Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 108-129).

Thesis (doctoral)--University of Lund, 1996. / Added t.p. with thesis statement added.

Orientador: Patricia da Silva Melo / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-05T14:49:01Z (GMT). No. of bitstreams: 1 Correa_DanielHenriquedoAmaral_M.pdf: 1457648 bytes, checksum: 6adbe0feca89d1798ac2084d68454c69 (MD5) Previous issue date: 2005 / Resumo: A droga desidrocrotonina (DHC), isolada da planta Croton cajucara, é conhecida pelas suas atividades biológicas, entretanto apresenta uma vasta e conhecida toxicidade. Neste trabalho foram preparadas formulações contendo DHC associada a polímeros (PLGA) ou ciclodextrinas (beta, metil-beta e hidroxipropil-beta) para obter nanopartículas poliméricas e complexos de inclusão, respectivamente. Objetivamos neste trabalho a preparação das formulações de veiculação de DHC, sua caracterização e avaliação de parâmetros de toxicidade in vitro visando o estudo comparativo dos efeitos da DHC livre e veiculada. Células V79 e hepatócitos de ratos, sistemas celulares bem estabelecidos, permitiram avaliar alvos celulares como lisossomos, mitocôndrias e ácidos nucléicos frente aos possíveis danos tóxicos causados pela DHC. Nos hepatócitos, além dos parâmetros citados acima, também foi avaliada a quantidade de GSH existente nessas células, bem como a quantificação da peroxidação lipídica através da formação de TBARS e a atividade enzimática de enzimas do sistema de metabolização de drogas após tratamento com DHC livre e veiculada nos sistemas de liberação controlada ¿ nanoesferas de PLGA e complexos de inclusão com ciclodextrinas. Nossos estudos em forma geral, indicam que os polímeros ou complexos de inclusão são estruturas que conferem estabilidade à DHC, provocam liberação controlada e diminuição dos efeitos tóxicos da DHC / Abstract: The drug dehydrocrotonin (DHC), isolated from Croton cajucara plant, is known for its biological activities, however presenting a well-known toxicity. In this work, we produced complexes containing DHC associated with polymers (PLGA) or with cyclodextrins (beta, methyl-beta and hydroxypropyl-beta) to obtain polymeric nanoparticles and inclusion complexes respectively. We aimed in this work to produce DHC-loaded formulations, to characterize these formulations and to evaluate the in vitro toxicity parameters through the comparative study of the effects of the free DHC and DHC-loaded. V79 cells and rat hepatocytes, well-established cellular systems, had allowed us to evaluate cellular targets as the lysosomes, mitochondrias and nucleic acids against the possible toxic effects caused by DHC. In hepatocytes, beyond the above-listed parameters, the amount of GSH in these cells was also evaluated, as well as the quantification of lipid peroxidation through the formation of TBARS and the enzymatic activity of the enzymes from the drug metabolization system after treatment with DHC in its free state and associated with controlled release systems ¿ nanospheres of PLGA and cyclodextrins inclusion complexes.Our studies indicates that the polymers or inclusion complexes are structures that confer stability to DHC, causing controlled release and reduction of the toxic effects of DHC / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

