Evolution of JAK-STAT pathway components : mechanisms and role in immune system development

Background
Lying downstream of a myriad of cytokine receptors, the Janus kinase (JAK) – Signal transducer and activator of transcription (STAT) pathway is pivotal for the development and function of the immune system, with additional important roles in other biological systems. To gain further insight into immune system evolution, we have performed a comprehensive bioinformatic analysis of the JAK-STAT pathway components, including the key negative regulators of this pathway, the SH2-domain containing tyrosine phosphatase (SHP), Protein inhibitors against Stats (PIAS), and Suppressor of cytokine signaling (SOCS) proteins across a diverse range of organisms.

Results
Our analysis has demonstrated significant expansion of JAK-STAT pathway components co-incident with the emergence of adaptive immunity, with whole genome duplication being the principal mechanism for generating this additional diversity. In contrast, expansion of upstream cytokine receptors appears to be a pivotal driver for the differential diversification of specific pathway components.

Conclusion
Diversification of JAK-STAT pathway components during early vertebrate development occurred concurrently with a major expansion of upstream cytokine receptors and two rounds of whole genome duplications. This produced an intricate cell-cell communication system that has made a significant contribution to the evolution of the immune system, particularly the emergence of adaptive immunity.

Microfluidic platforms for the investigation of intercellular signalling mechanisms

Intercellular signalling has been identified as a highly complex process, responsible for orchestrating many physiological functions. While conventional methods of investigation have been useful, their limitations are impeding further development. Microfluidics offers an opportunity to overcome some of these limitations. Most notably, microfluidic systems can emulate the in-vivo environments. Further, they enable exceptionally precise control of the microenvironment, allowing complex mechanisms to be selectively isolated and studied in detail. There has thus been a growing adoption of microfluidic platforms for investigation of cell signalling mechanisms. This review provides an overview of the different signalling mechanisms and discusses the methods used to study them, with a focus on the microfluidic devices developed for this purpose.

Cytokine networks and cancer stem cells

Cell-to-cell communication is an integral function of multicellular organisms. Many of these signals are received by a myriad of cell-surface receptors that utilize a range of intracellular signaling pathways to communicate this to the nucleus, rapidly impacting on the transcription of target genes in order to elicit the desired response, such as proliferation, differentiation, activation, and survival. Dysregulation of these important signaling pathways, and networks, often lead to pathological conditions due to inappropriate cell responses with negative consequences. The aberrant signaling pathways have been associated with many diseases, including cancer. Cytokines and chemokines convey a multitude of messages to the target cell, many of which are beneficial for cancers and cancer stem cells, such as proliferation, survival and migration. By hijacking this communication network, cancers and cancer stem cells can become invasive and more pathogenic. Furthermore, by using these communication systems, cancer stem cells are able to evade current therapies. Therefore, novel therapies may be developed to break the communication systems of the cancer stem cells. This chapter explores the role of the cytokines TGF-β, TNF-α, IL-1 and IL-6 and chemokine CXCL8 as well as NF-κB and their role in cancer stem cell survival and maintenance. Emerging therapies are beginning to target the cancer stem cell population, either specifically or synergistically with existing therapeutic options. These novel therapies may hold the key to breaking the communication network of cancer stem cells.

Prospecção e caracterização de genes associados ao processo de indução floral em cana-de-açúcar

The northeastern region is responsible to 14.32% of sugarcane national production. This lowered contribution is due to edaphoclimatic condition. Flowering is a vital process to plant which consumes lots of energy and it culminates in a process called isoporization. This one can give in a decreasing of 60% on alcohol and water production. It may consider that cropped sugarcane has a hibrid with octaploid genome, there are varieties with a flowering standard until of non flowering. Using this natural genetic potential on different croppings of sugarcane, the aim of this work was to understand as this process occurs by the usage of subtractive approaches. The total RNA was extracted using Trizol of peaks of merisematics of croppings with induced flowering and other with late flowering. From this total RNA were built four subtractives libraries (B1- induced early flowering subtracted on late flowering not induced; B2- late flowering not induced subtracted induced early flowering; B3- induced early flowering subtracted of not induced early flowering; B02- not induced early flowering subtracted from induced early flowering) using kits Super Smart cDNA synthesis and BD Clontech kit select cDNA subtraction (Clontech). This material was clone don vector pGEM T-easy(Promega) and changed in competent cells of E.coli DH10B. Given analysis sequence was carried out a program BLASTn against database of NCBI and genome of Arabidopsis thaliana, rice and maize. Clones were grouped in 9 different classes according to function. Some factors already related as couples of flower induction were identified at different libraries. And grouped proteins with cell cycle and it controls were presents, mainly kinases proteins. Related factors to proteic sinthesis, metabolism, defence, cell communication were also given in both libraries .Some identified genes did not show similarity on database or homology with hypothesis function, and it can represents new genes to be deposited in international database. These results offers that some identified on sugarcane, classified as on factors classes, cell cycle and cell communication, trough unknown genes, can be linked with genetic changing to the flowering process found in the northeastern region

