by Wong Chi Shing Micky. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 140-149). / Chapter Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- The Uterine Cervix --- p.1 / Chapter 1.2 --- Cervical Secretion and its Function --- p.3 / Chapter 1.3 --- Ion Transport System in Cell Volume Regulation --- p.6 / Chapter 1.3.1 --- "Regulatory Volume Decrease, RVD" --- p.8 / Chapter 1.3.2 --- "Regulatory Volume Increase, RVI" --- p.8 / Chapter 1.4 --- Signaling Pathways underlying RVD and RVI - The Role of Intracellular Free Calcium --- p.11 / Chapter 1.5 --- Swelling-induced Cl- Current --- p.12 / Chapter 1.6 --- Ca2+ activated K+ Channel --- p.14 / Chapter 1.7 --- Objectives of the Study --- p.15 / Chapter Chapter 2. --- Materials and Methods --- p.17 / Chapter 2.1 --- Materials --- p.17 / Chapter 2.1.1 --- Culture Media --- p.17 / Chapter 2.1.2 --- Chemicals --- p.17 / Chapter 2.2 --- Preparation of Solutions --- p.18 / Chapter 2.3 --- Cell Culture --- p.21 / Chapter 2.4 --- Patch-clamp Technique --- p.22 / Chapter 2.4.1 --- Preparation of Electrode --- p.27 / Chapter 2.4.1.1 --- Pulling and Polishing of Electrode --- p.27 / Chapter 2.4.1.2 --- Filling of the Electrode --- p.27 / Chapter 2.4.1.3 --- Coating of Electrode --- p.28 / Chapter 2.4.2 --- Patch-clamp Study --- p.29 / Chapter 2.4.2.1 --- Formation of Whole-cell Configuration --- p.29 / Chapter 2.4.2.2 --- Data Acquisition and Analysis --- p.31 / Chapter 2.5 --- Study of Cellular Volume Regulation by Confocal Laser Scanning Microscopy (CLSM) --- p.35 / Chapter 2.6 --- Determination of Intracellular Ca2+ by Confocal Laser Scanning Microscopy (CLSM) --- p.38 / Chapter Chapter 3. --- Results --- p.39 / Chapter 3.1 --- "Regulatory Volume Decrease, RVD" --- p.39 / Chapter 3.2 --- Responses of [Ca2+］i to swelling --- p.47 / Chapter 3.3 --- KC1 Efflux in RVD in HeLa Cells --- p.57 / Chapter 3.4 --- Swelling-induced Cl- Current --- p.67 / Chapter 3.4.1 --- Swelling-induced Anion and Cation Current --- p.67 / Chapter 3.4.2 --- Ca2+-independence of Swelling-induced Cl- Current --- p.71 / Chapter 3.4.3 --- Effect of Cl- Channel Blockers on Swelling-induced Cl- Current --- p.75 / Chapter 3.4.4 --- Anion Selectivity of Swelling-induced Cl- Current --- p.85 / Chapter 3.4.5 --- Cl- Dependence of Swelling-induced Cation Conductance --- p.85 / Chapter 3.4.6 --- K+ Independence of Swelling-induced Anion Conductance --- p.95 / Chapter 3.5 --- Ca2+ Activated K+ Current --- p.101 / Chapter 3.5.1 --- Ionomycin Induced Cell Shrinkage under Isotonic Condition --- p.101 / Chapter 3.5.2 --- Ionomycin Stimulated a Whole-cell K+ Conductance --- p.101 / Chapter 3.5.3 --- The Effect of Ionomycin on Intracellular Ca2+ Level --- p.111 / Chapter 3.5.4 --- Ca2+ Dependence of Ionomycin Stimulated K+ Current --- p.111 / Chapter Chapter 4. --- Discussion --- p.124 / Regulatory Volume Decrease in HeLa Cells --- p.124 / Role of Calcium in Regulatory Volume Decrease (RVD) --- p.126 / Swelling-induced Cation and Anion Conductance --- p.128 / Ca2+ Activated K+ Current in HeLa Cells --- p.134 / Chapter Chapter 5. --- Reference --- p.140

Stuttgart, Univ., Diss., 2005.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1966. / Typescript. Vita. Description based on print version record. Includes bibliographical references.

