High-throughput sequencing of small RNA transcriptomes reveals critical biological features targeted by microRNAs in cell models used for squamous cell cancer research

Abstract Background The implication of post-transcriptional regulation by microRNAs in molecular mechanisms underlying cancer disease is well documented. However, their interference at the cellular level is not fully explored. Functional in vitro studies are fundamental for the comprehension of their role; nevertheless results are highly dependable on the adopted cellular model. Next generation small RNA transcriptomic sequencing data of a tumor cell line and keratinocytes derived from primary culture was generated in order to characterize the microRNA content of these systems, thus helping in their understanding. Both constitute cell models for functional studies of microRNAs in head and neck squamous cell carcinoma (HNSCC), a smoking-related cancer. Known microRNAs were quantified and analyzed in the context of gene regulation. New microRNAs were investigated using similarity and structural search, ab initio classification, and prediction of the location of mature microRNAs within would-be precursor sequences. Results were compared with small RNA transcriptomic sequences from HNSCC samples in order to access the applicability of these cell models for cancer phenotype comprehension and for novel molecule discovery. Results Ten miRNAs represented over 70% of the mature molecules present in each of the cell types. The most expressed molecules were miR-21, miR-24 and miR-205, Accordingly; miR-21 and miR-205 have been previously shown to play a role in epithelial cell biology. Although miR-21 has been implicated in cancer development, and evaluated as a biomarker in HNSCC progression, no significant expression differences were seen between cell types. We demonstrate that differentially expressed mature miRNAs target cell differentiation and apoptosis related biological processes, indicating that they might represent, with acceptable accuracy, the genetic context from which they derive. Most miRNAs identified in the cancer cell line and in keratinocytes were present in tumor samples and cancer-free samples, respectively, with miR-21, miR-24 and miR-205 still among the most prevalent molecules at all instances. Thirteen miRNA-like structures, containing reads identified by the deep sequencing, were predicted from putative miRNA precursor sequences. Strong evidences suggest that one of them could be a new miRNA. This molecule was mostly expressed in the tumor cell line and HNSCC samples indicating a possible biological function in cancer. Conclusions Critical biological features of cells must be fully understood before they can be chosen as models for functional studies. Expression levels of miRNAs relate to cell type and tissue context. This study provides insights on miRNA content of two cell models used for cancer research. Pathways commonly deregulated in HNSCC might be targeted by most expressed and also by differentially expressed miRNAs. Results indicate that the use of cell models for cancer research demands careful assessment of underlying molecular characteristics for proper data interpretation. Additionally, one new miRNA-like molecule with a potential role in cancer was identified in the cell lines and clinical samples.

How low-level laser therapy can change mechanical properties of cells.

Low level laser therapy is used as a treatment of several conditions, including inflammatory processes and wound healing. Possible changes in mechanical properties of cells, caused by illumination, are investigated with optical magnetic twisting cytometry (OMTC), which is a technique used to evaluate mechanical properties in cell culture. Ferromagnetic micro beads are bound to cell cytoskeleton, the beads are magnetized vertically and a horizontal twisting magnetic field is applied causing a torque that moves the beads and deforms the cell, the beads rotate and displace. Based on the lateral displacement of the beads, elastic shear and loss moduli are obtained. Samples of human bronchial epithelial cell culture were divided in two groups: one was illuminated with a 660 nm red laser, 30 mW power, 0.75 W/cm2 irradiance, during different time intervals, and the other one, the control group, was not illuminated. The values of the mechanical constants of the cells of the control group showed a tendency of increasing with the time out of the incubator. On the other hand, the illuminated group showed constancy on the behavior of both moduli, keeping the normal conditions of the cell culture. Those results indicate that illumination can induce cells to homeostasis, and OMTC is sensitive to observe departures from the steady conditions. Hence, OMTC is an important technique which can be used to aggregate knowledge on the light effect in cell cytoskeleton and even on the low level laser therapy mechanisms in inflammatory processes and/or wound healing.

How low-level laser therapy can change mechanical properties of cells.

Low level laser therapy is used as a treatment of several conditions, including inflammatory processes and wound healing. Possible changes in mechanical properties of cells, caused by illumination, are investigated with optical magnetic twisting cytometry (OMTC), which is a technique used to evaluate mechanical properties in cell culture. Ferromagnetic micro beads are bound to cell cytoskeleton, the beads are magnetized vertically and a horizontal twisting magnetic field is applied causing a torque that moves the beads and deforms the cell, the beads rotate and displace. Based on the lateral displacement of the beads, elastic shear and loss moduli are obtained. Samples of human bronchial epithelial cell culture were divided in two groups: one was illuminated with a 660 nm red laser, 30 mW power, 0.75 W/cm2 irradiance, during different time intervals, and the other one, the control group, was not illuminated. The values of the mechanical constants of the cells of the control group showed a tendency of increasing with the time out of the incubator. On the other hand, the illuminated group showed constancy on the behavior of both moduli, keeping the normal conditions of the cell culture. Those results indicate that illumination can induce cells to homeostasis, and OMTC is sensitive to observe departures from the steady conditions. Hence, OMTC is an important technique which can be used to aggregate knowledge on the light effect in cell cytoskeleton and even on the low level laser therapy mechanisms in inflammatory processes and/or wound healing.

