Processos que envolvem sensibilização são extremamente importantes para diversas áreas do conhecimento, incluindo a biologia, a química e a medicina. A aplicação de sensibilização em medicina tem se destacado, especialmente, em face de uma modalidade alternativa de tratamento de câncer denominada terapia fotodinâmica (TFD). Uma das linhas de pesquisas fundamentais para a evolução da terapia fotodinâmica é o desenvolvimento de novos fotossensibilizadores (Fs) com composição definida, que absorvam na janela terapêutica (600- 800nm) e que apresentem maior eficiência na indução de apoptose. Os Fs que apresentam cargas positivas e que são relativamente lipofílicos, permeiam membranas e são atraídos pelo potencial negativo das mitocôndrias, que tem papel central no controle da vida e da morte celular. Neste trabalho foi realizado um estudo da influência dos grupos funcionais na atividade dos Fs, através da funcionalização da protoporfirina IX (Pp IX). Foram estudadas três diferentes rotas sintéticas. Na rota I (esquema 14), utilizou-se como composto de partida a Hematoporfina IX (Hp IX), a qual foi funcionalizada com grupos aminas e amidas, respectivamente, nas hidroxilas e nas carboxilas. Esta rota forneceu baixo rendimento global (20%), e compostos de difícil purificação, no entanto obteve-se 1 composto puro. Na rota II (esquema 15), utilizou-se como composto de partida a Pp IX, a qual foi funcionalizada nos grupos vinílicos com grupos aminas. Obteve-se 4 compostos, com rendimento global de reação superior a 50%, mas observou-se que ocorre uma reação de eliminação que impede a quartenarização das aminas localizadas nas posições α ao anel porfirínico. Na rota III (esquema 16), os grupos carboxílicos de Pp IX foram transformados em cloreto de ácido, que foram substituídos por compostos bifuncionais, amina primária e um segundo grupo. Esta rota possibilitou a obtenção de 7 compostos, inclusive compostos quaternários com rendimento global de reação superior a 70%. Dois derivados da clorofilina, que apresentam a banda QIV com um ε elevado em 650nm, foram também sintetizados. Todos os compostos, foram caracterizados estruturalmente de forma inequívoca através do espectro eletrônico (UV-vis e fluorescência), vibracional no infravermelho, RMN de 1H e 13C (1D e 2D) e espectrometria de massa. A série de compostos obtidos permitiu um estudo da relação entre a estrutura química do Fs com a sua fotoatividade. As propriedades fotofísicas foram caracterizadas por espectro de absorção e de emissão, fotólise de relâmpago a laser, eficiência quântica de fluorescência e de geração de oxigênio singlete. Estas determinações indicaram que as propriedades fotofísicas dos Fs não foram consideravelmente alteradas no processo sintético. Foi determinada a formação de agregados em solução aquosa e o equilíbrio monômero agregado foi deslocado no sentido da formação do monômero na presença de micelas de CTAB e SDS e de soro fetal. Observou-se, que a contribuição para desagregação é mais eficiente quando a carga da micela é oposta à do Fs. Foi determinado o coeficiente de partição octanol/água (logPo/a) em função do pH e constatou-se que os compostos que têm carga líquida apresentam valores de logPo/a entre -1 e 1 na faixa de pH entre 3 e 10. A incorporação destes compostos em lipossomos, seguiu perfil esperado considerando a carga, o logPo/a e a característica anfifílica dos compostos sintetizadas. Genericamente a eficiência de morte celular fotoinduzida seguiu o perfil de incorporação das drogas em células HeLa. Os estudos comparativos da citolocalização foram realizados por co-encubação das porfirinas sintetizadas com rodamina 123 (Rd), que se concentra em mitocôndrias. A localização em organelas citoplasmáticas foi determinada através de imagens obtidas por microscopia de fluorescência confocal. A sobreposição da emissão da Rd foi de 80% e 31% com o composto catiônico PpNpNI e aniônico PpNetPO3, respectivamente, comprovando