Intercellular pectic protuberances in Hymenaea stigonocarpa (Fabaceae, Caesalpinioideae): Occurrence and functional aspects

A study of the anatomy and ultrastructural aspects of leaf mesophyll and floral nectaries of Hymenaea stigonocarpa Mart. ex Hayne revealed the presence of intercellular pectic protuberances (IPPs) linking adjacent cells in both the leaf palisade cells and the secretory parenchyma of the floral nectary. Samples of the middle third of the leaf blade and of floral nectaries in anthesis were collected, fixed, and processed using standard procedures for light, transmission, and scanning electron microscopies. The IPPs of palisade cells of the mesophyll and the secretory parenchyma cells of the floral nectary take the form of scalae or strands, respectively. No evidence of the specific synthesis of these structures was observed, and they are apparently formed by the separation of adjacent cells due to cell expansion, when intercellular spaces develop. The IPPs observed in H. stigonocarpa increase cellular contact and probably act in apoplastic transport.

Secretion of tumor necrosis factor-alpha by mouse peritoneal macrophages in the presence of dental sealers, sealapex and endomethasone

After filling root canals, the healing process depends on the chemical composition or physical-chemical properties of the material used, among other factors. All root canal sealers, whether solid or plastic, are foreign matter for the body if they remain in permanent contact with apical and periapical tissues. As a result, the first organic reaction that occurs is an attempt to phagocytize the material. During phagocytosis, macrophages release a large number of cell mediators into the area, among which are cytokines that are essential in intercellular communication and in many physiological and pathophysiological processes. One of these cytokines is tumor necrosis factor-alfa (TNF-α), which acts through links to specific receptors on the cell membrane initiating a cascade of events leading to induction, activation, or inhibition of numerous cytokine-regulated genes in the cell nucleus. The release of TNF-α in a cell culture of mouse peritoneal macrophages incubated with three concentrations (25, 50, and 100 mg/ml) of two endodontic sealers was measured. The solutions containing the calcium hydroxide-based root canal sealer (Sealapex) released fewer units of TNF-α than solutions containing the zinc oxide and eugenol-based sealer (Endomethasone).

Chagas disease and triatomine biology (Heteroptera, Triatominae), with emphasis on aspects of spermatogenesis

A century after the discovery of Chagas disease, it is still one of the most important parasitic diseases affecting humans. The subfamily Triatominae is important in medical health, because these insects are vectors of Trypanosoma cruzi, the etiologic agent of Chagas disease. These insects are also of important cytological relevance because they have particular cell characteristics, such as persistence of nucleolar material in spermatogenesis. The germ cells of the animal kingdom have chromatoid bodies (CBs) in their cytoplasm that can originate from nucleolar material that is fragmented in the early stages of spermatogenesis and plays an important role in cellular communication between the spermatids during spermiogenesis. Currently, there are few studies on the function and formation of the CB in nucleologenesis, especially with emphasis on the ultrastructure of the cells involved in spermatogenesis of insects. Considering the importance of knowledge about the triatomine fauna, we conducted a study of the biogeography and reports of these insects and a survey of patients with Chagas disease in the northwestern region of São Paulo State. Data collected from 1995 to 2009 indicated 700 individuals with Chagas disease, demonstrating a range of 0 to 40 years, which shows that the disease may be active in this region. Moreover, of the 1150 patients treated for cardiomyopathy, 44% were chagasic. Regarding the triatomines noted and captured in the period from 2004 to 2009, the species were Triatoma sordida and Rhodnius neglectus, with T. sordida being the most abundant. In addition, some triatomines were infected by T. cruzi in various developmental stages. We also analyzed the nucleolar cycle and fibrillarin nucleolar protein expression in CB of spermatogenic cells of T. infestans and T. sordida, using histological, ultrastructural and immunocytochemical techniques. The results revealed fibrillarin nucleolar protein expression in the nucleus and in some cytoplasmic spots of germ cells during spermatogenesis in triatomines. These data suggest that fibrillarin could be a constituent of CB, which was most likely derived from nucleolar fragmentation. This is the first time that fibrillarin protein expression has been shown in CB during spermatogenesis progression in triatomines. Knowledge about the biology of triatomines was deepened in this study and, in particular, the structural and ultrastructural aspects of spermatogenesis in triatomines. This study showed that the disease may be active in the northwestern region of São Paulo and expanded our knowledge of the biology of triatomines, the main vectors of Chagas disease. © FUNPEC-RP.