Photobiological effect of low-level laser irradiation in bovine embryo production system

The objectives of this study were to evaluate the effect of low-level laser irradiation (LLLI) on bovine oocyte and granulosa cells metabolism during in vitro maturation (IVM) and further embryo development. Cumulus-oocytes complexes (COCs) were subjected (experimental group) or not (control group) to irradiation with LLLI in a 633-nm wavelength and 1 J/cm2 fluency. The COCs were evaluated after 30 min, 8, 16, and 24 h of IVM. Cumulus cells were evaluated for cell cycle status, mitochondrial activity, and viability (flow cytometry). Oocytes were assessed for meiotic progression status (nuclear staining), cell cycle genes content [real-time polymerase chain reaction (PCR)], and signal transduction status (western blot). The COCs were also in vitro fertilized, and the cleavage and blastocyst rates were assessed. Comparisons among groups were statistically performed with 5% significance level. For cumulus cells, a significant increase in mitochondrial membrane potential and the number of cells progressing through the cycle could be observed. Significant increases on cyclin B and cyclin-dependent kinase (CDK4) levels were also observed. Concerning the oocytes, a significantly higher amount of total mitogen-activated protein kinase was found after 8 h of irradiation, followed by a decrease in all cell cycle genes transcripts, exception made for the CDK4. However, no differences were observed in meiotic progression or embryo production. In conclusion, LLLI is an efficient tool to modulate the granulosa cells and oocyte metabolism

Study of apoptotic deletion mediated by Bifidobacterium longum with construction of recombinant strains for Serpin encoding gene and phenotypes comparison in a pig cell model

The first part of the research project of the Co-Advisorship Ph.D Thesis was aimed to select the best Bifidobacterium longum strains suitable to set the basis of our study. We were looking for strains with the abilities to colonize the intestinal mucosa and with good adhesion capacities, so that we can test these strains to investigate their ability to induce apoptosis in “damaged” intestinal cells. Adhesion and apoptosis are the two process that we want to study to better understand the role of an adhesion protein that we have previously identified and that have top scores homologies with the recent serpin encoding gene identified in B. longum by Nestlè researchers. Bifidobacterium longum is a probiotic, known for its beneficial effects to the human gut and even for its immunomodulatory and antitumor activities. Recently, many studies have stressed out the intimate relation between probiotic bacteria and the GIT mucosa and their influence on human cellular homeostasis. We focused on the apoptotic deletion of cancer cells induced by B. longum. This has been valued in vitro, performing the incubation of three B.longum strains with enterocyte-like Caco- 2 cells, to evidence DNA fragmentation, a cornerstone of apoptosis. The three strains tested were defined for their adhesion properties using adhesion and autoaggregation assays. These features are considered necessary to select a probiotic strain. The three strains named B12, B18 and B2990 resulted respectively: “strong adherent”, “adherent” and “non adherent”. Then, bacteria were incubated with Caco-2 cells to investigate apoptotic deletion. Cocultures of Caco-2 cells with B. longum resulted positive in DNA fragmentation test, only when adherent strains were used (B12 and B18). These results indicate that the interaction with adherent B. longum can induce apoptotic deletion of Caco-2 cells, suggesting a role in cellular homeostasis of the gastrointestinal tract and in restoring the ecology of damaged colon tissues. These results were used to keep on researching and the strains tested were used as recipient of recombinant techniques aimed to originate new B.longum strains with enhanced capacity of apoptotic induction in “damaged” intestinal cells. To achieve this new goal it was decided to clone the serpin encoding gene of B. longum, so that we can understand its role in adhesion and apoptosis induction. Bifidobacterium longum has immunostimulant activity that in vitro can lead to apoptotic response of Caco-2 cell line. It secretes a hypothetical eukaryotic type serpin protein, which could be involved in this kind of deletion of damaged cells. We had previously characterised a protein that has homologies with the hypothetical serpin of B. longum (DD087853). In order to create Bifidobacterium serpin transformants, a B. longum cosmid library was screened with a PCR protocol using specific primers for serpin gene. After fragment extraction, the insert named S1 was sub-cloned into pRM2, an Escherichia coli - Bifidobacterium shuttle vector, to construct pRM3. Several protocols for B. longum transformation were performed and the best efficiency was obtained using MRS medium and raffinose. Finally bacterial cell supernatants were tested in a dotblot assay to detect antigens presence against anti-antitrypsin polyclonal antibody. The best signal was produced by one starin that has been renamed B. longum BLKS 7. Our research study was aimed to generate transformants able to over express serpin encoding gene, so that we can have the tools for a further study on bacterial apoptotic induction of Caco-2 cell line. After that we have originated new trasformants the next step to do was to test transformants abilities when exposed to an intestinal cell model. In fact, this part of the project was achieved in the Department of Biochemistry of the Medical Faculty of the University of Maribor, guest of the abroad supervisor of the Co-Advisorship Doctoral Thesis: Prof. Avrelija Cencic. In this study we examined the probiotic ability of some bacterial strains using intestinal cells from a 6 years old pig. The use of intestinal mammalian cells is essential to study this symbiosis and a functional cell model mimics a polarised epithelium in which enterocytes are separated by tight junctions. In this list of strains we have included the Bifidobacterium longum BKS7 transformant strain that we have previously originated; in order to compare its abilities. B. longum B12 wild type and B. longum BKS7 transformant and eight Lactobacillus strains of different sources were co-cultured with porcine small intestine epithelial cells (PSI C1) and porcine blood monocytes (PoM2) in Transwell filter inserts. The strains, including Lb. gasseri, Lb. fermentum, Lb. reuterii, Lb. plantarum and unidentified Lactobacillus from kenyan maasai milk and tanzanian coffee, were assayed for activation of cell lines, measuring nitric oxide by Griess reaction, H202 by tetramethylbenzidine reaction and O2 - by cytochrome C reduction. Cytotoxic effect by crystal violet staining and induction on metabolic activity by MTT cell proliferation assay were tested too. Transepithelial electrical resistance (TER) of polarised PSI C1 was measured during 48 hours co-culture. TER, used to observe epithelium permeability, decrease during pathogenesis and tissue becomes permeable to ion passive flow lowering epithelial barrier function. Probiotics can prevent or restore increased permeability. Lastly, dot-blot was achieved against Interleukin-6 of treated cells supernatants. The metabolic activity of PoM2 and PSI C1 increased slightly after co-culture not affecting mitochondrial functions. No strain was cytotoxic over PSI C1 and PoM2 and no cell activation was observed, as measured by the release of NO2, H202 and O2 - by PoM2 and PSI C1. During coculture TER of polarised PSI C1 was two-fold higher comparing with constant TER (~3000 ) of untreated cells. TER raise generated by bacteria maintains a low permeability of the epithelium. During treatment Interleukin-6 was detected in cell supernatants at several time points, confirming immunostimulant activity. All results were obtained using Lactobacillus paracasei Shirota e Carnobacterium divergens as controls. In conclusion we can state that both the list of putative probiotic bacteria and our new transformant strain of B. longum are not harmful when exposed to intestinal cells and could be selected as probiotics, because can strengthen epithelial barrier function and stimulate nonspecific immunity of intestinal cells on a pig cell model. Indeed, we have found out that none of the strains tested that have good adhesion abilities presents citotoxicity to the intestinal cells and that non of the strains tested can induce cell lines to produce high level of ROS, neither NO2. Moreover we have assayed even the capacity of producing certain citokynes that are correlated with immune response. The detection of Interleukin-6 was assayed in all our samples, including B.longum transformant BKS 7 strain, this result indicates that these bacteria can induce a non specific immune response in the intestinal cells. In fact, when we assayed the presence of Interferon-gamma in cells supernatant after bacterial exposure, we have no positive signals, that means that there is no activation of a specific immune response, thus confirming that these bacteria are not recognize as pathogen by the intestinal cells and are certainly not harmful for intestinal cells. The most important result is the measure of Trans Epithelial Electric Resistance that have shown how the intestinal barrier function get strengthen when cells are exposed to bacteria, due to a reduction of the epithelium permeability. We have now a new strain of B. longum that will be used for further studies above the mechanism of apoptotic induction to “damaged cells” and above the process of “restoring ecology”. This strain will be the basis to originate new transformant strains for Serpin encoding gene that must have better performance and shall be used one day even in clinical cases as in “gene therapy” for cancer treatment and prevention.

