Structural variation-associated expression changes are paralleled by chromatin architecture modifications.

Copy number variants (CNVs) influence the expression of genes that map not only within the rearrangement, but also to its flanks. To assess the possible mechanism(s) underlying this "neighboring effect", we compared intrachromosomal interactions and histone modifications in cell lines of patients affected by genomic disorders and control individuals. Using chromosome conformation capture (4C-seq), we observed that a set of genes flanking the Williams-Beuren Syndrome critical region (WBSCR) were often looping together. The newly identified interacting genes include AUTS2, mutations of which are associated with autism and intellectual disabilities. Deletion of the WBSCR disrupts the expression of this group of flanking genes, as well as long-range interactions between them and the rearranged interval. We also pinpointed concomitant changes in histone modifications between samples. We conclude that large genomic rearrangements can lead to chromatin conformation changes that extend far away from the structural variant, thereby possibly modulating expression globally and modifying the phenotype. GEO SERIES ACCESSION NUMBER: GSE33784, GSE33867.

Hidden branches: developments in root system architecture.

The root system is fundamentally important for plant growth and survival because of its role in water and nutrient uptake. Therefore, plants rely on modulation of root system architecture (RSA) to respond to a changing soil environment. Although RSA is a highly plastic trait and varies both between and among species, the basic root system morphology and its plasticity are controlled by inherent genetic factors. These mediate the modification of RSA, mostly at the level of root branching, in response to a suite of biotic and abiotic factors. Recent progress in the understanding of the molecular basis of these responses suggests that they largely feed through hormone homeostasis and signaling pathways. Novel factors implicated in the regulation of RSA in response to the myriad endogenous and exogenous signals are also increasingly isolated through alternative approaches such as quantitative trait locus analysis.

An unusual uterine tumour with signet ring cell features

Objective: Lymphomas with signet ring cell features are rare, as is uterine dissemination of lymphomas. We report an exceptional case of a uterine tumor combining these two characteristics. Method: A 61-year-old female was diagnosed in 2004 with localized nodal grade 2 follicular lymphoma (stage IA). She received local radiation therapy, experienced total remission, and did well until 2009 when a systematic CT scan evidenced a pelvic anterior-lateral mass. Total enlarged hysterectomy was performed. Results: The anterior uterine wall contained a 4.8-cm fish flesh well-delineated mass corresponding to a mostly diffuse and focally nodular proliferation of medium to large cells with extensive signet ring cell changes. Tumor cells were CD20-, CD10-, Bcl2-, and Bcl6-positive with a low proliferation rate (<10-15%); CD21 underlined a focal follicular architecture. The vacuoles were PAS-negative and did not stain for immunoglobulin; ultrastructural analysis revealed nonspecific degenerative vacuoles. No lymph nodes were identified isolated from the surgical specimen. The tumor was considered as a secondary localization of the systemic follicular lymphoma, though no signet ring cells were evidenced in the cervical lymph node biopsy (reviewed). Follow-up showed retroperitoneal tissue infiltration (PET-CT) and normal medullar biopsy. She recently started R-CHOP chemotherapy. Conclusion: This case illustrates both an unusual site of dissemination and challenging cytological characteristics in a follicular lymphoma. The signet ring cell changes challenged the adequate classification of this lymphoma as either a large B cell or a follicular B cell lymphoma.

Preparation, maintenance, and use of serum-free aggregating brain cell cultures.

Serum-free aggregating brain cell cultures are free-floating three-dimensional primary cell cultures able to reconstitute spontaneously a histotypic brain architecture to reproduce critical steps of brain development and to reach a high level of structural and functional maturity. This culture system offers, therefore, a unique model for neurotoxicity testing both during the development and at advanced cellular differentiation, and the high number of aggregates available combined with the excellent reproducibility of the cultures facilitates routine test procedures. This chapter presents a detailed description of the preparation, maintenance, and use of these cultures for neurotoxicity studies and a comparison of the developmental characteristics between cultures derived from the telencephalon and cultures derived from the whole brain. For culture preparation, mechanically dissociated embryonic brain tissue is used. The initial cell suspension, composed of neural stem cells, neural progenitor cells, immature postmitotic neurons, glioblasts, and microglial cells, is kept in a serum-free, chemically defined medium under continuous gyratory agitation. Spherical aggregates form spontaneously and are maintained in suspension culture for several weeks. Within the aggregates, the cells rearrange and mature, reproducing critical morphogenic events, such as migration, proliferation, differentiation, synaptogenesis, and myelination. For experimentation, replicate cultures are prepared by the randomization of aggregates from several original flasks. The high yield and reproducibility of the cultures enable multiparametric endpoint analyses, including "omics" approaches.