Alocação de canais em sistemas de comunicação celular empregando algoritmo genético distribuído

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Identificação e análise da expressão de genes relacioanados com tolerância à seca em soja através de microarranjos de DNA e PCR em tempo real

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Vesicles as carriers of virulence factors in parasitic protozoan diseases

Different types of shed vesicles as, for example, exosomes, plasma-membrane-derived vesicles or microparticles, are the focus of intense research in view of their potential role in cell cell communication and under the perspective that they might be good tools for immunotherapy, vaccination or diagnostic purposes. This review discusses ways employed by pathogenic trypanosomatids to interact with the host by shedding vesicles that contain molecules important for the establishment of infection, as opposed to previous beliefs considering them as a waste of cellular metabolism. Trypanosomatids are compared with Apicomplexa, which circulate parasite antigens bound to vesicles shed by host cells. The knowledge of the origin and chemical composition of these different vesicles might lead to the understanding of the mechanisms that determine their biological function. (C) 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Implications of purinergic receptor-mediated intracellular calcium transients in neural differentiation

Purinergic receptors participate, in almost every cell type, in controlling metabolic activities and many physiological functions including signal transmission, proliferation and differentiation. While most of P2Y receptors induce transient elevations of intracellular calcium concentration by activation of intracellular calcium pools and forward these signals as waves which can also be transmitted into neighboring cells, P2X receptors produce calcium spikes which also include activation of voltage-operating calcium channels. P2Y and P2X receptors induce calcium transients that activate transcription factors responsible for the progress of differentiation through mediators including calmodulin and calcineurin. Expression of P2X2 as well as of P2X7 receptors increases in differentiating neurons and glial cells, respectively. Gene expression silencing assays indicate that these receptors are important for the progress of differentiation and neuronal or glial fate determination. Metabotropic receptors, mostly P2Y1 and P2Y2 subtypes, act on embryonic cells or cells at the neural progenitor stage by inducing proliferation as well as by regulation of neural differentiation through NFAT translocation. The scope of this review is to discuss the roles of purinergic receptor-induced calcium spike and wave activity and its codification in neurodevelopmental and neurodifferentiation processes.

Controllo molecolare del differenziamento osteoblestico per applicazioni nel campo della rigenerazione del tessuto osseo

The aim of the study was to identify expression signatures unique for specific stages of osteoblast differentiation in order to improve our knowledge of the molecular mechanisms underlying bone repair and regeneration. We performed a microarray analysis on the whole transcriptome of human mesenchymal stem cells (hMSCs) obtained from the femoral canal of patients undergoing hip replacement. By defining different time-points within the differentiation and mineralization phases of hMSCs, temporal gene expression changes were visualised. Importantly, the gene expression of adherent bone marrow mononuclear cells, being the undifferentiated progenitors of bone cells, was used as reference. In addition, only the cultures able to form mineral nodules at the final time-point were considered for the gene expression analyses. To obtain the genes of our interest, we only focused on genes: i) whose expression was significantly upregulated; ii) which are involved in pathways or biological processes relevant to proliferation, differentiation and functions of bone cells; iii) which changed considerably during the different steps of differentiation and/or mineralization. Among the 213 genes identified as differentially expressed by microarray analysis, we selected 65 molecular markers related to specific steps of osteogenic differentiation. These markers are grouped into various gene clusters according to their involvement in processes which play a key role in bone cell biology such as angiogenesis, ossification, cell communication, development and in pathways like TGF beta and Wnt signaling pathways. Taken together, these results allow us to monitor hMSC cultures and to distinguish between different stages of differentiation and mineralization. The signatures represent a useful tool to analyse a broad spectrum of functions of hMSCs cultured on scaffolds, especially when the constructs are conceived for releasing growth factors or other signals to promote bone regeneration. Morover, this work will enhance our understanding of bone development and will enable us to recognize molecular defects that compromise normal bone function as occurs in pathological conditions.