Elf magnetic field influence on ION Channels studied by Patch Clamp Technique: exposure set up and "Whole Cell" measurements on Potassium currents

The aim of this thesis was to study the effects of extremely low frequency (ELF) electromagnetic magnetic fields on potassium currents in neural cell lines ( Neuroblastoma SK-N-BE ), using the whole-cell Patch Clamp technique. Such technique is a sophisticated tool capable to investigate the electrophysiological activity at a single cell, and even at single channel level. The total potassium ion currents through the cell membrane was measured while exposing the cells to a combination of static (DC) and alternate (AC) magnetic fields according to the prediction of the so-called ‘ Ion Resonance Hypothesis ’. For this purpose we have designed and fabricated a magnetic field exposure system reaching a good compromise between magnetic field homogeneity and accessibility to the biological sample under the microscope. The magnetic field exposure system consists of three large orthogonal pairs of square coils surrounding the patch clamp set up and connected to the signal generation unit, able to generate different combinations of static and/or alternate magnetic fields. Such system was characterized in term of field distribution and uniformity through computation and direct field measurements. No statistically significant changes in the potassium ion currents through cell membrane were reveled when the cells were exposed to AC/DC magnetic field combination according to the afore mentioned ‘Ion Resonance Hypothesis’.

Bedeutung endogener Faktoren für Steady-State-Level und Reparatur oxidativer DNA-Modifikationen in Säugerzellen