Cardiovascular effects in patrol officers are associated with fine particulate matter from brake wear and engine emissions

Background: Exposure to fine particulate matter air pollutants (PM2.5) affects heart rate variability parameters, and levels of serum proteins associated with inflammation, hemostasis and thrombosis. This study investigated sources potentially responsible for cardiovascular and hematological effects in highway patrol troopers. Results: Nine healthy young non-smoking male troopers working from 3 PM to midnight were studied on four consecutive days during their shift and the following night. Sources of in-vehicle PM2.5 were identified with variance-maximizing rotational principal factor analysis of PM2.5-components and associated pollutants. Two source models were calculated. Sources of in-vehicle PM2.5 identified were 1) crustal material, 2) wear of steel automotive components, 3) gasoline combustion, 4) speed-changing traffic with engine emissions and brake wear. In one model, sources 1 and 2 collapsed to a single source. Source factors scores were compared to cardiac and blood parameters measured ten and fifteen hours, respectively, after each shift. The "speed-change" factor was significantly associated with mean heart cycle length (MCL, +7% per standard deviation increase in the factor score), heart rate variability (+16%), supraventricular ectopic beats (+39%), % neutrophils (+7%), % lymphocytes (-10%), red blood cell volume MCV (+1%), von Willebrand Factor (+9%), blood urea nitrogen (+7%), and protein C (-11%). The "crustal" factor (but not the "collapsed" source) was associated with MCL (+3%) and serum uric acid concentrations (+5%). Controlling for potential confounders had little influence on the effect estimates. Conclusion: PM2.5 originating from speed-changing traffic modulates the autonomic control of the heart rhythm, increases the frequency of premature supraventricular beats and elicits proinflammatory and pro-thrombotic responses in healthy young men. [Authors]

Development of a dose-controlled multiculture cell exposure chamber for efficient delivery of airborne and engineered nanoparticles

In order to study the various health influencing parameters related to engineered nanoparticles as well as to soot emitted b diesel engines, there is an urgent need for appropriate sampling devices and methods for cell exposure studies that simulate the respiratory system and facilitate associated biological and toxicological tests. The objective of the present work was the further advancement of a Multiculture Exposure Chamber (MEC) into a dose-controlled system for efficient delivery of nanoparticles to cells. It was validated with various types of nanoparticles (diesel engine soot aggregates, engineered nanoparticles for various applications) and with state-of-the-art nanoparticle measurement instrumentation to assess the local deposition of nanoparticles on the cell cultures. The dose of nanoparticles to which cell cultures are being exposed was evaluated in the normal operation of the in vitro cell culture exposure chamber based on measurements of the size specific nanoparticle collection efficiency of a cell free device. The average efficiency in delivering nanoparticles in the MEC was approximately 82%. The nanoparticle deposition was demonstrated by Transmission Electron Microscopy (TEM). Analysis and design of the MEC employs Computational Fluid Dynamics (CFD) and true to geometry representations of nanoparticles with the aim to assess the uniformity of nanoparticle deposition among the culture wells. Final testing of the dose-controlled cell exposure system was performed by exposing A549 lung cell cultures to fluorescently labeled nanoparticles. Delivery of aerosolized nanoparticles was demonstrated by visualization of the nanoparticle fluorescence in the cell cultures following exposure. Also monitored was the potential of the aerosolized nanoparticles to generate reactive oxygen species (ROS) (e.g. free radicals and peroxides generation), thus expressing the oxidative stress of the cells which can cause extensive cellular damage or damage on DNA.

