Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2017-04-03T21:43:19Z No. of bitstreams: 1 KleytonThiagoCostaDeCarvalho_DISSERT.pdf: 2993890 bytes, checksum: 64c5c1e9194dcf2ddec049dc4f793967 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2017-04-10T19:42:28Z (GMT) No. of bitstreams: 1 KleytonThiagoCostaDeCarvalho_DISSERT.pdf: 2993890 bytes, checksum: 64c5c1e9194dcf2ddec049dc4f793967 (MD5) / Made available in DSpace on 2017-04-10T19:42:28Z (GMT). No. of bitstreams: 1 KleytonThiagoCostaDeCarvalho_DISSERT.pdf: 2993890 bytes, checksum: 64c5c1e9194dcf2ddec049dc4f793967 (MD5) Previous issue date: 2016-07-27 / C?ncer cervical (CC) ? o terceiro tipo de c?ncer mais comum em mulheres no mundo todo e a quarta principal causa de morte em mulheres nos pa?ses em desenvolvimento. Os Papilomav?rus humano (HPV) de alto risco tais como HPV 16, 18, 31 e 33 s?o o principal fator de risco para esse tipo de c?ncer. Entre estes, o HPV-16 e -18 s?o respons?veis por quase 70% dos casos de CC. Quimioterapia com compostos ? base de platina em combina??o com a radioterapia ou a cirurgia ? o tratamento de escolha para CC, mas sua efic?cia ? limitada, especialmente em est?gios avan?ados da doen?a. Al?m disso, estes tratamentos podem facilmente levar a rea??es adversas e resist?ncia ?s drogas. Assim, a busca por novos agentes antitumorais seletivos e de alta efic?cia para o tratamento deste tipo de tumor ? necess?ria. Croton blanchetianus (CB), popularmente conhecida como ?marmeleiro preto?, ? um arbusto pertencente ? fam?lia Euphorbiaceae e amplamente disseminado no nordeste brasileiro. Alguns estudos t?m demonstrado a atividade citot?xica de plantas do g?nero Croton em linhagens tumorais humanas. Contudo, at? o momento, n?o h? nada descrito na literatura quanto ao efeito citot?xico da esp?cie Croton blanchetianus. Assim, o presente trabalho teve como objetivo investigar, in vitro, os efeitos de fra??es obtidas das folhas e raiz de CB nas linhagens de c?ncer cervical humano HeLa e SiHa. As fra??es foram obtidas pelo m?todo de varia??o do pH, a partir do qual foram obtidas duas fra??es ?cidas, uma das folhas (CBaF) e outra da raiz (CBaR), e duas b?sicas, tamb?m das folhas (CBbF) e raiz (CBbR). O perfil fitoqu?mico das fra??es foi avaliado por Cromatografia em camada delgada (CCD). A atividade citot?xica e a avalia??o do tipo de morte celular foram determinados pelos ensaios de MTT e Anexina V/PI, respectivamente, enquanto que para avalia??o de altera??es morfol?gicas nucleares e ensaio do ciclo celular foram utilizados, respectivamente, microscopia de fluoresc?ncia com o corante DAPI e citometria de fluxo. De acordo com os resultados, a maioria das fra??es apresentou terpenos, alcaloides e flavonoides. Al?m disso, todas as fra??es testadas foram capazes de diminuir significativamente a viabilidade celular de HeLa e SiHa de maneira concentra??o e tempo dependentes, promoveram modifica??es morfol?gicas celulares e nucleares, al?m de induzirem apoptose e parada do ciclo celular. Este ? o primeiro estudo que demonstrou os efeitos citot?xicos e pr?-apopt?ticos de CB em linhagens de c?ncer cervical humano. Portanto, CB parece ser uma fonte natural promissora para o desenvolvimento de agentes para o tratamento do CC. No entanto, mais estudos s?o necess?rios para isolar, purificar e elucidar os poss?veis mecanismos de a??o dos compostos ativos. / Cervical cancer (CC) is the third most common cancers in women worldwide and the fourth major cause of cancer death in the woman in developing countries. High-risk human papilloma viruses (HPVs) such as HPV 16, 18, 31 and 33 have been attributed to be the major risk factors for cervical cancer, out of which HPV-16 and -18 account for almost 70% of the cancers. Platinum-based chemotherapy in combination with radiotherapy or surgery is now mainly used to treat CC, but the efficacy is limited especially in advanced-stage disease. Furthermore, these treatments could easily lead to adverse reactions and drug resistance. Thus, it is necessary to seek antitumor drugs of high efficacy for the treatment of this kind of tumor. Croton blanchetianus (CB), known as ?black marmeleiro?, belongs to the family Euphorbiaceae and it is a widely disseminated shrub found in northeast Brazil. Some studies have demonstrated cytotoxic activity of plants of this genus against human tumor cell lines. However, to date, there is nothing described in the literature as to the cytotoxic effect of the Croton blanchetianus. Therefore, the present study aimed to investigate, in vitro, the effects of leaves and root fractions from Croton blanchetianus (CB) against human cervical cancer HeLa and SiHa cells. Fractions were obtained by pH variation method, from which were obtained two acidic fractions, one of the leaves (CBaF) and root (CBaR) and two basic also obtained from leaves (CBbF) and root (CBbR). Phytochemical screening was evaluated by thin layer chromatography. Cytotoxic activity and cell death evaluation were determined with MTT and annexin V/PI assays by flow cytometry, respectively. Nuclear morphological changes were evaluated by fluorescence with DAPI stanning and flow cytometry was used to cycle assay. According to results, most of the fractions exhibited terpenoids, alkaloids and flavonoids. All fractions decreased significantly cell viability of HeLa and SiHa cells in a concentration- and time-dependent manner, promoted cellular and nuclear morphological changes and induced apoptosis and cell cycle arrest. This is the first study that demonstrated cytotoxic and pro apoptotic effects of CB on HeLa and SiHa cells. Therefore, CB appears to be a valuable natural source for the development of agents for the treatment of CC. However, more studies are needed to isolate, purify and elucidate the possible action mechanisms of the active compounds.

Orientador: Maria Luiza Silveira Mello / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-20T21:08:55Z (GMT). No. of bitstreams: 1 Felisbino_MarinaBarreto_M.pdf: 5744960 bytes, checksum: 792f3ed20bbe7d53c917089733be679f (MD5) Previous issue date: 2012 / Resumo: O ácido valproico (VPA) é um potente anti-convulsante conhecido como inibidor de deacetilases de histonas (HDACi) de classe I em diversos tipos celulares. Buscando conhecer se a estrutura cromatínica se alteraria quando da ação de HDACi, investigamos a supraorganização cromatínica de células tumorais HeLa e de células NIH 3T3, estas últimas caracterizadas por apresentarem áreas de heterocromatina conspícuas, sob tratamento com VPA. Essas informações foram associadas a da atividade enzimática de HDACs assim como do nível de acetilação das histonas H3 nesses modelos celulares tratados por VPA. As frequências de anomalias cromossômicas, morte celular e índices mitóticos também foram investigados. As células tratadas com VPA nas concentrações 0,05, 0,5 e 1,0 mM por 1-24 h foram submetidas à reação de Feulgen e analisadas através de microespectrofotometria de varredura automática e microscopia óptica. Western blots, análises enzimáticas e ensaio TUNEL também foram utilizados neste estudo. Células tratadas com tricostatina A (TSA), uma HDACi de atividade mais ampla do que o VPA, foram utilizadas como controles positivos. Em todas as condições de tratamento com VPA e TSA foi demonstrada descompactação cromatínica acompanhada de diminuição na atividade de HDACs e aumento na acetilação de histona H3. Essa alteração textural cromatínica também atingiu áreas heterocromáticas de células NIH 3T3. Nenhuma alteração nas frequências de anomalias cromossômicas, índices mitóticos e morte celular foi observada nesses modelos celulares nas condições relatadas, embora tenha ocorrido um aumento de fragmentação de DNA em células HeLa tratadas com VPA por 24 h e por TSA a partir de 4 h. Diminuição na proliferação celular nas células HeLa ocorreu apenas sob tratamento com VPA 5,0 mM por 48 h. Os resultados indicam que o VPA e a TSA promovem remodelação cromatínica em células tumorais HeLa e em células fibroblásticas NIH 3T3, que pode ser atribuída à sua ação de HDACi. Não se pôde descartar, porém, que o VPA atue sobre outras proteínas nucleares, cuja expressão poderia se apresentar diminuída sob sua ação / Abstract: Valproic acid (VPA) is a potent anticonvulsant that inhibits class I histone deacetylases (HDACi) in several cell types. Seeking to know whether the chromatin structure would change when the action of HDACi, we investigated whether VPA would affect chromatin supraorganization of tumoral HeLa cells and NIH 3T3 cells, this latter characterized by presenting areas of conspicuos heterochromatin. This information was associated with enzymatic activity of HDACs as well as the level of H3 histone acetylation in these cell models treated with VPA. The frequency of chromosome abnormalities and cell death and mitotic indices were also investigated. VPA-treated cells at concentration 0.05, 0.5 and 1.0 mM for 1-24 h were subjected to the Feulgen reaction and analysed by automatic scanning microspectrophotometry and optical microscopy. Western blots, enzymatic analysis and TUNEL assay were also performed in this study. Trichostatin A (TSA)-treated cells, an HDACi whose activity is broader than VPA, were used as positive control. Chromatin decondensation was demonstrated under all TSA and VPA treatments and was associated with decrease in HDAC activity and with increase in the level of H3 histone acetylation. This chromatin textural change also affected heterochromatic areas of NIH 3T3 cells. No changes in chromosome abnormalities, mitotic indices or morphologically identified cell death were found in both cellular models with the VPA treatment conditions mentioned above, although there was an increase of DNA fragmentation after a 24 h-VPA treatment and a 4 h-TSA treatment in HeLa cells. Decrease in cell proliferation in HeLa cells ocurred only under a 5.0 mM 48 h-VPA treatment. The results indicate that VPA and TSA promote chromatin remodeling in tumoral HeLa cells and fibroblastic NIH 3T3 cells, which may be attrituted to their HDACi action. It may not be discarded, however, that VPA acts on other nuclear proteins whose expression could be reducted under its action / Mestrado / Biologia Celular / Mestre em Biologia Celular e Estrutural

Bakgrund och problematisering: Miljömässig hållbarhet är ett ämne som på senare tid har uppmärksammats i allt större utsträckning. Miljömässig hållbarhet har fått större betydelse för både externa men även interna intressenter vilket därmed har fått företag att uppmärksamma ämnet. Det finns flera bakomliggande orsaker till varför företag väljer att arbeta med detta samt ett antal styrmedel som kan användas för att involvera hela organisationen i det miljömässiga hållbarhetsarbetet. En svårighet för utvalda företag är att få samtliga medarbetare att känna sig involverade och veta hur de kan bidra genom sitt dagliga arbete.   Syfte: Syftet med denna uppsats är att undersöka samt analysera hur två utvalda företag använder sig av olika styrmedel för att involvera samtliga medarbetare inom organisationen i det miljömässiga hållbarhetsarbetet. Samt undersöka de bakomliggande faktorerna till varför utvalda företag väljer att arbeta med miljömässig hållbarhet och dess koppling till valt styrmedel.   Metod: Studien har utformats utifrån en kvalitativ undersökningsmetod samt en abduktiv ansats. Insamlad empiri kommer bestå av sex semistrukturerade intervjuer på två välkända företag inom livsmedels- respektive möbelbranschen. Det har utförts totalt tre intervjuer på respektive företag för att lättare kunna besvara uppsatsens forskningsfrågor samt uppnå dess syfte.   Slutsats: Uppsatsen främsta slutsats är att utvalda företag använder en mix av formella, informella och informativa styrmedel för att involvera hela organisationen i det miljömässiga hållbarhetsarbetet. Vi kan urskilja likheter i att båda företagen använder miljöpolicy som styrmedel, därefter finns vissa skillnader där det ena företaget främst använder miljömärkningar medan det andra till stor del styr med värderingar. Värderingar är även något båda företagen lyfter fram som ett betydelsefullt verktyg, framför allt på högre nivåer, för att förmedla hållbarhetsarbetet till medarbetarna. Det mest fördelaktiga sättet att förmedla hållbarhetsarbetet anser medarbetarna dock vara genom information.