Die endogene Bildung reaktiver Sauerstoffspezies (ROS) - wie beispielsweise Hydroxyl-Radikale, Superoxid-Radikalanionen, Wasserstoffperoxid und Singulett-Sauerstoff - bei essentiellen Stoffwechselreaktionen in allen aeroben Lebewesen stellt eine potentielle Gefahr für die Integrität der DNA in jeder Zelle dar. ROS generieren in der DNA unter anderem oxidative DNA-Modifikationen (zum größten Teil wahrscheinlich 8-Hydroxyguanin (8-oxoG)), welche wiederum zu einem Teil zu Mutationen führen.In dieser Arbeit wurden Untersuchungen vorgenommen, in welchem Ausmaß zum einen die Steady-State-Level oxidativer DNA-Schäden in Säugerzellen zum anderen die Reparaturgeschwindig-keiten solcher DNA-Modifikationen durch verschiedene endogene Faktoren beeinflußt werden.Im Mittelpunkt der Arbeit stand dabei die Charakterisierung der 8-Hydroxyguaninglykosylase der Säugerzellen. Sie ist das Produkt des OGG1-Gens, das erst 1997 kloniert wurde. In transfizierten Zellinien konnte durch eine konstitutive Überexpression des menschlichen OGG1-Gens demonstriert werden, daß die Reparatur von induzierten oxidativen Basenmodifikationen bis zu dreifach beschleunigt wird und daß eine Korrelation zwischen dem Grad der Überexpression und der Reparaturrate besteht. Dagegen waren die Steady-State-Level der oxidativen DNA-Schäden durch die Überexpression unbeeinflußt. Sowohl bei den spontanen Mutationsraten als auch bei den durch oxidative Schädigungen induzierten Mutationsfrequenzen konnte keine Erniedrigung bedingt durch die hOGG1-Überexpression beobachtet werden.Weitere Untersuchungen zur Bedeutung von Ogg1-Protein konnten in Mäusezellen durchgeführt werden, in denen das OGG1-homologe Mäusegen, mOGG1, homozygot inaktiviert (mOGG1(-/-)) worden war. Hierbei konnte gezeigt werden, daß in den mOGG1-defizienten Zellen im Vergleich zu den entsprechenden Wildtyp-Zellen (mOGG1(+/+)) eine Reparatur induzierter oxidativer Basenmodifikationen erst nach 8 h einsetzt, während in den Kontrollzellen schon nach 3-4 h 50 % der Modifikationen repariert waren. Die Steady-State-Level oxidativer Modifikationen in mOGG1(-/-)-Zellen waren in immortalisierten, schnell proliferierenden Mäusefibroblasten nur um den Faktor 1.4, in primären Mäusehepatocyten jedoch um den Faktor 2.5 gegenüber den Wildtyp-Zellen erhöht.Inwieweit das menschliche Reparaturprotein Xrcc1 (X-ray repair cross complementing group 1) auch an der Prozessierung oxidativer DNA-Modifikationen beteiligt ist, und ob dabei möglicherweise eine Interaktion mit Ogg1 vorliegt, wurde in der XRCC1-defizienten CHO-Zellinie EM9 untersucht. Dabei wurde ermittelt, daß weder die Steady-State-Level noch die Reparaturkinetiken der oxidativen Basenmodifikationen durch die XRCC1-Defizienz beeinflußt werden. Aufgrund weiterer Ergebnisse kann jedoch nicht ausgeschlossen werden, daß das Xrcc1-Protein zumindest am Ligationsschritt während der Reparatur oxidativer DNA-Schäden beteiligt ist.In einem weiteren Schwerpunkt der Arbeit wurde untersucht, ob Unterschiede im Steady-State-Level in Abhängigkeit von Organ-, Gewebe- und Zelltyp auftreten. Dazu wurden Untersuchungen in Bronchialkarzinom-Zellinien verschiedener Subtypen durchgeführt. Des weiteren wurde zur Frage der Zelltyp-Abhängigkeit in der menschlichen Zellinie HL60 der Einfluß des Zelldifferenzierungsstadiums auf die Steady-State-Level untersucht.

Multi-level models and infrastructures for simulating biological system development

The hierarchical organisation of biological systems plays a crucial role in the pattern formation of gene expression resulting from the morphogenetic processes, where autonomous internal dynamics of cells, as well as cell-to-cell interactions through membranes, are responsible for the emergent peculiar structures of the individual phenotype. Being able to reproduce the systems dynamics at different levels of such a hierarchy might be very useful for studying such a complex phenomenon of self-organisation. The idea is to model the phenomenon in terms of a large and dynamic network of compartments, where the interplay between inter-compartment and intra-compartment events determines the emergent behaviour resulting in the formation of spatial patterns. According to these premises the thesis proposes a review of the different approaches already developed in modelling developmental biology problems, as well as the main models and infrastructures available in literature for modelling biological systems, analysing their capabilities in tackling multi-compartment / multi-level models. The thesis then introduces a practical framework, MS-BioNET, for modelling and simulating these scenarios exploiting the potential of multi-level dynamics. This is based on (i) a computational model featuring networks of compartments and an enhanced model of chemical reaction addressing molecule transfer, (ii) a logic-oriented language to flexibly specify complex simulation scenarios, and (iii) a simulation engine based on the many-species/many-channels optimised version of Gillespie’s direct method. The thesis finally proposes the adoption of the agent-based model as an approach capable of capture multi-level dynamics. To overcome the problem of parameter tuning in the model, the simulators are supplied with a module for parameter optimisation. The task is defined as an optimisation problem over the parameter space in which the objective function to be minimised is the distance between the output of the simulator and a target one. The problem is tackled with a metaheuristic algorithm. As an example of application of the MS-BioNET framework and of the agent-based model, a model of the first stages of Drosophila Melanogaster development is realised. The model goal is to generate the early spatial pattern of gap gene expression. The correctness of the models is shown comparing the simulation results with real data of gene expression with spatial and temporal resolution, acquired in free on-line sources.