Transmission electron microscopy in cell biology: sample preparation techniques and image information

Transmission electron microscopy is a proven technique in the field of cell biology and a very useful tool in biomedical research. Innovation and improvements in equipment together with the introduction of new technology have allowed us to improve our knowledge of biological tissues, to visualizestructures better and both to identify and to locate molecules. Of all the types ofmicroscopy exploited to date, electron microscopy is the one with the mostadvantageous resolution limit and therefore it is a very efficient technique fordeciphering the cell architecture and relating it to function. This chapter aims toprovide an overview of the most important techniques that we can apply to abiological sample, tissue or cells, to observe it with an electron microscope, fromthe most conventional to the latest generation. Processes and concepts aredefined, and the advantages and disadvantages of each technique are assessedalong with the image and information that we can obtain by using each one ofthem.

Destruction of lymphoid organ architecture and hepatitis caused by CD4 T cells.

Immune responses have the important function of host defense and protection against pathogens. However, the immune response also causes inflammation and host tissue injury, termed immunopathology. For example, hepatitis B and C virus infection in humans cause immunopathological sequel with destruction of liver cells by the host's own immune response. Similarly, after infection with lymphocytic choriomeningitis virus (LCMV) in mice, the adaptive immune response causes liver cell damage, choriomeningitis and destruction of lymphoid organ architecture. The immunopathological sequel during LCMV infection has been attributed to cytotoxic CD8(+) T cells. However, we now show that during LCMV infection CD4(+) T cells selectively induced the destruction of splenic marginal zone and caused liver cell damage with elevated serum alanin-transferase (ALT) levels. The destruction of the splenic marginal zone by CD4(+) T cells included the reduction of marginal zone B cells, marginal zone macrophages and marginal zone metallophilic macrophages. Functionally, this resulted in an impaired production of neutralizing antibodies against LCMV. Furthermore, CD4(+) T cells reduced B cells with an IgM(high)IgD(low) phenotype (transitional stage 1 and 2, marginal zone B cells), whereas other B cell subtypes such as follicular type 1 and 2 and germinal center/memory B cells were not affected. Adoptive transfer of CD4(+) T cells lacking different important effector cytokines and cytolytic pathways such as IFNγ, TNFα, perforin and Fas-FasL interaction did reveal that these cytolytic pathways are redundant in the induction of immunopathological sequel in spleen. In conclusion, our results define an important role of CD4(+) T cells in the induction of immunopathology in liver and spleen. This includes the CD4(+) T cell mediated destruction of the splenic marginal zone with consecutively impaired protective neutralizing antibody responses.

Messaging Application Engine for Symbian Platform

Tämä diplomityö kuvaa viestintä sovelluksen ytimen kehitystyön Symbian-alustalle. Koko sovelluksen vaatimuksena oli vastaamattomiin puheluihin vastaaminen ennalta määritellyillä tekstiviesteillä käyttäjän määrittelemien sääntöjen mukaisesti. Ei-toiminnallisia vaatimuksia olivat resurssien käytön vähentäminen ja uudelleenkäytön mahdollistaminen. Täten tämän työn tavoitteena oli kehittää ydin, joka kapseloi sovelluksen sellaisen toiminnallisuuden, joka on käyttöliittymästä riippumatonta ja uudelleenkäytettävää. Kehitystyössä ohjasi Unified Process, joka on iteroiva, käyttötapauksien ohjaama ja arkkitehtuurikeskeinen ohjelmistoprosessi. Se kannusti käyttämään myös muita teollisuudenalan vakiintuneita menetelmiä, kuten suunnittelumalleja ja visuaalista mallintamista käyttäen Unified Modelling Languagea. Suunnittelumalleja käytettiin kehitystyön aikana ja ohjelmisto mallinnettiin visuaalisesti suunnittelun edistämiseksi ja selkiyttämiseksi. Alustan palveluita käytettiin hyväksi kehitysajan ja resurssien käytön minimoimiseksi. Ytimen päätehtäviksi määrättiin viestien lähettäminen sekä sääntöjen talletus ja tarkistaminen. Sovelluksen eri alueet, eli sovelluspalvelin ja käyttöliittymää, pystyivät käyttämään ydintä ja sillä ei ollut riippuvuuksia käyttöliittymätasolle. Täten resurssien käyttö väheni ja uudelleenkäytettävyys lisääntyi. Viestien lähettäminen toteutettiin Symbian-alustan menetelmin. Sääntöjen tallettamiseen tehtiin tallennuskehys, joka eristää sääntöjen sisäisen ja ulkoisen muodon. Tässä tapauksessa ulkoiseksi tallennustavaksi valittiin relaatiotietokanta. Sääntöjen tarkastaminen toteutettiin tavanomaisella olioiden yhteistoiminnalla. Päätavoite saavutettiin. tämä ja muut hyviksi arvioidut lopputulokset, kuten uudelleenkäytettävyys ja vähentynyt resurssien käyttö, arveltiin juontuvan suunnittelumallien ja Unified Processin käytöstä. Kyseiset menetelmät osoittivat mukautuvansa pieniinkin projekteihin. Menetelmien todettiin myös tukevan ja kannustavan kehitystyön aikaista oppimista, mikä oli välttämätöntä tässä tapauksessa.