Cell penetrating peptides have been the focus of drug delivery research for 15 years due to their apparent ability to deliver cargoes inside cells more readily than many other carriers. Using two of the most commonly studied peptides (tat47-57 and R9), the present study differs from previous work by deliberately choosing to observe uptake with lower peptide concentrations closer to potential therapeutic doses, and by implementing raster image correlation spectroscopy (RICS) on a commercial microscope to quantify uptake in parallel to other techniques such as fluorescence correlation spectroscopy (FCS), confocal microscopy, and mass spectroscopy.An initial study using mass spectrometry and ExPASy (Expert Protein Analysis System) revealed that the peptides are stable for at least one hour in PBS. Based on this initial information and other experimental conditions, the study took two main directions with regards to the peptide: the membrane interaction and accumulation in the cell.The peptides interaction with the cell membrane revealed that neither tat-TAMRA nor R9-TAMRA disrupts the membrane of cells: incorporation of FM2-10 in the membrane was not modified in K562 cells whilst it was in presence of the control lytic peptides GALA and mellitin. Based on this information confocal microscopy was utilised to assess the localisation on the cell membrane. Peptide binding to the membrane appeared to be heterogeneous in distribution at 1µM bulk concentration.Accumulation in cells of the peptides was observed incubated at 37°C, confocal microscopy showed punctuated distribution with intracellular aggregations of fluorescence measuring between 2.5-3.5µm in diameter. Co-staining with a nuclear dye revealed these aggregations to be focused around the nucleus of the cell. Initial FCS experiments indicated a concentration dependent accumulation of the peptide in the cells and a decrease of the intracellular diffusion coefficients at high concentration possibly corresponding with molecular crowding. Interestingly the anomalous diffusion model did not statistically improve the results.RICS was implemented to study the kinetics of entry of TAMRA labelled cell penetrating peptides in both Caco-2 and HeLa cells lines at concentrations between 500nM and 2µM. Concentrations above 1µM exhibited higher final intracellular concentrations, yet the measured diffusion coefficients were similarly independent of extracellular concentration. Both peptides appeared to enter the cell quickly with a fast initial uptake over the first 10 minutes, reaching a concentration maxima after 30 minutes.Overall, the study reveals that many published studies may be incorrect as they may only be reporting the presence of a fluorescent dye inside the cell not the peptide. The fast binding of the peptide to the membrane is likely to cause false positive results when traditionally studying internalisation kinetics such as using flow cytometry and confocal microscopy. Correlation spectroscopy techniques such as FCS, provide useful information on internalisation of the peptides, but the single spot measurement is limited when providing information on the entire cell. RICS is a progression of correlation spectroscopy and provides a more representative picture of the cell.

Denna studie undersöker fördelar och nackdelar med att genomföra utdragsundervisning respektive undervisning med hela verk i litteraturundervisning, på gymnasienivå i Sverige. Syftet med studien är att undersöka lärarnas attityder gentemot användningen av utdrag och hela verk, och hur deras attityder korrelerar med den tidigare forskningen i ämnet. Undersökningen syftar till att besvara frågorna; Hur kan svensklärares attityder till användningen av textutdrag respektive hela verk i litteraturundervisningen beskrivas? och hur korrelerar lärarnas attityder till den litteraturdidaktiska forskningen om utdragsundervisning och undervisning med hela verk? med stöd i fenomenologi. Studien använder sig av en kvalitativ metod och genomför intervjuer med sex legitimerade gymnasielärare. Studien undersöker deras användning av utdrag respektive hela verk i litteraturundervisning samt deras uppfattning om vilka för- och nackdelar som finns med utdrag respektive hela verk. Lärarna uppgav att de använder båda metoder i sin litteraturundervisning, men de särskilde på deras användningsområden i kurserna svenska 1, 2 och 3. Resultatet visar att det finns både fördelar och nackdelar med utdrag respektive hela verk; hela verk anses vara mer lämpligt när eleverna läser för berättelsen och tolkar innehållet eftersom det möjliggör en djupare förståelse medan utdrag är mer lämpligt när man studerar litteraturhistoria eftersom det möjliggör en bredare förståelse av de litterära epokerna. / This essay examines the advantages and disadvantages of using excerpts or an entire works in literature teaching at upper secondary schools in Sweden. The objective is to see teachers’ attitudes towards the use of excerpts and entire literary works, and how the correlate with the previous research about the matter. The research aims to answer the questions; How can Swedish teachers' attitudes to the use