a localização mitocondrial do composto positivo. / Processes that involve sensitization are extremely important for several areas of knowledge, including biology, chemistry and medicine. The application of sensitization in medicine is especially important, in face of an alternative modality of cancer treatment called Photodynamic Therapy (PDT). One of the lines of basic research important for the evolution of PDT is the development of new photosensitizers (PS) with defined composition, that absorb in the therapeutical window (600-800nm) and that present greater efficiency in the induction of apoptosis. Positively charged PS with the proper lipophilic/hidrophilic balance permeate membranes and are attracted by the negative potential of mitochondria, which is an organelle that has central roles in the control of cell life and death. In this work the influence of the functional groups in the activity of PS was carried out, through the functionalization of protoporphyrin IX (Pp IX). Three different synthetic routes were used. In route I (scheme 14), Hematoporphyrin IX (Hp IX), was used as departure compound and it was modified with amino and amide groups in hydroxyls and carboxyls, respectively. We found low yield in the synthetic (20%) and purification steps. However 1 pure PS was obtained. In route II (scheme 15), Pp IX was used as departure compound, which was modified in the vinilic groups with amino groups. We obtained 4 compounds, with global yield superior than 50%, but it was observed that an elimination reaction occurs that hinders the quarternization of the amino groups located in position α to the porphyrin ring. Into route III (scheme 16), the carboxilic groups of Pp IX had been transformed into chloride acid, which was substituted by bi- functional groups, primary amine in one side and another group in the other. This route made it possible to obtain 7 compounds, including quaternary ammonium compounds with global yield superior than 70%. Two derivatives of chlorophyllin, that present QIV band with large extinction coefficient in 650nm, also were synthesized. All compounds were characterized structurally through the electronic and vibrational spectra (UV-vis and fluorescence, IR), RMN of 1H and 13C (1D and 2D) and mass spectrometry. This series of compounds allowed us to study the relation between the chemical structure of the Fs with its photoactivity. The photophysical properties were characterized by emission and absorption spectra, laser flash photolysis, fluorescence emission in the visible and NIR regions (generation of singlet oxygen). These determinations indicated that the photophysical properties of the PS were not modified in the synthetic process. The formation of aggregates were characterized in aqueous solution and the balance between aggregate and monomer species was dislocated in the direction of the formation of monomers in the presence of CTAB, SDS micelles and fetal serum. It was observed, that the disaggregating efficient is larger with micelles that have the opposite charges to that of the PS. The Octanol water partition coefficient (logPo/a), was determined as a function of pH. logPo/a values between -1 and 1 were observed for charged PS in the pH range of 3-10. The incorporation of these compounds in liposomes, followed the expected profile considering the charge, logPo/a and the amphifilic nature. Generically, the photoinduced cell death efficiency followed the profile of incorporation of the PS in HeLa cells. Comparative studies of cytolocalization were carried out by co-incubation of the cells with porphyrins and Rhodamine 123 (Rd), which localizes in mitochondria. The localization in cytoplasmic organelles was determined through fluorescence confocal microscopy images. The overlapping emission of the Rd was of 80% and 31% with the cationic PpNpNI and the anionic PpNetPO3 compounds, respectively, proving the mitochondrial localization of the positive PS.