Alopecia areata (AA) is a chronic inflammatory disease of hair follicles manifesting as patchy areas of hair loss on the scalp and body. Development of AA is associated with pen- and intra-follicular inflammation of anagen stage hair follicles, primarily by CD4+ and CD8+ cells. We hypothesized that if cell-mediated cytotoxicy against hair follicles is to be a component of the hair loss disease mechanism, increased expression of genes and products typical of cytotoxic cells, as well as increased apoptosis activity within affected hair follicles, would be expected to occur in the lesional skin compared to the normal skin. Furthermore, we studied gene expression levels of multiple cytokines and characteristic chemokines, using the C3FI/HeJ mouse model of AA. mRNA expression levels of granzyme A, granzyme B, perform Fas, Fas ligand, TNF-cL, TNF-aRl and R2, TRAIL, TRAILR, TRAMP, Thi-, Th2-, and Th17-associated cytokines, as well as multiple chemokines were compared between the skin, draining lymph nodes, thymus and spleens of normal and AA-affected mice using quantitative reverse transcriptase PCR. FasL, granzyme A, granzyme B, pro- and anti-inflammatory cytokines were all highly up-regulated in the skin of AA-affected mice. Immunohistochemical studies of the skin revealed that, although greater numbers of granzyme B and FasL expressing cells were present in AA affected skin, the cells were morphologically diffusely distributed and not exclusively located within the focal pen- and intrafollicular infiltrate. The majority of these cells were further characterized as mast cells, which were also found in substantially greater numbers in the skin of mice with AA compared to their normal haired controls. Almost no perform expressing cells were identified in AA affected mouse skin and TUNEL staining suggested relatively limited apoptosis activity in hair follicle keratinocytes. In conclusion, while granzymes and FasL may play important roles in disease development, the profiles and patterns of expression are not consistent with direct cell-mediated cytotoxic action against the follicular epithelium in chronic mouse AA. Potentially, hair growth inhibiting cytokines may play a more dominant role in AA development than previously thought. Furthermore, mast cells, with their increased presence around hair follicles in the AA affected mouse skin and their ability to express granzyme B and FasL, are suggested as potential key players in the pathogenesis of AA. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

No description available.

Treatment of acute myeloid leukemia, an aggressive hematopoietic malignancy of myeloid progenitors, has remained rather stagnant over the course of several decades. Infusions of cytarabine and anthracycline antibiotics have dominated the landscape of AML therapy, with minor changes to dosing schedule occasionally making slight adjustments to efficacy or tolerability. Improvements in prognosis have been bittersweet, with most progress seen in younger populations less likely to get the disease, and already more likely to achieve remission and to meet survival milestones. Much of this progress is attributed to other factors, such as improved supportive care and availability of hematopoietic stem cell and platelet transfusion. In most patients, occupying the 60-and-above demographic, improvements in survival have not been significant. In turn, the population impact of AML has changed little over time. While accounting for about one-third of total leukemia cases and one percent of total cancer cases, AML accounts for about one half of total leukemia deaths and two percent of total cancer deaths. Most advances straying away from standard treatment have been in important pathways that could be impactful in subsets of the overall AML patient population. Tyrosine kinases are implicated in numerous cancers including AML, with activity-enhancing mutations conferring growth advantages to malignant cells. About one-third of AML patients have mutations in one such kinase, FLT3, and may benefit from inhibitors to tyrosine kinases overall and from FLT3- specific agents. Mutations in isocitrate dehydrogenases highlight another subpopulation, about one-fifth of AML patients, who might benefit from emerging agents that inhibit these pathways from creating a leukemia-favoring environment in the bone marrow. Other pathways similarly implicated in numerous cancers including AML are being targeted with new agents that can benefit some AML patients, such as Hedgehog signaling and apoptotic regulation. Still, breakthroughs are needed that can help most AML patients, particularly in the cases of relapsed leukemia that occurs in most patients within a year or two after remission is achieved. CD33 is among a few molecular targets for AML, though it is just as ubiquitously expressed on healthy myeloid cells. Antibody-drug conjugates like Mylotarg have made progress in this approach, though hematopoietic toxicities have made treatment difficult in older populations. Clever techniques such as ablation of CD33 from healthy myeloid progenitors may be supportive in CD33-based approaches, and immunotherapy involving CD33-targeting is a rapidly growing research focus. This dissertation describes a new type of bispecific antibody that binds CD33 on AML and CD3 on cytotoxic T cells in a proof-of-concept study. Various formats for bifunctional molecules have been created and used clinically, including antibody-drug conjugates and bispecific antibodies that simultaneously engage antigens on two different types of cells. Those like the one described here, bispecific T-cell engagers, have typically taken the form of single-chain fusion proteins containing the variable regions binding to both antigens of interest. Other bispecific antibodies have imitated naturally-occurring immunoglobulin structures, boasting superior pharmacokinetics while facing steep obstacles in large-scale production. The single-chain fusions, easier to produce, can face difficulties in full engagement, with loss of function sometimes seen in fusion partners at the C-terminus. We propose a new format, believed to present two antigen-binding domains in N-terminal positions on a two-chain heterodimeric structure. Capitalizing on an elegantly designed system of hydrophobic cores and hydrogen-bonding networks generating an orthogonal heterodimer, we added an immunoglobulin hinge region to secure a permanently-bound heterodimer, and attached domains binding to CD3 and CD33. We hypothesized that this design, ensured to present its antibody components at N-termini, could bind two antigens at a distance appropriate for facilitating T cell cytotoxicity to AML. After expressing and purifying these proteins in mammalian cells, we demonstrated their ability to persist as a bispecific heterodimer. We showed in vitro that our bispecific heterodimers could bind both CD3+ and CD33+ cells, and that they bolstered T cell cytotoxicity to AML cell lines in a dose-dependent manner. Monomeric components bound only CD3+ or CD33+ cells depending on antibody variable domain present, and had no effect on T cell cytotoxicity. In a mouse model of minimal residual disease, T cells alone did not have a significant effect on the growth of AML, nor did they have an effect on overall survival. T cells with bispecific heterodimer greatly extended survival, and mice of this treatment group were free of leukemia. These findings suggest that this format for bispecific proteins allows for robust simultaneous engagement with both antigens of interest in a manner conducive to T cell cytotoxicity against AML. We believe this presents a compelling modular system for bispecific antibodies, where CD3- and CD33-binding domains can be readily swapped with domains binding to other cancer- or immune cell-specific antigens, and can be further developed into a trispecific system engaging other immune cells or extending half-life with anti-albumin or Fc domains.