Differential cell type-specific transcriptional regulation of the CYP1A1 gene

Cytochrome P450 1A1 (CYP1A1) monooxygenase plays an important role in the metabolism of environmental pollutants such as polycyclic aromatic hydrocarbons (PAHs) and halogenated polycyclic aromatic hydrocarbons (HAHs). Oxidation of these compounds converts them to the metabolites that subsequently can be conjugated to hydrophilic endogenous entities e.g. glutathione. Derivates generated in this way are water soluble and can be excreted in bile or urine, which is a defense mechanism. Besides detoxification, metabolism by CYP1A1 may lead to deleterious effects since the highly reactive intermediate metabolites are able to react with DNA and thus cause mutagenic effects, as it is in the case of benzo(a) pyrene (B[a]P). CYP1A1 is normally not expressed or expressed at a very low level in the cells but it is inducible by many PAHs and HAHs e.g. by B[a]P or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Transcriptional activation of the CYP1A1 gene is mediated by aryl hydrocarbon receptor (AHR), a basic-helix-loop-helix (bHLH) transcription factor. In the absence of a ligand AHR stays predominantly in the cytoplasm. Ligand binding causes translocation of AHR to the nuclear compartment, its heterodimerization with another bHLH protein, the aryl hydrocarbon nuclear translocator (ARNT) and binding of the AHR/ARNT heterodimer to a DNA motif designated dioxin responsive element (DRE). This process leads to the transcriptional activation of the responsive genes containing DREs in their regulatory regions, e.g. that coding for CYP1A1. TCDD is the most potent known agonist of AHR. Since it is not metabolized by the activated enzymes, exposure to this compound leads to a persisting activation of AHR resulting in diverse toxic effects in the organism. To enlighten the molecular mechanisms that mediate the toxicity of xenobiotics like TCDD and related compounds, the AHR-dependent regulation of the CYP1A1 gene was investigated in two cell lines: human cervix carcinoma (HeLa) and mouse hepatoma (Hepa). Study of AHR activation and its consequence concerning expression of the CYP1A1 enzyme confirmed the TCDD-dependent formation of the AHR/ARNT complex on DRE leading to an increase of the CYP1A1 transcription in Hepa cells. In contrast, in HeLa cells formation of the AHR/ARNT heterodimer and binding of a protein complex containing AHR and ARNT to DRE occurred naturally in the absence of TCDD. Moreover, treatment with TCDD did not affect the AHR/ARNT dimer formation and binding of these proteins to DRE in these cells. Even though the constitutive complex on DRE exists in HeLa, transcription of the CYP1A1 gene was not increased. Furthermore, the CYP1A1 level in HeLa cells remained unchanged in the presence of TCDD suggesting repressional mechanism of the AHR complex function which may hinder the TCDD-dependent mechanisms in these cells. Similar to the native, the mouse CYP1A1-driven reporter constructs containing different regulatory elements were not inducible by TCDD in HeLa cells, which supported a presence of cell type specific trans-acting factor in HeLa cells able to repress both the native CYP1A1 and CYP1A1-driven reporter genes rather than species specific differences between CYP1A1 genes of human and rodent origin. The different regulation of the AHR-mediated transcription of CYP1A1 gene in Hepa and HeLa cells was further explored in order to elucidate two aspects of the AHR function: (I) mechanism involved in the activation of AHR in the absence of exogenous ligand and (II) factor that repress function of the exogenous ligand-independent AHR/ARNT complex. Since preliminary studies revealed that the activation of PKA causes an activation of AHR in Hepa cells in the absence of TCDD, the PKA-dependent signalling pathway was the proposed endogenous mechanism leading to the TCDD-independent activation of AHR in HeLa cells. Activation of PKA by forskolin or db-cAMP as well as inhibition of the kinase by H89 in both HeLa and Hepa cells did not lead to alterations in the AHR interaction with ARNT in the absence of TCDD and had no effect on binding of these proteins to DRE. Moreover, the modulators of PKA did not influence the CYP1A1 activity in these cells in the presence and in the absence of TCDD. Thus, an involvement of PKA in the regulation of the CYP1A1 Gen in HeLa cells was not evaluated in the course of this study. Repression of genes by transcription factors bound to their responsive elements in the absence of ligands has been described for nuclear receptors. These receptors interact with protein complex containing histone deacetylase (HDAC), enzyme responsible for the repressional effect. Thus, a participation of histone deacetylase in the transcriptional modulation of CYP1A1 gene by the constitutively DNA-bound AHR/ARNT complex was supposed. Inhibition of the HDAC activity by trichostatin A (TSA) or sodium butyrate (NaBu) led to an increase of the CYP1A1 transcription in the presence but not in the absence of TCDD in Hepa and HeLa cells. Since amount of the AHR and ARNT proteins remained unchanged upon treatment of the cells with TSA or NaBu, the transcriptional upregulation of CYP1A1 gene was not due to an increased expression of the regulatory proteins. These findings strongly suggest an involvement of HDAC in the repression of the CYP1A1 gene. Similar to the native human CYP1A1 also the mouse CYP1A1-driven reporter gene transfected into HeLa cells was repressed by histone deacetylase since the presence of TSA or NaBu led to an increase in the reporter activity. Induction of reporter gene did not require a presence of the promoter or negative regulatory regions of the CYP1A1 gene. A promoter-distal fragment containing three DREs together with surrounding sequences was sufficient to mediate the effects of the HDAC inhibitors suggesting that the AHR/ARNT binding to its specific DNA recognition site may be important for the CYP1A1 repression. Histone deacetylase is recruited to the specific genes by corepressors, proteins that bind to the transcription factors and interact with other members of the HDAC complex. Western blot analyses revealed a presence of HDAC1 and the corepressors mSin3A (mammalian homolog of yeast Sin3) and SMRT (silencing mediator for retinoid and thyroid hormone receptor) in both cell types, while the corepressor NCoR (nuclear receptor corepressor) was expressed exclusively in HeLa cells. Thus the high inducibility of CYP1A1 in Hepa cells may be due to the absence of NCoR in these cells in contrast to the non-responsive HeLa cells, where the presence of NCoR would support repression of the gene by histone deacetylase. This hypothesis was verified in reporter gene experiments where expression constructs coding for the particular members of the HDAC complex were cotransfected in Hepa cells together with the TCDD-inducible reporter constructs containing the CYP1A1 regulatory sequences. An overexpression of NCoR however did not decrease but instead led to a slight increase of the reporter gene activity in the cells. The expected inhibition was observed solely in the case of SMRT that slightly reduced constitutive and TCDD-induced reporter gene activity. A simultaneous expression of NCoR and SMRT shown no further effects and coexpression of HDAC1 with the two corepressors did not alter this situation. Thus, additional factors that are likely involved in the repression of CYP1A1 gene by HDAC complex remained to be identified. Taking together, characterisation of an exogenous ligand independent AHR/ARNT complex on DRE in HeLa cells that repress transcription of the CYP1A1 gene creates a model system enabling investigation of endogenous processes involved in the regulation of AHR function. This study implicates HDAC-mediated repression of CYP1A1 gene that contributes to the xenobiotic-induced expression in a tissue specific manner. Elucidation of these processes gains an insight into mechanisms leading to deleterious effects of TCDD and related compounds.