of text excerpts and entire works in literary education be described? and how do teachers' attitudes correlate to literary didactic research on excerpt teaching and teaching with whole works? with a support in phenomenology. The study is conducted with the qualitative method interviews. Six teachers, certified for upper secondary school, were interviewed about their current use of excepts and entire works in their literary teachings, as well as their attitudes toward the advantages and disadvantages of them. The teachers stated that they use both methods in their literary teachings, however they differentiated their use between the different courses Swedish 1, 2 and 3. The result show that there are advantages and disadvantages with both methods, however, entire works are considered more suitable when students are reading for the story and to interpret the content as it allows for a deeper understanding. Furthermore, excerpts are considered more suitable when studying history of literature as it allows for a broader understanding of the literary eras

Tidigare forskning har gång på gång visat att grundskolans elever inte kontinuerligt genom hela skoltiden får studie- och yrkesvägledning (SYV) trots att styrdokumenten kräver detta. Denna studie syftar till att få en djupare förståelse för vilka årskurser som får SYV i grundskolan och i vilken utsträckning de får vägledning i snäv- respektive vid bemärkelse, vilka befattningshavare som ansvarar för vägledningen och vad dessa aktörer anser om SYV-verksamheten vid sin skola. De teorier som använts för analys är Gottfredsons teori om hur vi redan från unga år väljer bort vissa yrken och Savickas teori som pekar på att vi ofta reproducerar våra föräldrars yrken och behöver vägledning för att få perspektiv och se andra möjligheter. Vidare användes även bland annat Lovéns forskning som stöd för analys. Studien har en kvalitativ ansats och datainsamlingen har genomförts genom en webbaserad enkät som 22 informanter, Rektorer, Lärare samt Studie- och yrkesvägledare har besvarat. Resultat och analys visar på samma tendenser som tidigare forskning och kvalitetsgranskningar, att tyngdpunkten ligger i årskurs nio och att det till störst del är studie-och yrkesvägledaren som ansvarar för både snäv och vid vägledning, något som lärarna är nöjda med medan SYV gärna skulle se mer samarbete. Flertalet av informanterna menar att verksamheten fungerar bra. Samtidigt har många lärare på flera frågor svarat att de inte vet vilket pekar på att alla inte själva deltar i SYV- verksamheten. De tidigare årskurserna kan alltså sedan en lång tid tillbaka beträffande SYV, betraktas som de glömda eleverna. En slutsats är också att många tar SYV för givet, att SYV av tradition ligger i årskurs nio och att studie- och yrkesvägledaren är den som förväntas ansvara för verksamheten.

I en granskning av Skolinspektionen framkom att ansvaret för vägledningen i skolan ligger på den enskilda studie- och yrkesvägledaren, vilket betonas i de styrdokument som reglerar vägledning i skolan. Syftet med studien är att få en inblick i hur studie- och yrkesvägledare inom grundskolan uppfattar och beskriver skolans arbete med vägledning i vid bemärkelse. Trots tydliga styrdokument som framhäver den vida vägledningens betydelse för eleverna, finns det stora brister i hur skolorna arbetar med detta. Forskning visar att vägledning i vid bemärkelse fungerar bäst när den är förankrad på alla nivåer i kommunen med tydliga mål. Kvalitativa intervjuer genomfördes med sex studie- och yrkesvägledare och resultatet analyserades med stöd av karriärteorier samt styrdokument och tidigare forskning. Resultaten visar att för att elevernas valkompetens ska kunna utvecklas behövs vägledning i vid bemärkelse där flera professioner samverkar med varandra. Framtida forskning i ämnet skulle kunna vara att skapa en likvärdig nationell plan för studie- och yrkesvägledare som alla skolor arbetar utifrån. / A review by the Swedish Schools Inspectorate revealed that the responsibility for guidance in the school lies with the individual study and career counsellor, which is emphasized in the governing documents that regulate guidance in the school. The purpose of the study is to gain an insight into how study and career counsellors in compulsory school perceive and describe the school's work with guidance in a broad sense. Despite clear governing documents that emphasize the importance of broad guidance for students, there are major shortcomings in how the schools work with this. Research shows that guidance in the broadest sense works best when it is anchored at all levels in the municipality with clear goals. Qualitative interviews were conducted with six study and career counsellors and the results were analysed with the support of career theories as well as governing documents and previous research. The results show that in order for the students' choice competence to be developed, guidance is needed in a broad sense where several professions collaborate with each other. Future research in the subject could be to create an equivalent national plan for study and career counselors from which all schools work.