Lau Wing-Ngar Vivian. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 135-144). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / Abbreviations --- p.vi / List of figures --- p.viii / List of tables --- p.xi / Contents --- p.xiii / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- General introduction --- p.1 / Chapter 1.2 --- Roadside air pollution worldwide and in Hong Kong --- p.2 / Chapter 1.2.1 --- Air quality in Hong Kong --- p.3 / Chapter 1.3 --- Characteristics of particulate matter --- p.9 / Chapter 1.4 --- Composition and sources of particulate matter --- p.11 / Chapter 1.5 --- Toxic effects of particulate matter --- p.12 / Chapter 1.5.1 --- Lung injury --- p.12 / Chapter 1.5.2 --- Cardiovascular injury --- p.15 / Chapter 1.5.3 --- Mutagenesis and carcinogenesis --- p.16 / Chapter 1.6 --- Aims of my study --- p.16 / Chapter 2 --- Toxic Effects of Heavy Metals Ions on Selected Cultured Cell-lines --- p.18 / Chapter 2.1 --- Introduction --- p.18 / Chapter 2.1.1 --- Metals --- p.18 / Chapter 2.1.1.1 --- Cadmium --- p.22 / Chapter 2.1.1.2 --- Chromium --- p.23 / Chapter 2.1.1.3 --- Lead --- p.25 / Chapter 2.1.1.4 --- Zinc --- p.26 / Chapter 2.1.2 --- Metallothioneins --- p.28 / Chapter 2.1.3 --- p53 --- p.31 / Chapter 2.1.4 --- Tumor Necrosis Factor-alpha (TNF-α) --- p.32 / Chapter 2.1.5 --- Aims of this chapter --- p.32 / Chapter 2.2 --- Materials and methods --- p.35 / Chapter 2.2.1 --- Reagents --- p.35 / Chapter 2.2.2 --- Cultured Cell lines --- p.35 / Chapter 2.2.2.1 --- PU5-18 --- p.36 / Chapter 2.2.2.2 --- LL24 --- p.36 / Chapter 2.2.2.3 --- HBE4-E6/E7 --- p.37 / Chapter 2.2.3 --- Cytotoxicity assays --- p.37 / Chapter 2.2.4 --- ELISA assays --- p.40 / Chapter 2.2.4.1 --- ELISA assay ofp53 levels --- p.41 / Chapter 2.2.4.2 --- ELISA assay of TNF-α levels --- p.43 / Chapter 2.2.5 --- MT gene expression studies by Luciferase assay --- p.44 / Chapter 2.2.5.1 --- PCR amplification --- p.44 / Chapter 2.2.5.2 --- 5´ة End modification of PCR amplified DNA --- p.44 / Chapter 2.2.5.3 --- Ligation of DNA fragment to linearized vector --- p.46 / Chapter 2.2.5.4 --- E. coli. transformation by heat shock --- p.46 / Chapter 2.2.5.5 --- PCR sequencing --- p.47 / Chapter 2.2.5.6 --- Transfection of plasmid into HBE4-E6/E7 cells --- p.49 / Chapter 2.2.5.7 --- Data analysis --- p.50 / Chapter 2.3 --- Results and discussion --- p.51 / Chapter 2.3.1 --- Cytotoxicity assays --- p.51 / Chapter 2.3.2 --- Combination effects of metals on cytotoxicity --- p.61 / Chapter 2.3.3 --- p53 --- p.65 / Chapter 2.3.4 --- TNF-α --- p.68 / Chapter 2.3.5 --- MT gene expression studies by Luciferase assay --- p.69 / Chapter 2.4 --- Conclusion --- p.74 / Chapter 