Leaf level VOC emissions of single plants from Amazonian and Mediterranean ecosystems: Ontogeny and flooding as stress factor for VOC emissions

Die Vegetation ist die wichtigste Quelle von organischen flüchtigen Verbindungen (auf Englisch volatile organic compounds,VOCs), die einen bemerkenswerten Einfluss auf der Chemie und Physik der Atmosphäre haben. VOCs beeinflussen die oxidative Kapazität der Atmosphäre und tragen zu der Bildung und zum Wachstum von sekundären organischen Aerosolen bei, welche einerseits eine Streuung und Reflektierung der Energie verursachen und andererseits sich an der Bildung und Entwicklung von Wolken beteiligen. Ziel dieser Arbeit war die Beschreibung und der Vergleich von VOC Emissionen aus Pflanzen aus zwei verschiedenen Ökosystemen: Mediterranes Ökosystem und Tropisches Ökosystem. Für diese Aufgabe wurden gewöhnliche Pflanzen von beiden Ökosystemen untersucht. Siebzehn Pflanzenspezies aus der Mittelmeergebiet, welches bekannt ist für seine Vielfalt an VOC emittierenden Pflanzen, wurden in die Untersuchungen einbezogen. Im Gegensatz zum mediterranen Ökosystem sind nur wenig Information verfügbar über VOC Emissionen aus Blättern tropischer Baumspezies. Vor diesem Hintergrund wurden sechsundzwanzig Baumspezies aus verschiedenen Ökotypen des Amazonasbeckens (Terra firme, Várzea und Igapó) wurden auf VOC Emissionen auf Blattebene mit einem Küvetten-System untersucht. Analysen von flüchtigen organischen Verbindungen wurden online mit PTR-MS und offline mittels Sammlung auf entsprechenden Adsorbern (Kartuschen) und nachfolgender GC-FID Analyse untersucht. Die höchsten Emissionen wurden für Isoprene beobachtete, gefolgt durch Monoterpene, Methanol und Aceton. Die meisten Mittelmeer Spezies emittierten eine hohe Vielfalt an Monoterpenspezies, hingegen zeigten nur fünf tropische Pflanzenspezies eine Monoterpene mit einen sehr konservativen Emissionsprofil (α-Pinen>Limonen>Sabinen >ß-Pinen). Mittelmeerpflanzen zeigten zusätzlich Emissionen von Sesquiterpenen, während bei der Pflanzen des Amazonas Beckens keine Sesquiterpenemissionen gefunden wurden. Dieser letzte Befund könnte aber auch durch eine niedrigere Sensitivität des Messsystems während der Arbeiten im Amazonasgebiet erklärt werden. Zusätzlich zu den Isoprenoidemissionen waren Methanolemissionen als Indikator für Wachtumsvorgänge sehr verbreitet in den meisten Pflanzenspezies aus tropischen und mediterranen Gebieten. Einige Pflanzenspezies beider Ökosystemen zeigten Acetonemissionen. rnrnVOC Emissionen werde durch eine große Vielfalt an biotischen und abiotischen Faktoren wie Lichtintensität, Temperatur, CO2 und Trockenheit beeinflusst. Ein anderer, öfter übersehener Faktor, der aber sehr wichtig ist für das Amazonas Becken, ist die regelmäßige Überflutung. In dieser Untersuchung wir fanden heraus, dass am Anfang einer Wurzelanoxie, die durch die Überflutung verursacht wurde, Ethanol und Acetaldehyd emittiert werden können, vor allem in Pflanzenspezies, die schlechter an eine unzureichende Sauerstoffversorgung bei Flutung adaptiert sind, wie z.B. Vatairea guianensis. Die Spezies Hevea spruceana, welche besser an Überflutung adaptiert ist, könnte möglicherweise der gebildete Ethanol sofort remetabolisieren ohne es zu emittieren. Nach einer langen Periode einer Überflutung konnte allerdings keine Emission mehr beobachtet werden, was auf eine vollständige Adaptation mit zunehmender Dauer schließen lässt. Als Reaktion auf den ausgelösten Stress können Isoprenoidemissionen ebenfalls kurzfristig nach einigen Tage an Überflutung zunehmen, fallen dann aber dann nach einer langen Periode zusammen mit der Photosynthese, Transpiration und stomatäre Leitfähigkeit deutlich ab.rnrnPflanzen Ontogenese ist anscheinend von Bedeutung für die Qualität und Quantität von VOC Emissionen. Aus diesem Grund wurden junge und erwachsene Blätter einiger gut charakterisierten Pflanzen Spezies aus dem Mittelmeerraum auf VOC Emissionen untersucht. Standard Emissionsfaktoren von Isopren waren niedriger in jungen Blättern als in erwachsene Blätter. Hingegen wurden höhere Monoterpen- und Sesquiterpenemissionen in jungen Blätter einiger Pflanzenspezies gefunden. Dieser Befund deutet auf eine potentielle Rolle dieser VOCs als Abwehrkomponenten gegen Pflanzenfresser oder Pathogene bei jungen Blätter hin. In einigen Fällen variierte auch die Zusammensetzung der Monoterpen- und Sesquiterpenspezies bei jungen und erwachsenen Blättern. Methanolemissionen waren, wie erwartet, höher in jungen Blättern als in ausgewachsenen Blättern, was mit der Demethylierung von Pectin bei der Zellwandreifung erklärt werden kann. Diese Befunde zu Änderungen der Emissionskapazität der Vegetation können für zukünftige Modellierungen herangezogen werden. rn

