Oncogene c-KIT: promoter region and downstream effectors as novel therapeutic targets

An attractive anticancer strategy deals with the impairment of the functions of selected genes relevant for cell proliferation. Among them an interesting target is represented by KIT. c-KIT - V-Kit Hardy-Zuckerman 4 Feline Sarcoma Viral Oncogene Homolog - is a tyrosine kinase receptor involved in the regulation of cell survival and proliferation as well as in progression of some tumors. Its expression has been confirmed to be crucial for leukemias, mastocytosis, gastrointestinal stromal tumor, and lung carcinomas often associated to c-kit mis-regulation. Also the most common skin cancer in domestic dog, mast cell tumor (MCT), is linked to a mutation and/or to an over-expression of c-kit, thus supporting dog as an excellent animal model. In this work we explore two strategies to suppress the cell growth activity of this oncogene. The first one was based on the downregulation of gene expression by induction of non-canonical DNA structures at promotorial level. Indeed, in this region of the human oncogene KIT two guanine-rich sequences (KIT1_man and KIT2_man) were identified. Here, we characterized the canine KIT promoter region in terms of sequence and conformational equilibria in physiologically relevant conditions. Our results demonstrate that the conformational features of the canine KIT1 sequence are substantially shared with the human one whereas an altered distribution among several folded conformations characterizes the two isoforms identified in dog-derived KIT2 sequences. These structural diversities identified KIT1 and KIT2 as two potential distinct targets, which means that G-4 ligands can be designed to preferentially bind human/dog sequences. Thus, an “in house” library of compounds belonging to different chemical scaffolds (i.e., anthracene, anthracenedione, indole, quinoline, isoquinoline, fluorenone and heterocyclic diamidines derivatives) was screened for the recognition of these particular folding. Three promising G4-ligands, belonging to anthracenedione (Bal1,5 – Bapl2,6) and anthracene (Bis1,8) families, were selected and different techniques (Fluorescent Intercalator Displacement, Fluorescence melting studies, CD titrations, Surface Plasmon Resonance and Polymerase Stop Assay) were applied to fully dissect their interaction with KIT-related sequences. Our results showed that Bal1,5 is an efficient G-4 binder towards all the tested sequences, while Bapl2,6 and Bis1,8 preferentially recognize KIT2 sequences. Interestingly, a parallel level of cytotoxicity and suppression of KIT production was observed in cells treated with Bal1,5. In the second approach, downstream effectors of c-KIT pathway were considered as putative therapeutic targets. In particular we focused on a cytoplasmic tyrosine kinase FER. During the third year of my PhD I spent 7 months at Centre de Recherche en Cancérologie de Marseille (France) in the Dr P. Dubreuil’s team. In this period we worked to confirm a potential tumor suppressor role of FER. For this purpose mouse embryonic fibroblasts MEFs wild type and knockout for fer immortalized with SV40 were used. Removing fer, an uncontrolled growth was observed for KO MEFs in the contact inhibition test. However, at the moment a detailed description of the molecular events was not designed. Indeed no defined differences between molecular mechanisms, related to protein complexes involved in cell adhesion mechanisms, were observed between wt and KO MEFs. In conclusion, present results suggest that G-quadruplex structures at the promotorial level represent a promising therapeutic target in order to prevent oncogene KIT transcription. As far as FER is concerned, further studies are necessary to better define its possible tumor-suppressor role

Pedagogia materna e relaçõoes sociais

Dissertação (mestrado) - Universidade Federal de Santa Catarina. Centro de Ciencias da EducaçãoO trabalho tem como objeto de estudo a relação educativa entre mãe e filho, enquanto expressão da totalidade do social. Foi realizado a partir do convívio e de entrevistas orientadas, segundo os temas: Educação, Trabalho e Sociedade, a mães da chamada classe subalterna, direta e indiretamente relacionadas ao trabalho produtivos, seguidos de discussões aprofundadas com profissionais envolvidos com programas de educação infantil de quatro municípios do Estado de Santa Catarina. O estudo permitiu compreender a forma que toma em nossa sociedade, a relação pedagógica do grupo familiar, cuja síntese se vê expressa no discurso e repertório educacional materno. Traz os resultados e os pressupostos das relações sociais que caracterizam como naturais os processos de educação materna, que são sociais e culturalmente determinados