Orientador: Marcelo Brocchi / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-19T03:47:25Z (GMT). No. of bitstreams: 1 Gregoracci_GustavoBueno_D.pdf: 6197096 bytes, checksum: 05eb5e18d8084e5c5f742c6ec5afd63d (MD5) Previous issue date: 2010 / Resumo: Bacteriófagos são vírus que infectam bactérias e arqueias, representando as entidades biológicas mais abundantes do mundo e influenciando de maneira marcante populações naturais de seus hospedeiros. A terapia com bacteriófagos, que representa uma das primeiras formas modernas de combate a infecções bacterianas, foi recentemente redescoberta e vem sendo reavaliada seguindo metodologias atuais quanto à sua viabilidade terapêutica. Para completar a caracterização dos fagos de nossa coleção, sequenciamos completamente o genoma da maior parte destes, através da metodologia multiplex pair-ended utilizando a plataforma Illumina. Visando contribuir para verificação da viabilidade terapêutica de bacteriófagos testamos os efeitos protetor e terapêutico dos fagos Shfl1, Saen1v2 e Saen1v4, pertencentes à coleção de nosso laboratório, em modelos biológicos relevantes. O fago Shfl1, lítico contra Shigella flexneri, foi testado em ensaio de invasão em células HeLa. A redução de bactérias intracelulares foi mensurada independentemente através de plaqueamento e citometria de fluxo, além da observação direta por microscopia de fluorescência. O fago Saen1v2, lítico contra Salmonella Typhimurium, foi estudado quanto à biodistribuição e meia-vida em modelo murino, e uma variante viral com maior persistência in vivo foi selecionada. Essa variante, denominada Saen1v2p5, e o fago Saen1v4, lítico contra Salmonella Typhi, foram testados em modelo murino de infecção tifoide, contra seus respectivos hospedeiros. Encontramos similaridade genômica a fagos conhecidos, como T4, T7, T1 entre outros, em maior ou menor grau. Obtivemos um efeito protetor e terapêutico contra Shigella flexneri utilizando o fago Shfl1 em ensaio de invasão em cultura de células HeLa, verificado por todas as metodologias empregadas. Não verificamos efeito antimicrobiano in vivo do fago Saen1v2p5 em modelo murino de infecção por Salmonella Typhimurium. Por outro lado, observamos efeito terapêutico e protetor dose dependente utilizando o fago Saen1v4 em modelo murino de infecção por Salmonella Typhi. O sucesso obtido com baixas multiplicidades de infecção sugere um possível efeito indireto ou estimulação imune inespecífica / Abstract: Bacteriophages are viruses that infect Bacteria and Achaea, representing the most abundant biological entities in the world and markedly influencing natural host populations. Phage therapy, which represents one of the first modern ways to fight bacterial infections, was recently rediscovered and is being re-evaluated according to current methodologies regarding its therapeutic viability. In order to complete phage characterization in our collection, we sequenced completely the genomes of most of these, through the multiplex pair-ended methodology using the Illumina platform. Aiming to contribute to the therapeutic viability verification of bacteriophages we tested phage protective and therapeutic effects of Shfl1, Saen1v2 and Saen1v4, which belong to our collection, in biologically relevant models. Phage Shfl1, lytic against Shigella flexneri, was tested in a HeLa invasion assay. Intracellular bacteria reduction was measured independently through plating and flow cytometry, besides direct observation through fluorescent microscopy. Phage Saen1v2, lytic against Salmonella Typhimurium, was studied about its bio-distribution and half-life in murine model, and a viral variant with longer in vivo persistence was selected. This variant, denominated Saen1v2p5, and phage Saen1v4, lytic against Salmonella Typhi, were tested in murine typhoid model, against their respective hosts. Genomic similarity to known phages such as T4, T7, T1 among others, was found, in various degrees. We obtained both protective and therapeutic effect against Shigella flexneri using phage Shfl1 in the HeLa invasion assay, through all methodologies utilized. We could not verify in vivo antimicrobial effect of phage Saen1v2p5 in the murine model of Salmonella Typhimurium infection. On the other hand, we observed both therapeutic and protective dose dependent effect using phage Saen1v4 in Salmonella Typhi murine infection model. The success obtained with low multiplicities of infection may suggest a possible indirect effect or unspecific immune stimulation / Doutorado / Microbiologia / Doutor em Genetica e Biologia Molecular