Physical layer development in third generation protocol software architecture

Diplomityön tavoitteena oli kehittää kolmannen sukupolven fyysistä protokollakerrosta matkapuhelimen ohjelmistoarkkitehtuurille. Kolmannen sukupolven matkapuhelinjärjestelmät ovat aikaisempia järjestelmiä monimutkaisempia. Ohjelmiston koon ja monimutkaisuuden sekä aikataulujen kiireellisyyden vuoksi on tullut tarve ottaa käyttöön formaaleja menetelmiä ohjelmiston kehitystyöhön. Formaalit kuvauskielet mahdollistavat tarkan, yksiselitteisen ja simuloitavissa olevan järjestelmäkuvauksen muodostamisen. Fyysinen protokollakerros tarjoaa tiedon siirtoa ylemmille protokollakerroksille. Tämän tiedonsiirron hallinta vaatii protokollakerrosten välistä viestinvälitystä. Formaaleja kuvauskieliä käyttämällä voidaan viestinvälityksen toteutusta automatisoida ja siinä tarvittavaa logiikkaa havainnollistaa. Työssä suunniteltiin, toteutettiin ja testattiin ylempien protokollakerrosten kanssa kommunikoivaa osaa fyysisestä protokollakerroksesta. Tuloksena saatiin solunvalintatoiminnallisuuden vaatiman kommunikoinnin ja tilakoneen toteutus ohjelmistoarkkitehtuurissa. Ohjelmistonkehityksen alkuvaiheiden havaittiin olevan fyysisen kerroksen suorituskyvyn kannalta merkittävässä asemassa, koska tällöin viestinvälityksen optimointi on helpointa. Formaalit kuvauskielet eivät ole sellaisenaan täysin soveltuvia tarkoin määritellyn ohjelmistoarkkitehtuurin osien kehitykseen.

Multimedia Messaging Service Center in a Component Based Architecture

Multimedia-sanomanvälityspalvelu (MMS) on matkapuhelinten väliseen viestintään kehitetty palvelu, joka mahdollistaa yhteyden Internet maailmaan. Multimedia-sanomanvälityspalvelua voidaan käyttää luomaan yhteys matkapuhelimen käyttäjän ja ulkoisen sovelluspalvelimen välille. MMS voidaan nähdä sovelluksena, joka yhdistää multimediaviestin luonnin, käsittelyn sekä toimituksen monelle eri sisältö tyypille. Multimedia-viestikeskus (MMSC) on uusi verkkoelementti, joka on vastuussa multimediaviestien varastoinnista ja toimituksesta. Multimedia-viestikeskuksella on kolme loogista elementtiä, jotka ovat välityspalvelin, sovellusrajapinnat ja matkapuhelinverkkorajapinta. Operaattorit sekä kolmannen osapuolen sovelluskehittäjät voivat kehittää lisäarvopalveluita multimedia-sanomanvälityspalvelulle hyödyntämällä sovellusrajapintoja. Sovellusrajapinnat perustuvat olemassa oleviin Internet protokolliin. Tämä diplomityö tutkii Multimedia-sanomanvälityspalvelun verkkoelementtien rajapintoja. Tulevaisuudessa on tarkoitus lisätä Multimedia-sanomanvälityspalvelun verkkoelementtejä sähköisen kaupankäynnin kehysarkkitehtuuriin, joka perustuu komponentteihin.