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

<p> This thesis is a study of the adhesive behaviour of HeLa cells using a novel instrument designed for measuring both the shearing and compression force applied to the cells. For these experiments a micropipette forged as a double cantilever is used to grasp and manoeuvre a cell onto a silicon or Poly-L-Lysine (PLL) coated substrate. The substrate is then moved perpendicularly with respect to the micropipette tip thereby sliding and shearing the cell across the surface. The perpendicular and parallel deflection of the cantilever enables us to directly measure the friction and normal force. A new approach for calibrating both sections of the cantilever has been developped and will also be presented in this work. As a proof of concept, the experiment is also performed with a polystyrene bead. The polystyrene bead, a simpler system, manifests some of the typical results expected from friction experiments. </p> / Thesis / Master of Science (MSc)

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 &#945; 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 &#949; 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 &#945; 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.

Thesis (M.Sc. (Biochemistry) -- University of Limpopo, 2018 / Dicerocaryum senecioides is a plant widely used as a nutritional source. It is used also for treatment of measles, wounds and to facilitate birth in domestic animal and humans in many parts of southern Africa (Mampuru et al., 2012). Findings in our laboratory have shown that a dichloromethane fraction of D. senecioides possesses antiinflammatory properties in human t-lymphocytes (Madiga, 2009), while the methanol crude extract possesses anti-proliferative and proapoptotic properties against Jurkat T cancer cells (Mphahlele, 2008). In this study, the probable anti-cancer effect of D. senecioides crude methanol leaf extract was investigated on cervical HeLa cancer cells. Dried powdered leaves of D. senecioides were extracted with absolute methanol to obtain a crude extract. To assess the cytotoxicity effect of the extract, KMST-6 and HeLa cell cultures were exposed to various extract concentrations (0 to 600 µg/ml) for 24 and 48 hours and subjected to the MTT assay. The results showed the extract to have no significant increase in the viability inhibition of HeLa cells at all tested concentrations after 24 hours of treatment. However, treatment with 400, 500 and 600 µg/ml of the extract for 48 hours revealed significantly increased HeLa cell viability inhibition. Furthermore, the extract showed to have no effect on the viability of normal human fibroblast KMST-6 cells at concentrations below 600 µg/ml, after 24 and 48 hours of treatment, thus showing selective cytotoxicity of the extract. To determine the mode of cell death associated with the increase in HeLa cell viability inhibition, the Hoechst 33258 nuclear staining assay and inverted light microscopy were employed. The data proposed apoptosis as the mode of cell death associated with the inhibition of HeLa cell viability. This was evidenced by changes in cell morphology such as the loss of HeLa cell radial extensions, cell shrinkage, as well as nuclear morphological features such as chromatin condensation. Apoptosis induction was further confirmed by the annexin-V/PI and multicaspase assays, using flow cytometry. The results showed an increase in the percentage of cells stained with annexin-V/PI, as well as increased caspase activity in extract-treated HeLa cells. To elucidate proapoptotic mechanisms of the extract, Western blotting analysis as well as the human apoptosis antibody array kit were used. This was to measure the expression profile of a number of apoptosis regulatory proteins. The results demonstrated modulation of some anti- and pro-apoptotic proteins, as well as the release of mitochondrial proteins required xiii for initiation of apoptosis, in the cytoplasm. The D. senecioides extract showed to have no effect on the cell division cycle of HeLa cells as determined by the PI staining assay. In conclusion, D. senecioides crude methanol leaf extract induced some degree of apoptosis in cervical HeLa cancer cells via the intrinsic apoptosis pathway. This was by modulating some of the members of the Bcl-2 family of proteins, which, facilitated the release of cytochrome C and activation of a caspase cascade. / South African Medical Research Council (SAMRC)