published_or_final_version / Biological Sciences / Master / Master of Philosophy

Syftet med examensarbetet var att undersöka på vilket sätt lärare tolkar sitt uppdrag att arbeta med studie- och yrkesvägledning i vid bemärkelse i förhållande till rådande styrdokument. Vi har använt oss av en kvalitativ metod med semistrukturerade intervjufrågor och med ett urval av totalt sex behöriga lärare inom tre grundskolor på två olika orter. Intervjuerna utgick från frågeställningar om hur lärare arbetar med studie – och yrkesvägledning i den ordinarie undervisningen samt hur aktuella styrdokument och riktlinjer efterföljs. Resultatet visade att lärares uppfattning av vad vägledning i vid bemärkelse innebär är av varierande karaktär. Den skiftande uppfattningen avspeglades även i lärarnas vägledningsarbete i undervisningen samt på vilket sätt de arbetar i förhållande till rådande styrdokument. Det centrala är att arbeta med vägledning i vid bemärkelse i tidig ålder för att motverka begränsningar och komproisser, vilket kan förhindra att elevens studie- och yrkesdrömmar skyms.

Sammanfattning Det finns många anti-mobbningsprogram och metoder för att förhindra förekomsten av mobbning. Det svenska anti-mobbningsprogrammet Friendsprogrammet använder sig av en hela-skolan-ansats för att reducera förekomsten av mobbning. Vidare fokuserar Friends-programmet på normkritisk pedagogik för att reducera mobbning med utgångspunkt i diskrimineringsgrunderna/normer och värderingar. Det primära syftet med Friendsprogrammet är ett främjande anti-mobbningsarbete, men fokus ligger även på förebyggande och åtgärdande arbete. Eftersom mobbning syns på tre nivåer, sker anti-mobbningsarbete på individ-, grupp- samt organisationsnivå. Friendsprogrammet inkluderar samtliga i ett anti-mobbningsarbete. För att utvärdera huruvida Friendsprogrammet är verksamt, har programteori använts som en metod. I denna studie har gruppintervjuer utförts med lärare på en grundskola som är belägen i en liten tätort i mellansverige för att undersöka huruvida Friendsprogrammet är verksamt i en liten-skol-miljö. Resultatet visar att kontextuella förutsättningar påverkar implementeringen av Friendsprogrammet samt ett främjande arbete, vilket innebär att ett främjande anti-mobbningsarbete inte alltid är mest relevant. Vidare framgår av resultatet att den undersökta Friendsskolan inte tillämpar Friendsprogrammet fullt ut, utan mer som en inspiration. Skolan kombinerar sina egna metoder med metoder från Friendsprogrammet. Trots att skolans anti-mobbningsarbete skiljer sig från Friendsprogrammets arbetssätt, inkluderas ett anti-mobbningsarbete på samtliga tre nivåer: individ-, grupp- samt organisationsnivå.

The Wnt signaling pathway is highly conserved and participates in many important cellular functions including differentiation, embryonic development and tissue generations. £]-catenin, the central mediator of the Wnt signaling, interacts with the TCF/LEF family of transcription factors in the nucleus and initiates downstream gene transcription. In addition, £]-catenin is known as a proto-oncogene implicated in numerous cancers including colorectal, cervical, endometrial and skin cancer. Chibby (Cby) is evolutionarily conserved in many species and acts as a repressor of Wnt/£]-catenin signaling. In our previous study, we have established that Cby over-expression attenuated £]-catenin translocation to nucleus and its transcriptional activity. Thus, it was hypothesized that Cby may possess potential tumor suppressing capabilities. In the present study, we first explored endogenous Cby expression status in human cervical cancer cells: HeLa and SiHa cell lines. It was observed that Cby mRNA and protein levels were significantly down-regulated in both cancer lines compared with primary cervical cells. We then conducted functional assays of tumorigenicity on both cells using adenoviral-encoded Cby and its NLS (nuclear localization signaling) deleted variant (Cby∆NLS). It was found that gene delivery of Cby or Cby∆NLS inhibited the proliferation, invasiveness, and colony forming in HeLa and SiHa cells. Immunofluorescent analysis revealed that Cby or Cby∆NLS gene transfer reduced the expression of Ki-67, a cell proliferative marker. Furthermore, Cby or Cby∆NLS restoration induced apoptosis and perturbed cell cycle progression in both cervical cancer cells. Finally, Cby over-expression decreases the expression of £]-catenin/TCF4 regulated genes such as c-myc and PCNA, which might contributed to the anti-neoplastic mechanism for Cby in cervical cancer cell lines. Our results strongly suggest that Cby may serve as a tumor suppressor gene during cervical carcinogenesis, and may facilitate in creation of new therapeutic methods.

ABSTRACT Chibby (or PIGEA-14) is a novel antagonist of the Beta-catenin pathway in nucleus. However, the tumor-suppressing function of Chibby and the importance of nuclear targeting to the cellular functions of Chibby have not been validated. By fusion of Chibby cDNA with green fluorescent protein (GFP) or Flag-tag, it was found that exogenous Chibby expression was detected in the nucleus as well as cytoplasm of transfected HeLa cells, but with a preferential nuclear localization (more than 50% cells with nuclear Chibby expression). Chibby overexpression significantly abrogated the cellular Beta¡Vcatenin activities and induced apoptosis in HeLa cells. Moreover, Chibby gene delivery attenuated the proliferation, migration, and anchorage-independent growth of HeLa cells, supporting the tumor suppressor function of Chibby. Mutation or deletion of the predicted nuclear localization sequence (NLS), at residues 123-126, significantly promoted the cytoplasmic localization of Chibby, indicating residues 123-126 is the NLS domain of Chibby. Interestingly, ecotopic expression of Chibby NLS mutants remained capable of inducing apoptosis and inhibiting Beta¡Vcatenin activities in HeLa cells. Besides, overexpression Chibby NLS mutants effectively attenuated the viability, motility and colonies formation of HeLa cells. Expression analysis revealed that Chibby NLS mutants retained Beta-catenin in the cytoplasm and prevented its nuclear entry, thereby inhibiting the Beta-catenin transcriptional activities. In summary, Chibby shuttles between nucleus and cytoplasm, and possesses the functions of tumor suppressor and Beta-catenin antagonist.

