control strategies for PMWS and PCV2SI. The results demonstrate the economic value of PCV2 vaccination, and highlight that on highly affected farms biosecurity measures are required to achieve optimal profitability. The model developed has potential as a farm-level decision support tool for the control of this economically important syndrome.

A review of dendrogeomorphological research applied to flood risk analysis in Spain

Over the last forty years, applying dendrogeomorphology to palaeoflood analysis has improved estimates of the frequency and magnitude of past floods worldwide. This paper reviews the main results obtained by applying dendrogeomorphology to flood research in several case studies in Central Spain. These dendrogeomorphological studies focused on the following topics: (1) anatomical analysis to understand the physiological response of trees to flood damage and improve sampling efficiency; (2) compiling robust flood chronologies in ungauged mountain streams, (3) determining flow depth and estimating flood discharge using two-dimensional hydraulic modelling, and comparing them with other palaeostage indicators; (4) calibrating hydraulic model parameters (i.e. Manning roughness); and (5) implementing stochastic-based, cost–benefit analysis to select optimal mitigation measures. The progress made in these areas is presented with suggestions for further research to improve the applicability of dendrogeochronology to palaeoflood studies. Further developments will include new methods for better identification of the causes of specific types of flood damage to trees (e.g. tilted trees) or stable isotope analysis of tree rings to identify the climatic conditions associated with periods of increasing flood magnitude or frequency.

Electromyographic activity of back muscles during stochastic whole body vibration

OBJECTIVES: Stochastic resonance whole body vibrations (SR-WBV) may reduce and prevent musculoskeletal problems (MSP). The aim of this study was to evaluate how activities of the lumbar erector spinae (ES) and of the ascending and descending trapezius (TA, TD) change in upright standing position during SR-WBV. METHODS: Nineteen female subjects completed 12 series of 10 seconds of SR-WBV at six different frequencies (2, 4, 6, 8, 10, 12Hz) and two types of "noise"-applications. An assessment at rest had been executed beforehand. Muscle activities were measured with EMG and normalized to the maximum voluntary contraction (MVC%). For statistical testing a three-factorial analysis of variation (ANOVA) was applied. RESULTS: The maximum activity of the respective muscles was 14.5 MVC% for the ES, 4.6 MVC% for the TA (12Hz with "noise" both), and 7.4 MVC% for the TD (10Hz without "noise"). Furthermore, all muscles varied significantly at 6Hz and above (p⋜0.047) compared to the situation at rest. No significant differences were found at SR-WBV with or without "noise". CONCLUSIONS: In general, muscle activity during SR-WBV is reasonably low and comparable to core strength stability exercises, sensorimotor training and "abdominal hollowing" in water. SR-WBV may be a therapeutic option for the relief of MSP.

Interim analysis of clinical trials: Simulation studies of fractional Brownian motion

Interim clinical trial monitoring procedures were motivated by ethical and economic considerations. Classical Brownian motion (Bm) techniques for statistical monitoring of clinical trials were widely used. Conditional power argument and α-spending function based boundary crossing probabilities are popular statistical hypothesis testing procedures under the assumption of Brownian motion. However, it is not rare that the assumptions of Brownian motion are only partially met for trial data. Therefore, I used a more generalized form of stochastic process, called fractional Brownian motion (fBm), to model the test statistics. Fractional Brownian motion does not hold Markov property and future observations depend not only on the present observations but also on the past ones. In this dissertation, we simulated a wide range of fBm data, e.g., H = 0.5 (that is, classical Bm) vs. 0.5< H <1, with treatment effects vs. without treatment effects. Then the performance of conditional power and boundary-crossing based interim analyses were compared by assuming that the data follow Bm or fBm. Our simulation study suggested that the conditional power or boundaries under fBm assumptions are generally higher than those under Bm assumptions when H > 0.5 and also matches better with the empirical results. ^

Functional data analysis approaches for genotype-phenotype association studies from next-generation sequencing