The Glitter of Carbon Nanostructures in Hybrid/Composite Hydrogels for Medicinal Use

In recent years, we have witnessed to fast developments in the medicinal field of hydrogels containing various forms of integrated nanostructured carbon that adds interesting mechanical, thermal, and electronic properties. Besides key advances in tissue engineering (especially for conductive tissue, such as for the brain and the heart), there has been innovation also in the area of drug delivery on-demand, with engineered hydrogels capable of repeated response to light, thermal, or electric stimuli. This mini-review focusses on the most promising developments as applied to the gelation of protein/ peptide (including self-assembling amino acids and low-molecular-weight gelators), polysaccharide, and/or synthetic polymer components in medicine. The emerging field of graphene-only hydrogels is also briefly discussed, to give the reader a full flavor of the rising new paradigms in medicine that are made possible through the integration of nanostructured carbon (e.g., carbon nanotubes, nanohorns, nanodiamonds, fullerene, etc.). Nanocarbons are offering great opportunities to bring on a revolution in therapy that the modern medicinal chemist needs to master, to realise their full potential into powerful therapeutic solutions for the patient

Evaluation of the efficacy of carbon nanotubes for delivering peptides into mitochondria

Mitochondrial (mt) diseases are devastating neurodegenerative pathologies due tomutations in nuclear or mt genes. Among mtDNA pathogenic mutations, more than one half have been identified in transfer RNA (tRNA) genes. These are responsible for a wide range of pathologies including myopathies, encephalopathies, cardiomyopathies and deafness for which no effective treatment is available at present. Therefore, new strategies to suppress their damaging effects are required to envisage therapeutic approaches for these diseases. Here we report data for carbon nanotube (CNT) derivatives showing that the conjugates bearing a specific peptide sequence are able to target the mitochondria in yeast and human monocyte cells while the control derivative without the peptide diffuses into the cytoplasm. Moreover the compounds do not affect cellular viability and cytotoxicity both in vitro and in vivo. Toxicity of the constructs is also assessed on the simple pluricellular model Caenorhabditis elegans

Screening of candidate G-quadruplex ligands for the human c-KIT promotorial region and their effects in multiple in-vitro models

Stabilization of G-quadruplex (G4) structures in promoters is a novel promising strategy to regulate gene expression at transcriptional and translational levels. c-KIT proto-oncogene encodes for a tyrosine kinase receptor. It is involved in several physiological processes, but it is also dysregulated in many diseases, including cancer. Two G-rich sequences able to fold into G4, have been identified in c-KIT proximal promoter, thus representing suitable targets for anticancer intervention. Herein, we screened an \u201cin house\u201d library of compounds for the recognition of these G4 elements and we identified three promising ligands. Their G4-binding properties were analyzed and related to their antiproliferative, transcriptional and post-transcriptional effects in MCF7 and HGC27 cell lines. Besides c-KIT, the transcriptional analysis covered a panel of oncogenes known to possess G4 in their promoters. From these studies, an anthraquinone derivative (AQ1) was found to efficiently downregulate c-KIT mRNA and protein in both cell lines. The targeted activity of AQ1 was confirmed using c-KIT\u2013dependent cell lines that present either c-KIT mutations or promoter engineered (i.e., \u3b1155, HMC1.2 and ROSA cells). Present results indicate AQ1 as a promising compound for the target therapy of c-KIT-dependent tumors, worth of further and in depth molecular investigations

Iron-related toxicity of single-walled carbon nanotubes and crocidolite fibres in human mesothelial cells investigated by Synchrotron XRF microscopy