No description available.

Gene expression at the level of mRNA translation is critical for cells to respond to external signals; it allows changes in protein synthesis without triggering transcription of a new set of genes. Control of mRNA translation and stability is important in several cellular processes including cell growth and differentiation. Thus regulation of the cellular machinery involved in mRNA translation is crucial. Poly (A) binding protein (PABP1), eukaryotic elongation factor 1A (eEF1A) and ribosomal protein S6 (RPS6) are important members of the cellular mRNA translation machinery, the mRNAs that encode these proteins belong to the terminal oligo pyrimidine tract (TOP) containing family. Translation of the TOP mRNAs is regulated by growth signals and usually codes for several proteins involved in mRNA translation. Our laboratory has previously reported up regulation of PABP1 mRNA translation during recovery from heat shock. It was also shown that the terminal oligopyrmidine tract (TOP) cis-element of PABP1 mRNA is responsible for the preferential increase of PABP1 mRNA translation; however the mechanism for achieving this is unknown. In the studies reported here, we showed that translation of eEF1A and RPS6 expression was similarly enhanced during recovery from heat shock. Analyses of samples of in vivo cross linked RNA– protein complexes, immunoprecipitated by ZNF9 antibody, for the presence of specific mRNAs showed that the cellular nucleic acid binding protein ZNF9 binds not only to TOP mRNAs but also mRNA that lack the TOP element such as to β-actin mRNA. To elucidate the mechanism of activation of TOP mRNA translation, as a candidate trans acting factor, siRNA was used to deplete the cellular level of ZNF9 from heat shocked HeLa cells to examine its potential role in stimulation of TOP mRNA translation during recovery from heat shock. Results show that the knock down of ZNF9 disallowed the preferred stimulation of PABP1, eEF1A and RPS6 expression during recovery from heat shock. There was no detectable effect on the constitutive expression of either β-actin or PABP1, eEF1A and RPS6 in exponentially growing HeLa cells. These results suggest that binding of ZNF9 to TOP mRNAs per se does not inhibit translation, but more likely it acts as a general facilitator of mRNA translation. It is possible that modification of the interaction between ZNF9 with other unknown protein factors is responsible for its preferred effect on all three TOP mRNAs studied here. Additionally, results also suggest that a different TOP sequences amongst the observed TOP mRNAs responds similarly to ZNF9.

The correction of human genetic disorders by transfer of genetic material to cells is under intensive investigation in a number of 1aboratories. One possible way of trying to achieve the transfer of nucleic acid is by attaching DNA to a protein which has specific receptors on cells and which undergoes receptor-mediated endocytosis. In order to make use of the ligand protein-receptor approach for DNA transfer, iron-loaded human serum transferrin has been modified with the water soluble carbodiimides N-ethy1-N I -(3-dilllethy1aminopropyl) carbodiimide (CDI) and its quaterary analogue (ECDI) to give modified N-acy1urea transferrins. N-Acy1urea CDI (Fe 3+) transferrin and N-acy1urea CDI (Fe ) transferrin have been found to interact with and bind DNA in a reversible manner which i! dependent on ionic strength. [1251] N-Acy1urea CDI+(Fe3+) transferrin binds to transferrin receptors on Hea cells in culture and undergoes internalization through receptor-mediated endocytosis. Binding of the modified transferrin in the presence of calf thymus DNA to transferrin receptors also takes place. However, although internalization in the presence of DNA doe! appear to take place, the results of the internalization are not fully understood. Transfection studies with N-acy1urea CDI (Fe ) transferrin and plasmid pBR322 DNA as well as plasmid ptkNEO DNA complexes in the HeLa cell system are reported. The results of a number of transfection experiments suggests that N-acy1urea transferrins are capable of transfecting DNA (ptkNEO DNA), carrying genes for resistance to the antibiotic Geneticin (G41S) in the HeLa cell system. However, further development of the transfection system is necessary in order that consistantly reproducible results may be achievd. / Thesis (M.Sc.)-University of Durban-Westville, 1986.