A SMO inhibitor drug reduces the progenitor cell's maintenance in the Drosophila hematopoietic organ: a novel model to study Chronic Myelogenous Leukemia relapse

The chronic myeloid leukemia complexity and the difficulties of disease eradication have recently led to the development of drugs which, together with the inhibitors of TK, could eliminate leukemia stem cells preventing the occurrence of relapses in patients undergoing transplantation. The Hedgehog (Hh) signaling pathway positively regulates the self-renewal and the maintenance of leukemic stem cells and not, and this function is evolutionarily conserved. Using Drosophila as a model, we studied the efficacy of the SMO inhibitor drug that inhibit the human protein Smoothened (SMO). SMO is a crucial component in the signal transduction of Hh and its blockade in mammals leads to a reduction in the disease induction. Here we show that administration of the SMO inhibitor to animals has a specific effect directed against the Drosophila ortholog protein, causing loss of quiescence and hematopoietic precursors mobilization. The SMO inhibitor induces in L3 larvae the appearance of melanotic nodules generated as response by Drosophila immune system to the increase of its hemocytes. The same phenotype is induced even by the dsRNA:SMO specific expression in hematopoietic precursors of the lymph gland. The drug action is also confirmed at cellular level. The study of molecular markers has allowed us to demonstrate that SMO inhibitor leads to a reduction of the quiescent precursors and to an increase of the differentiated cells. Moreover administering the inhibitor to heterozygous for a null allele of Smo, we observe a significant increase in the phenotype penetrance compared to administration to wild type animals. This helps to confirm the specific effect of the drug itself. These data taken together indicate that the study of inhibitors of Smo in Drosophila can represent a useful way to dissect their action mechanism at the molecular-genetic level in order to collect information applicable to the studies of the disease in humans.

Drugs down-regulating E2F-1 expression hinders cell proliferation through a p53-indipendent mechanism

E2F-1 is a transcription factor that plays a key role in cell-cycle control at G1/S check-point level by regulating the timely expression of many target genes whose products are required for S phase entry and progression. In mammalian cells, E2F-1 is negatively regulated by hypo-phosphorylated Retinoblastoma protein (pRb) whereas it is protected against degradation by its binding to Mouse Double Minute 2 protein (MDM2). In this study we experimented a drug combination in order to obtain a strong down-regulation of E2F-1 by acting on two different mechanisms of E2F-1 regulation mentioned above. This was achieved by combining drugs inhibiting the phosphorylation of pRb with drugs inactivating the MDM2 binding capability. The mechanism of action of these drugs in down-regulating E2F-1 level and activity is p53 independent. As expected, when combined, these drugs strongly inhibits E2F-1 and hinder cell proliferation in p53-/- and p53-mutated cells by blocking them in G1 phase of cell cycle, suggesting that E2F-1 down-regulation may represent a valid chemotherapeutic approach to inhibit proliferation in tumors independently of p53 status.