Machine-learning methods for structure prediction of β-barrel membrane proteins

Different types of proteins exist with diverse functions that are essential for living organisms. An important class of proteins is represented by transmembrane proteins which are specifically designed to be inserted into biological membranes and devised to perform very important functions in the cell such as cell communication and active transport across the membrane. Transmembrane β-barrels (TMBBs) are a sub-class of membrane proteins largely under-represented in structure databases because of the extreme difficulty in experimental structure determination. For this reason, computational tools that are able to predict the structure of TMBBs are needed. In this thesis, two computational problems related to TMBBs were addressed: the detection of TMBBs in large datasets of proteins and the prediction of the topology of TMBB proteins. Firstly, a method for TMBB detection was presented based on a novel neural network framework for variable-length sequence classification. The proposed approach was validated on a non-redundant dataset of proteins. Furthermore, we carried-out genome-wide detection using the entire Escherichia coli proteome. In both experiments, the method significantly outperformed other existing state-of-the-art approaches, reaching very high PPV (92%) and MCC (0.82). Secondly, a method was also introduced for TMBB topology prediction. The proposed approach is based on grammatical modelling and probabilistic discriminative models for sequence data labeling. The method was evaluated using a newly generated dataset of 38 TMBB proteins obtained from high-resolution data in the PDB. Results have shown that the model is able to correctly predict topologies of 25 out of 38 protein chains in the dataset. When tested on previously released datasets, the performances of the proposed approach were measured as comparable or superior to the current state-of-the-art of TMBB topology prediction.

Molecular and functional characterization of the chemotactic genes in the PCBs-degrader Pseudomonas pseudoalcaligenes KF707

Although bacteria represent the simplest form of life on Earth, they have a great impact on all living beings. For example the degrader bacterium Pseudomonas pseudoalcaligenes KF707 is used in bioremediation procedures for the recovery of polluted sites. Indeed, KF707 strain is know for its ability to degrade biphenyl and polychlorinated biphenyls - to which is chemotactically attracted - and to tolerate the oxydative stress due to toxic metal oxyanions such as tellurite and selenite. Moreover, in bioremediation processes, target compounds can be easily accessible to KF707 through biofilm formation. All these considerations suggest that KF707 is such a unique microorganism and this Thesis work has been focused on determining the molecular nature of some of the peculiar physiological traits of this strain. The genome project provided a large set of informations: putative genes involved in the degradation of aromatic and toxic compounds and associated to stress response were identified. Notably, multiple chemotactic operons and cheA genes were also found. Deleted mutants in the cheA genes were constructed and their role in motility, chemotaxis and biofilm formation were assessed and compared to those previously attributed to a cheA1 gene in a KF707 mutant constructed by a mini-Tn5 transposon insertion and which was impaired in motility and biofilm development. The results of this present Thesis work, taken together, were interpreted to suggest that in Pseudomonas pseudoalcaligenes KF707 strain, multiple factors are involved in these networks and they might play different roles depending on the environmental conditions. The ability of KF707 strain to produce signal molecules possibly involved in cell-to-cell communication, was also investigated: lack of a lux-like QS system - which is conversely widely present in Gram negative bacteria – keeps open the question about the actual molecular nature of KF707 quorum sensing mechanism.

Interactions of inhaled nanoparticles with the alveolar-capillary barrier of the human respiratory tract