Carbon nanotubes (CNTs) are promising products in industry and medicine, but there are several human health concerns since their fibrous structure resembles asbestos. The presence of transition metals, mainly iron, in the fibres seems also implicated in the pathogenetic mechanisms. To unravel the role of iron at mesothelial level, we compared the chemical changes induced in MeT-5A cells by the exposure to asbestos (crocidolite) or CNTs at different content of iron impurities (raw-SWCNTs, purified- and highly purified-SWCNTs). We applied synchrotron-based X-Ray Fluorescence (XRF) microscopy and soft X-ray imaging (absorption and phase contrast images) to monitor chemical and morphological changes of the exposed cells. In parallel, we performed a ferritin assay. X-ray microscopy imaging and XRF well localize the crocidolite fibres interacting with cells, as well as the damage-related morphological changes. Differently, CNTs presence could be only partially evinced by low energy XRF through carbon distribution and sometimes iron co-localisation. Compared to controls, the cells treated with raw-SWCNTs and crocidolite fibres showed a severe alteration of iron distribution and content, with concomitant stimulation of ferritin production. Interestingly, highly purified nanotubes did not altered iron metabolism. The data provide new insights for possible CNTs effects at mesothelial/pleural level in humans

Asiakaslähtöisen munasolunsaajien hoitoprosessin kehittäminen lapsettomuusklinikalla