Regulatory T cell-mediated suppression of Th9 cell development and effector function

T helper (Th) 9 cells are an important subpopulation of the CD4+ T helper cells. Due to their ability to secrete Interleukin-(IL-)9, Th9 cells essentially contribute to the expulsion of parasitic helminths from the intestinal tract but they play also an immunopathological role in the course of asthma. Recently, a beneficial function of Th9 cells in anti-tumor immune responses was published. In a murine melanoma tumor model Th9 cells were shown to enhance the anti-melanoma immune response via the recruitment of CD8+ T cells, dendritic cells and mast cells. In contrast to Th9 effector cells regulatory T cells (Tregs) are able to control an immune response with the aid of different suppressive mechanisms. Based on their ability to suppress an immune response Tregs are believed to be beneficial in asthma by diminishing excessive allergic reactions. However, concerning cancer they can have a detrimental function because Tregs inhibit an effective anti-tumor immune reaction. Thus, the analysis of Th9 suppression by Tregs is of central importance concerning the development of therapeutic strategies for the treatment of cancer and allergic diseases and was therefore the main objective of this PhD thesis.rnIn general it could be demonstrated that the development of Th9 cells can be inhibited by Tregs in vitro. The production of the lineage-specific cytokine IL-9 by developing Th9 cells was completely suppressed at a Treg/Th9 ratio of 1:1 on the transcriptional (qRT-PCR) as well as on the translational level (ELISA). In contrast, the expression of IRF4 that was found to strongly promote Th9 development was not reduced in the presence of Tregs, suggesting that IRF4 requires additional transcription factors to induce the differentiation of Th9 cells. In order to identify such factors, which regulate Th9 development and therefore represent potential targets for Treg-mediated suppressive mechanisms, a transcriptome analysis using “next-generation sequencing” was performed. The expression of some genes which were found to be up- or downregulated in Th9 cells in the presence of Tregs was validated with qRT-PCR. Time limitations prevented a detailed functional analysis of these candidate genes. Nevertheless, the analysis of the suppressive mechanisms revealed that Tregs probably suppress Th9 cells via the increase of the intracellular cAMP concentration. In contrast, IL-9 production by differentiated Th9 cells was only marginally affected by Tregs in vitro and in vivo analysis (asthma, melanoma model). Hence, Tregs represent very effective inhibitors of Th9 development whereas they have only a minimal suppressive influence on differentiated Th9 cells.rn

The CFTR frameshift mutation 3905insT and its effect at transcript and protein level

Cystic fibrosis (CF) is one of the most common genetic diseases in the Caucasian population and is characterized by chronic obstructive pulmonary disease, exocrine pancreatic insufficiency, and elevation of sodium and chloride concentrations in the sweat and infertility in men. The disease is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, which encodes a protein that functions as chloride channel at the apical membrane of different epithelia. Owing to the high genotypic and phenotypic disease heterogeneity, effects and consequences of the majority of the CFTR mutations have not yet been studied. Recently, the frameshift mutation 3905insT was identified as the second most frequent mutation in the Swiss population and found to be associated with a severe phenotype. The frameshift mutation produces a premature termination codon (PTC) in exon 20, and transcripts bearing this PTC are potential targets for degradation through nonsense-mediated mRNA decay (NMD) and/or for exon skipping through nonsense-associated alternative splicing (NAS). Using RT-PCR analysis in lymphocytes and different tissue types from patients carrying the mutation, we showed that the PTC introduced by the mutation does neither elicit a degradation of the mRNA through NMD nor an alternative splicing through NAS. Moreover, immunocytochemical analysis in nasal epithelial cells revealed a significantly reduced amount of CFTR at the apical membrane providing a possible molecular explanation for the more severe phenotype observed in F508del/3905insT compound heterozygotes compared with F508del homozygotes. However, further experiments are needed to elucidate the fate of the 3905insT CFTR in the cell after its biosynthesis.

Effects of TLR agonists on the hypoxia-regulated transcription factor HIF-1alpha and dendritic cell maturation under normoxic conditions

Dendritic cells (DC) are professional antigen presenting cells that represent an important link between innate and adaptive immunity. Danger signals such as toll-like receptor (TLR) agonists induce maturation of DC leading to a T-cell mediated adaptive immune response. In this study, we show that exogenous as well as endogenous inflammatory stimuli for TLR4 and TLR2 induce the expression of HIF-1alpha in human monocyte-derived DC under normoxic conditions. On the functional level, inhibition of HIF-1alpha using chetomin (CTM), YC-1 and digoxin lead to no consistent effect on MoDC maturation, or cytokine secretion despite having the common effect of blocking HIF-1alpha stabilization or activity through different mechanisms. Stabilization of HIF-1alpha protein by hypoxia or CoCl(2) did not result in maturation of human DC. In addition, we could show that TLR stimulation resulted in an increase of HIF-1alpha controlled VEGF secretion. These results show that stimulation of human MoDC with exogenous as well as endogenous TLR agonists induces the expression of HIF-1alpha in a time-dependent manner. Hypoxia alone does not induce maturation of DC, but is able to augment maturation after TLR ligation. Current evidence suggests that different target genes may be affected by HIF-1alpha under normoxic conditions with physiological roles that differ from those induced by hypoxia.