In dieser Arbeit wurden zytotoxische Effekte sowie die inflammatorische Reaktionen des distalen respiratorischen Traktes nach Nanopartikelexposition untersucht. Besondere Aufmerksamkeit lag auch auf der Untersuchung unterschiedlicher zellulärer Aufnahmewege von Nanopartikeln wie z.B. Clathrin- oder Caveolae-vermittelte Endozytose oder auch Clathrin- und Caveolae-unabhängige Endozytose (mit möglicher Beteiligung von Flotillinen). Drei unterschiedliche Nanopartikel wurden hierbei gewählt: amorphes Silica (aSNP), Organosiloxan (AmorSil) und Poly(ethyleneimin) (PEI). Alle unterschiedlichen Materialien gewinnen zunehmend an Interesse für biomedizinische Forschungsrichtungen (drug and gene delivery). Insbesondere finden aSNPs auch in der Industrie vermehrt Anwendung, und stellen somit ein ernstzunehmendes Gesundheitsrisiko dar. Dieser wird dadurch zu einem begehrten Angriffsziel für pharmazeutische Verabreichungen von Medikamenten über Nanopartikel als Vehikel aber bietet zugleich auch eine Angriffsfläche für gesundheitsschädliche Nanomaterialien. Aus diesem Grund sollten die gesundheitsschädigenden Risiken, sowie das Schicksal von zellulär aufgenommenen NPs sorgfältig untersucht werden. In vivo Studien an der alveolaren-kapillaren Barriere sind recht umständlich. Aus diesem Grund wurde in dieser Arbeit ein Kokulturmodel benutzt, dass die Alveolar-Kapillare Barrier in vivo nachstellt. Das Model besteht aus dem humanen Lungenepithelzelltyp (z.B. NCI H441) und einem humanen microvasculären Endothelzelltyp (z.B. ISO-HAS-1), die auf entgegengesetzten Seiten eines Transwell-Filters ausgesät werden und eine dichte Barriere ausbilden. Die NP Interaktion mit Zellen in Kokultur wurde mit denen in konventioneller Monokultur verglichen, in der Zellen 24h vor dem Experiment ausgesät werden. Diese Studie zeigt, dass nicht nur die polarisierte Eigenschaft der Zellen in Kokultur sondern auch die unmittelbare Nähe von Epithel und Endothelzelle ausschlaggebend für durch aSNPs verursachte Effekte ist. Im Hinblick auf inflammatorische Marker (sICAM, IL-6, IL8-Ausschüttung), reagiert die Kokultur auf aSNPs empfindlicher als die konventionelle Monokultur, wohingegen die Epithelzellen in der Kokultur auf zytotoxikologischer Ebene (LDH-Ausschüttung) unempfindlicher auf aSNPs reagierten als die Zellen in Monokultur. Aufnahmestudien haben gezeigt, dass die Epithelzellen in Kokultur entschieden weniger NPs aufnehmen. Somit zeigen die H441 in der Kokultur ähnliche epitheliale Eigenschaften einer schützenden Barriere, wie sie auch in vivo zu finden sind. Obwohl eine ausreichende Aufnahme von NPs in H441 in Kokultur erreicht werden konnte, konnte ein Transport von NPs durch die epitheliale Schicht und eine Aufnahme in die endotheliale Schicht mit den gewählten Inkubationszeiten nicht gezeigt werden. Eine Clathrin- oder Caveolae-vermittelte Endozytose von NPs konnte mittels Immunfluoreszenz weder in der Mono- noch in der Kokultur nachgewiesen werden. Jedoch zeigte sich eine Akkumulation von NPs in Flotillin-1 und-2 enthaltende Vesikel in Epithelzellen aus beiden Kultursystemen. Ergebnisse mit Flotillin-inhibierten (siRNA) Epithelzellen, zeigten eine deutlich geringere Aufnahme von aSNPs. Zudem zeigte sich eine eine reduzierte Viabilität (MTS) von aSNP-behandelten Zellen. Dies deutet auf eine Beteiligung von Flotillinen an unbekannten (Clathrin oder Caveolae -unabhängig) Endozytosemechanismen und (oder) endosomaler Speicherung. Zusammenfassend waren die Aufnahmemechanismen für alle untesuchten NPs in konventioneller Monokultur und Kokultur vergleichbar, obwohl sich die Barriereeigenschaften deutlich unterscheiden. Diese Arbeit zeigt deutlich, dass sich die Zellen in Kokultur anders verhalten. Die Zellen erreichen hierbei einen höheren Differenzierungsgrad und eine Zellkommunikation mit anderen relevanten Zelltypen wird ermöglicht. Durch das Einbringen eines dritten relevanten Zelltyps in die Kokultur, des Alveolarmakrophagen (Zelllinie THP-1), welcher die erste Verteidigungsfront im Alveolus bildet, wird diese Aussage weiter bekräftigt. Erste Versuche haben gezeigt, dass die Triplekultur bezüglich ihrer Barriereeigenschaften und IL-8-Ausschüttung sensitiver auf z.B. TNF- oder LPS-Stimulation reagiert als die Kokultur. Verglichen mit konventionellen Monokulturen imitieren gut ausgebildete, multizelluräre Kokulturmodelle viel präziser das zelluläre Zusammenspiel im Körper. Darum liefern Nanopartikelinteraktionen mit dem in vitro-Triplekulturmodel aufschlussreichere Ergebnisse bezüglich umweltbedingter oder pharmazeutischer NP-Exposition in der distalen Lung als es uns bisher möglich war.