Tämä opinnäytetyö oli työelämä lähtöinen kehittämistyö, joka toteutettiin Väestöliiton lapsettomuus klinikoiden Helsingin yksikössä. Tämän kehittämistyön tarkoituksena oli tuottaa uusi asiakaslähtöinen munansolunsaajien hoitoprosessi ja sen myötä parantaa asiakkaiden tyytyväisyyttä saamaansa hoitoon. Kehittämishankkeen tavoitteena oli kartoittaa haastattelemalla munasolunluovutushoidon läpi käyneiden asiakkaiden näkemyksiä nykyisestä hoitoprosessista ja sen asiakaslähtöisyydestä. Tavoitteena oli myös huomioida klinikan kokeneen henkilökunnan ajatuksia hoitoprosessin parantamiseksi. Uuden hoitoprosessin luominen aloitettiin määrittelemällä nykyinen munasolunsaajien hoitoprosessi. Tämän jälkeen kartoitettiin munasolunluovutushoidon läpi käyneiden asiakkaiden näkemyksiä nykyisestä hoitoprosessista, sen asiakaslähtöisyydestä ja heidän ehdotuksia hoitoprosessin parantamiseksi. Analysoitujen haastattelu aineistojen avulla tehtiin hahmotelma uudesta hoitoprosessista. Hahmotelma hoitoprosessista ja haastatteluiden tulokset esiteltiin klinikan henkilökunnalle, joka ideoi näiden pohjalta kehittämiskohteita uuteen hoitoprosessiin. Edellisten vaiheiden jälkeen kehitettiin lopullinen versio uudesta hoitoprosessista. Munasolunluovutushoito on otettu käyttöön Väestöliiton klinikoilla vuonna 1991 ja se on nykyisin yksi standardi lapsettomuudenhoito menetelmä. Munasolunluovutushoito on saajille iso panostus henkisesti ja taloudellisesti. Hoitoprosessin kehittämisellä pyritään siihen, että hoitoprosessin toimimattomuuden takia he eivät joutuisi uusien pettymysten eteen. Terveydenhuollossa on ymmärretty viime vuosina asiakaslähtöisyyden tärkeys niin Suomessa, kuin maailmallakin. Opinnäytetyön lähestymistapana oli toimintatutkimus. Aineistonkeruu menetelmänä oli munasolunluovutushoidossa olleiden asiakkaiden teemahaastattelu. Tämän lisäksi henkilökunnan ehdotuksia hoitoprosessin kehittämiseksi kartoitettiin aivoriihi ideoinnin avulla. Teemahaastatteluun osallistui kahdeksan asiakasta ja haastatteluaineisto analysoitiin induktiivisella sisällön analyysilla. Aivoriihi ideoinnin tuloksena syntyneet ideat pisteytettiin, ryhmiteltiin ja yhdisteltiin samaa tarkoittaviin ideoihin. Tämän jälkeen aineistosta muodostettiin uusi hoitoprosessi. Haastatteluaineiston tulosten mukaan asiakaslähtöisyys on empaattista vuorovaikutusta ja asiakkaan kokonaisuuden huomioimista. Asiakkaat kokivat nykyisen hoitoprosessin sujuvana, mutta toisaalta kokivat siihen sisältyvän hallitsemattomuutta. Hyvänä nykyisessä hoitoprosessissa he kokivat tulosten mukaan olevan empaattisen ja yksilöllisen kohtaamisen ja hoitoprosessin yksilöllisyyden. Monipuolisiin palveluihin, hoitoprosessin yksilölliseen suunnitteluun ja psyykkiseen taakkaan tulisi tulosten mukaan kiinnittää huomiota uutta hoitoprosessia laadittaessa. Suomessa ei ole tehty tutkimuksia asiakaslähtöisyydestä lapsettomuushoitoihin liittyen. Ulkomaisista tutkimuksista on saatu samansuuntaisia tuloksia asiakaslähtöisyydestä lapsettomuushoitoihin liittyen kuin tässä opinnäytetyössä. Se osoittaa samojen asioiden olevan tärkeitä lapsettomuuspotilaille maan rajoista huolimatta. Jatkossa Väestöliiton klinikoilla tulisi tehdä seurantaa, siitä miten uuden hoitoprosessin käyttöönotto on mennyt.This thesis is the result of a workplace based development project implemented at the Väestöliitto fertility clinic in Helsinki. The goal of the project was to develop a new patient centred ovum donation process which would increase patients’ satisfaction with their care. The aim of the project was to survey thoughts of patients who had gone through the ovum donation process with regard to the current treatment process and its patient centeredness. The expertise of the more experienced employees of the clinic was to be taken into consideration throughout the development project. The development of the new process began by defining the current care process for ovum donation recipients. Following this, the ovum donation recipients were interviewed for their thoughts of the current care process and its patient centeredness. The patients were asked for suggestions for the improvement of the care process. Following an analysis of the intelligence gathered from the interviews, an outline for a new patient care process was drafted. The analysis of the intelligence and the new patient care process were presented to the clinic employees at a staff event. The goal of the event was to encourage the employees to brainstorm ideas for improving patient centeredness and thus enabling them to take part in developing the improved patient care process. Following the event, with the new ideas gathered, the final care process was developed. The ovum donation treatments started in 1991 at Väestöliitto fertility clinic and it is today one of the standard treatment methods for infertility. The treatment method requires a significant financial and psychological input from the recipients. The patients treated by this method have usually suffered from infertility for several years already, and have experienced many disappointments whilst going through previous infertility treatments. The aim of developing the new, improved patient care process, is that a poor design of the care process would not add to the disappointments that the patient may experience. The value of patient centeredness has been realised in Finland and all over the world in the recent years. The approach for this thesis was an action research method. The data was collected by interviewing patients and by executing a brainstorming event with the personnel. Eight patients who had undergone ovum donation were interviewed by an individual theme interview method. The data was analysed by inductive content analysis. The new ideas collected during the brainstorming ses-sion were categorised into groups based on their similarities and differences. By using a points system, the ideas and categories were analysed and the most significant ideas were utilized in the design of the new care process. The results of the patient interviews show that patient centeredness is empathic interaction and entire consideration of the patient. The patients felt that the current care process was fluent, but uncontrollable. Positive things in the current treatment process were empathic and individual encounter with the caregivers and the individuality of the treatment process. According to analysed data, a diverse range of treatment methods, individual planning of the treatment process, and the psychological burden of the treatments are things which should be taken carefully into account when planning new treatment processes. Previous studies show similar results about patient centerednes

A comparison between the responses of neutral red and acridine orange: Acridine Orange should be preferential and alternative to neutral red as a dye for the monitoring of contaminants by means of biological sensors

The acridine orange (AO) and neutral red (NR) dyes, commonly used as probes to measure the internal pH in acidic vesicles, are compared in this article. The comparison between the two dyes (arising from calculations taking into account their analytical constants) illustrated that the use of AO is preferential to that of NR because the AO response is sensitive over the whole pH range between 4.0 and 7.4, whereas the NR response is effective only between pHs 4.0 and 6.0. In addition, it became evident from the mitochondrial respiration response that NR, unlike AO, is a protonophore. When taken into consideration, these two properties suggest that AO is more suitable than NR as an indicator of toxicity measurements in water samples because the environmental toxic compounds induce pH changes in the acidic vesicles of biological structures that are used as environmental biosensors