A capacidade de complexos oxindolimínicos de cobre (II) de inibir células cancerígenas HeLa foi determinada no Laboratório de Bioinorgânica, Catálise e Farmacologia (IQ-USP), coordenado pela Profa. Dra. Ana Maria da Costa Ferreira. Nesta dissertação um dos estudos foi obter o imageamento SERS desses compostos no interior de células HeLa vivas com o objetivo de identificar o alvo intracelular desses complexos. Devido a intensificação SERS desses complexos ser baixa e causarem a agregação das nanopartículas de ouro (AuNP) foi necessária a utilização de um marcador Raman (MR) nestes estudos, sendo escolhido o composto DTNB (Ácido 5,5\'-ditiobis-(2-nitrobenzóico)). Portanto a sonda SERS desse estudo consiste de AuNP esféricas juntamente com o MR e os complexos de cobre (AuNP/MR/Cu(isatp) ou Cu(nisatp)). No imageamento das células HeLa foram utilizadas duas radiações excitantes: 532 nm, onde não ocorre intensificação SERS originando uma imagem Raman dos constituintes celulares em maior quantidade e 785 nm, que possibilita o monitoramento dos sistemas AuNP/MR/Cu(isatp) ou Cu(nisatp) no interior das células. Para a análise dos espectros desse estudo (cerca de 2000 espectros para cada imageamento) a linha base foi corrigida por WLS (Weighted Least Squares) para os espectros SERS e por EMSC (Extended Multiplicative Scatter Correction) para os espectros Raman, além de ser feito alisamento utilizando um filtro Savitzky-Golay. Os espectros Raman foram submetidos à uma análise multivariada MCR-ALS (Multivariate Curve Resolution - Asymmetric Least Squares) para se obter as imagens de diferentes componentes celulares e uma análise univariada dos espectros SERS, baseada na banda do MR, possibilitando obter as imagens dos sistemas AuNP/MR/Cu(isatp) e AuNP/MR/Cu(nisatp). O imageamento SERS e Raman das células HeLa mostraram que os complexos têm a capacidade de penetrar nas células, juntamente com as nanopartículas de ouro, e causar dano ao núcleo celular, sem a entrada de água na célula, característica não observada ao tratar as células com outros sistemas sem a presença dos complexos. Outro estudo realizado nesta dissertação foi a caracterização do complexo de superfície formado entre a 5-nitroisatina em nanopartículas de prata, através da espectroscopia SERS e Raman ressonante. Foi observado que o complexo de superfície apresenta uma grande deslocalização eletrônica frente às espécies moleculares estudadas para fins comparativos. Essa deslocalização gera um cromóforo distinto no complexo de superfície que possui uma transição eletrônica na região do verde do espectro visível, enquanto que as espécies moleculares apresentam transições na região do azul e ultravioleta. / The inhibition of cancer HeLa cells by oxindoliminic copper (II) complexes was determined in the Bioinorganic, Catalysis and Pharmacology Laboratory (IQ-USP), coordinated by Profa. Dra. Ana Maria da Costa Ferreira. In this work one of the studies was to identify the intracellular targets of these complexes by SERS imaging of live HeLa cells. Due to the low SERS intensification of these complexes and the aggregation that they cause in the gold nanoparticles (AuNP), it was necessary to use a Raman reporter (MR) in these studies. The reporter chosen was the DTNB (5,5\'-dithiobis-(2-nitrobenzoic acid)). The SERS probe was constituted by spherical AuNP together with the MR and the copper complexes. In the imaging of live HeLa cells two excitation radiations were used: 532 nm, to acquire the normal Raman information of the cells and 785 nm, to acquire the localization of the modified AuNP inside the cells. To analyze the spectra (about 2000 spectra to each imaging) the baseline, for the SERS spectra, was corrected by WLS (Weighted Least Squares) and the EMSC (Extended Multiplicative Scatter Correction) was used to correct the Raman spectra. A Savitzky-Golay filter was used for smoothing. The Raman images of the cellular components were obteined by multivariate analysis MCR-ALS (Multivariate Curve Resolution - Asymmetric Least Squares), and the SERS images were obtained by univariate analysis based on the band of the MR. The SERS and Raman images showed that the complexes are capable of entering the cells together with the AuNP, and cause damage to the nuclei, without the entrance of water into the cell, a characteristic not observed in other studied systems. Another realized study was the characterization of a surface complex formed between 5-nitroisatin and silver nanoparticles (AgNP), using SERS and resonant Raman. The surface complex showed a massive electron delocalization when compared to the molecular species. This electron delocalization in the surface complex generated a new chromophore, not seen in the molecular complex. This chromophore has an electronic transition in the green region of the visible spectrum, while the molecular species transitions occur in higher energies.

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted. Includes bibliographical references.

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted. Includes bibliographical references.