Next-generation DNA sequencing platforms can effectively detect the entire spectrum of genomic variation and is emerging to be a major tool for systematic exploration of the universe of variants and interactions in the entire genome. However, the data produced by next-generation sequencing technologies will suffer from three basic problems: sequence errors, assembly errors, and missing data. Current statistical methods for genetic analysis are well suited for detecting the association of common variants, but are less suitable to rare variants. This raises great challenge for sequence-based genetic studies of complex diseases.^ This research dissertation utilized genome continuum model as a general principle, and stochastic calculus and functional data analysis as tools for developing novel and powerful statistical methods for next generation of association studies of both qualitative and quantitative traits in the context of sequencing data, which finally lead to shifting the paradigm of association analysis from the current locus-by-locus analysis to collectively analyzing genome regions.^ In this project, the functional principal component (FPC) methods coupled with high-dimensional data reduction techniques will be used to develop novel and powerful methods for testing the associations of the entire spectrum of genetic variation within a segment of genome or a gene regardless of whether the variants are common or rare.^ The classical quantitative genetics suffer from high type I error rates and low power for rare variants. To overcome these limitations for resequencing data, this project used functional linear models with scalar response to develop statistics for identifying quantitative trait loci (QTLs) for both common and rare variants. To illustrate their applications, the functional linear models were applied to five quantitative traits in Framingham heart studies. ^ This project proposed a novel concept of gene-gene co-association in which a gene or a genomic region is taken as a unit of association analysis and used stochastic calculus to develop a unified framework for testing the association of multiple genes or genomic regions for both common and rare alleles. The proposed methods were applied to gene-gene co-association analysis of psoriasis in two independent GWAS datasets which led to discovery of networks significantly associated with psoriasis.^

IDENTIFICATION AND ANALYSIS OF A NOVEL ROLE FOR THE TOUSLED-LIKE KINASE IN REGULATING MITOTIC SPINDLE DYNAMICS

Deregulation of kinase activity is one example of how cells become cancerous by evading evolutionary constraints. The Tousled kinase (Tsl) was initially identified in Arabidopsis thaliana as a developmentally important kinase. There are two mammalian orthologues of Tsl and one orthologue in C. elegans, TLK-1, which is essential for embryonic viability and germ cell development. Depletion of TLK-1 leads to embryonic arrest large, distended nuclei, and ultimately embryonic lethality. Prior to terminal arrest, TLK-1-depleted embryos undergo aberrant mitoses characterized by poor metaphase chromosome alignment, delayed mitotic progression, lagging chromosomes, and supernumerary centrosomes. I discovered an unanticipated requirement for TLK-1 in mitotic spindle assembly and positioning. Normally, in the newly-fertilized zygote (P0) the maternal pronucleus migrates toward the paternal pronucleus at the posterior end of the embryo. After pronuclear meeting, the pronuclear-centrosome complex rotates 90° during centration to align on the anteroposterior axis followed by nuclear envelope breakdown (NEBD). However, in TLK-1-depleted P0 embryos, the centrosome-pronuclear complex rotation is significantly delayed with respect to NEBD and chromosome congression, Additionally, centrosome positions over time in tlk-1(RNAi) early embryos revealed a defect in posterior centrosome positioning during spindle-pronuclear centration, and 4D analysis of centrosome positions and movement in newly fertilized embryos showed aberrant centrosome dynamics in TLK-1-depleted embryos. Several mechanisms contribute to spindle rotation, one of which is the anchoring of astral microtubules to the cell cortex. Attachment of these microtubules to the cortices is thought to confer the necessary stability and forces in order to rotate the centrosome-pronuclear complex in a timely fashion. Analysis of a microtubule end-binding protein revealed that TLK-1-depleted embryos exhibit a more stochastic distribution of microtubule growth toward the cell cortices, and the types of microtubule attachments appear to differ from wild-type embryos. Additionally, fewer astral microtubules are in the vicinity of the cell cortex, thus suggesting that the delayed spindle rotation could be in part due to a lack of appropriate microtubule attachments to the cell cortex. Together with recently published biochemical data revealing the Tousled-like kinases associate with components of the dynein microtubule motor complex in humans, these data suggest that Tousled-like kinases play an important role in mitotic spindle assembly and positioning.