Nuclear factor kappa B (NFκB) is an inflammatory transcription factor that plays an important role in the host immune response to infection. The potential for chlamydiae to activate NFκB has been an area of interest, however most work has focused on chlamydiae impacting human health. Given that inflammation characteristic of chlamydial infection may be associated with severe disease outcomes or contribute to poor overall fitness in farmed animals, we evaluated the ability of porcine chlamydiae to induce NFκB activation in vitro. C. pecorum infection induced both NFκB nuclear translocation and activation at 2 hours post infection (hpi), an effect strongly enhanced by suppression of host de novo protein synthesis. C. suis and C. trachomatis showed less capacity for NFκB activation compared to C. pecorum, suggesting a species-specific variation in NFκB activation. At 24 hpi, C. pecorum induced significant NFκB activation, an effect not abolished by penicillin (beta lactam)-induced chlamydial stress. C. pecorum-dependent secretion of interleukin 6 was also detected in the culture supernatant of infected cells at 24 hpi, and this effect, too, was unchanged by penicillin-induced chlamydial stress. Taken together, these results suggest that NFκB participates in the early inflammatory response to C. pecorum and that stressed chlamydiae can promote inflammation.

A common model for studying Chlamydia trachomatis and growing chlamydial stocks uses Lymphogranuloma venereum serovar L2 and non-polarized HeLa cells. However, recent publications indicate that the growth rate and progeny yields can vary considerably for a particular strain depending on the cell line/type used, and seem to be partially related to cell tropism. In the present study, the growth of invasive serovar L2 was compared in endometrial HEC-1B and endocervical HeLa cells polarized on collagen-coated microcarrier beads, as well as in HeLa cells grown in tissue culture flasks. Microscopy analysis revealed no difference in chlamydial attachment/entry patterns or in inclusion development throughout the developmental cycle between cell lines. Very comparable growth curves in both cell lines were also found using real-time PCR analysis, with increases in chlamydial DNA content of 400-500-fold between 2 and 36 h post-inoculation. Similar progeny yields with comparable infectivity were recovered from HEC-1B and HeLa cell bead cultures, and no difference in chlamydial growth was found in polarized vs. non-polarized HeLa cells. In conclusion, unlike other C. trachomatis strains such as urogenital serovar E, invasive serovar L2 grows equally well in physiologically different endometrial and endocervical environments, regardless of the host cell polarization state.

Denna studie behandlar hur samhällskunskapslärare i sex svenska grundskolor bedriver sin undervisning med koppling till vägledning. Studiens syfte var att studera hur lärarna jobbar med vägledning som hela skolans ansvar. Samtliga av de aktuella skolorna har i sina riktlinjer tydliga mål med att vägledning skall inkludera all personal. Vi ville undersöka lärarnas personliga upplevelser av detta och eventuellt vilka verktyg dem använder för att lyckas med sitt uppdrag. Metoden som användes var kvalitativ och det empiriska materialet inhämtades genom semi-strukturerade intervjuer. I studien deltog sex informanter varav samtliga var utbildade samhällskunskapslärare. Resultatet analyserades med hjälp av Careership och Theory of action. Resultatet visade på att enbart en del av de tillfrågade bedrev undervisningen enligt framtagna riktlinjer. Samtidigt var det bara några av dem som arbetade utifrån en strukturerad och genomtänkt plan i sina respektive skolor där en kontinuerlig kontakt med skolans studie- och yrkesvägledare fanns. Resterande informanter bedrev visserligen en medveten undervisning med inslag av bred vägledning. Men dessa moment förekom istället mer spontant utan någon större planering.