Dynamic single cell measurements of kinase activity by synthetic kinase activity relocation sensors.

BACKGROUND: Mitogen activated protein kinases (MAPK) play an essential role in integrating extra-cellular signals and intra-cellular cues to allow cells to grow, adapt to stresses, or undergo apoptosis. Budding yeast serves as a powerful system to understand the fundamental regulatory mechanisms that allow these pathways to combine multiple signals and deliver an appropriate response. To fully comprehend the variability and dynamics of these signaling cascades, dynamic and quantitative single cell measurements are required. Microscopy is an ideal technique to obtain these data; however, novel assays have to be developed to measure the activity of these cascades. RESULTS: We have generated fluorescent biosensors that allow the real-time measurement of kinase activity at the single cell level. Here, synthetic MAPK substrates were engineered to undergo nuclear-to-cytoplasmic relocation upon phosphorylation of a nuclear localization sequence. Combination of fluorescence microscopy and automated image analysis allows the quantification of the dynamics of kinase activity in hundreds of single cells. A large heterogeneity in the dynamics of MAPK activity between individual cells was measured. The variability in the mating pathway can be accounted for by differences in cell cycle stage, while, in the cell wall integrity pathway, the response to cell wall stress is independent of cell cycle stage. CONCLUSIONS: These synthetic kinase activity relocation sensors allow the quantification of kinase activity in live single cells. The modularity of the architecture of these reporters will allow their application in many other signaling cascades. These measurements will allow to uncover new dynamic behaviour that previously could not be observed in population level measurements.

Population Variation and Genetic Control of Modular Chromatin Architecture in Humans.

Chromatin state variation at gene regulatory elements is abundant across individuals, yet we understand little about the genetic basis of this variability. Here, we profiled several histone modifications, the transcription factor (TF) PU.1, RNA polymerase II, and gene expression in lymphoblastoid cell lines from 47 whole-genome sequenced individuals. We observed that distinct cis-regulatory elements exhibit coordinated chromatin variation across individuals in the form of variable chromatin modules (VCMs) at sub-Mb scale. VCMs were associated with thousands of genes and preferentially cluster within chromosomal contact domains. We mapped strong proximal and weak, yet more ubiquitous, distal-acting chromatin quantitative trait loci (cQTL) that frequently explain this variation. cQTLs were associated with molecular activity at clusters of cis-regulatory elements and mapped preferentially within TF-bound regions. We propose that local, sequence-independent chromatin variation emerges as a result of genetic perturbations in cooperative interactions between cis-regulatory elements that are located within the same genomic domain.

Macroporous Scaffolds for Bone Engineering. Studies on Cell Culture and Ectopic Bone Formation

Bone engineering is a rapidly developing area of reconstructive medicine where bone inducing factors and/or cells are combined with a scaffold material to regenerate the structure and function of the original tissue. The aim of this study was to compare the suitability of different macroporous scaffold types for bone engineering applications. The two scaffold categories studied were a) the mechanically strong and stable titanium fiber meshes and b) the elastic and biodegradable porous polymers. Furthermore, bioactive modifications were applied to these basic scaffold types, and their effect on the osteogenic responses was evaluated in cell culture and ectopic bone formation studies. The osteogenic phenotype of cultured cell-scaffold constructs was heightened with a sol-gel derived titania coating, but not with a mixed titania-silica coating. The latter coating also resulted in delayed ectopic bone formation in bone marrow stromal cell seeded scaffolds. However, the better bone contact in early implantation times and more even bone tissue distribution at later times indicated enhanced osteoconductivity of both the coated scaffold types. Overall, the most promising bone engineering results were obtained with titania coated fiber meshes. Elastic and biodegradable poly(ε-caprolactone/D,L-lactide) based scaffolds were also developed in this study. The degradation rates of the scaffolds in vitro were governed by the hydrophilicity of the polymer matrix, and the porous architecture was controlled by the amount and type of porogen used. A continuous phase macroporosity was obtained using a novel CaCl2 • 6H2O porogen. Dynamic culture conditions increased cell invasion, but decreased cell numbers and osteogenicity, within the scaffolds. Osteogenic differentiation in static cultures and ectopic bone formation in cell seeded scaffolds were enhanced in composites, with 30 wt-% of bioactive glass filler.

JNK regulates dendrite and spine architecture in the central nervous system influencing spatial learning and motor tasks

JNK1 is a MAP-kinase that has proven a significant player in the central nervous system. It regulates brain development and the maintenance of dendrites and axons. Several novel phosphorylation targets of JNK1 were identified in a screen performed in the Coffey lab. These proteins were mainly involved in the regulation of neuronal cytoskeleton, influencing the dynamics and stability of microtubules and actin. These structural proteins form the dynamic backbone for the elaborate architecture of the dendritic tree of a neuron. The initiation and branching of the dendrites requires a dynamic interplay between the cytoskeletal building blocks. Both microtubules and actin are decorated by associated proteins which regulate their dynamics. The dendrite-specific, high molecular weight microtubule associated protein 2 (MAP2) is an abundant protein in the brain, the binding of which stabilizes microtubules and influences their bundling. Its expression in non-neuronal cells induces the formation of neurite-like processes from the cell body, and its function is highly regulated by phosphorylation. JNK1 was shown to phosphorylate the proline-rich domain of MAP2 in vivo in a previous study performed in the group. Here we verify three threonine residues (T1619, T1622 and T1625) as JNK1 targets, the phosphorylation of which increases the binding of MAP2 to microtubules. This binding stabilizes the microtubules and increases process formation in non-neuronal cells. Phosphorylation-site mutants were engineered in the lab. The non-phosphorylatable mutant of MAP2 (MAP2- T1619A, T1622A, T1625A) in these residues fails to bind microtubules, while the pseudo-phosphorylated form, MAP2- T1619D, T1622D, Thr1625D, efficiently binds and induces process formation even without the presence of active JNK1. Ectopic expression of the MAP2- T1619D, T1622D, Thr1625D in vivo in mouse brain led to a striking increase in the branching of cortical layer 2/3 (L2/3) pyramidal neurons, compared to MAP2-WT. The dendritic complexity defines the receptive field of a neuron and dictates the output to the postsynaptic cells. Previous studies in the group indicated altered dendrite architecture of the pyramidal neurons in the Jnk1-/- mouse motor cortex. Here, we used Lucifer Yellow loading and Sholl analysis of neurons in order to study the dendritic branching in more detail. We report a striking, opposing effect in the absence of Jnk1 in the cortical layers 2/3 and 5 of the primary motor cortex. The basal dendrites of pyramidal neurons close to the pial surface at L2/3 show a reduced complexity. In contrast, the L5 neurons, which receive massive input from the L2/3 neurons, show greatly increased branching. Another novel substrate identified for JNK1 was MARCKSL1, a protein that regulates actin dynamics. It is highly expressed in neurons, but also in various cancer tissues. Three phosphorylation target residues for JNK1 were identified, and it was demonstrated that their phosphorylation reduces actin turnover and retards migration of these cells. Actin is the main cytoskeletal component in dendritic spines, the site of most excitatory synapses in pyramidal neurons. The density and gross morphology of the Lucifer Yellow filled dendrites were characterized and we show reduced density and altered morphology of spines in the motor cortex and in the hippocampal area CA3. The dynamic dendritic spines are widely considered to function as the cellular correlate during learning. We used a Morris water maze to test spatial memory. Here, the wild-type mice outperformed the knock-out mice during the acquisition phase of the experiment indicating impaired special memory. The L5 pyramidal neurons of the motor cortex project to the spinal cord and regulate the movement of distinct muscle groups. Thus the altered dendrite morphology in the motor cortex was expected to have an effect on the input-output balance in the signaling from the cortex to the lower motor circuits. A battery of behavioral tests were conducted for the wild-type and Jnk1-/- mice, and the knock-outs performed poorly compared to wild-type mice in tests assessing balance and fine motor movements. This study expands our knowledge of JNK1 as an important regulator of the dendritic fields of neurons and their manifestations in behavior.