3 --- Effects of Polycyclic Aromatic Hydrocarbons (PAHs) on Cultured Cell-lines --- p.75 / Chapter 3.1 --- Introduction --- p.75 / Chapter 3.2 --- Materials and methods --- p.79 / Chapter 3.2.1 --- Reagents --- p.79 / Chapter 3.2.2 --- Cell culture --- p.79 / Chapter 3.2.3 --- AlamarBlue assay --- p.80 / Chapter 3.2.4 --- EROD assay --- p.80 / Chapter 3.3 --- Results and discussion --- p.84 / Chapter 3.4 --- Conclusion --- p.88 / Chapter 4 --- Chemical and Biological Assays on Roadside Dust --- p.89 / Chapter 4.1 --- Introduction --- p.89 / Chapter 4.1.1 --- Composition of particulate matter in Hong Kong --- p.89 / Chapter 4.1.2 --- Metal contents of particulate matter in Hong Kong --- p.91 / Chapter 4.1.3 --- Possible adverse health impacts of particulate matter --- p.94 / Chapter 4.1.3.1 --- In vitro studies using different cell models --- p.94 / Chapter 4.1.3.2 --- In vivo studies using rodents --- p.97 / Chapter 4.1.3.3 --- Epidemiological studies --- p.98 / Chapter 4.1.4 --- Aims of this chapter --- p.100 / Chapter 4.2 --- Materials and methods --- p.101 / Chapter 4.2.1 --- Sampling of roadside dust --- p.101 / Chapter 4.2.2 --- Chemical analysis of roadside dust --- p.104 / Chapter 4.2.2.1 --- Reagents --- p.104 / Chapter 4.2.2.2 --- Total metal contents --- p.105 / Chapter 4.2.2.3 --- Extractable metal contents --- p.105 / Chapter 4.2.3 --- Biological assays --- p.105 / Chapter 4.2.3.1 --- Cell models --- p.106 / Chapter 4.2.3.2 --- Pretreatment of roadside dust --- p.106 / Chapter 4.2.3.3 --- AlamarBlue assay --- p.106 / Chapter 4.2.3.4 --- ELISA assays --- p.108 / Chapter 4.2.3.5 --- Luciferase assay --- p.108 / Chapter 4.3 --- Results and discussion --- p.110 / Chapter 4.3.1 --- Total metal contents --- p.110 / Chapter 4.3.2 --- Extractable metal contents --- p.113 / Chapter 4.3.3 --- AlamarBlue assay --- p.116 / Chapter 4.3.4 --- p53 --- p.122 / Chapter 4.3.5 --- TNF-α --- p.122 / Chapter 4.3.6 --- Luciferase assay --- p.126 / Chapter 4.4 --- Conclusion --- p.129 / Chapter 5 --- General discussion and conclusion --- p.130 / Chapter 6 --- References --- p.135

A dissertation submitted to the Faculty of Science, University of Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science. March 2015 / Lung cancer is currently the most deadly form of cancer due to the fact that metastasis occurs in the lymph nodes making it difficult to remove by surgical means. Chemotherapy has been the most successful method of treatment, although it has been harmful to human health as a consequence of non-specific cytotoxicity. There has been, therefore, a growing interest in cancer research to develop alternative cancer treatments, which are less toxic. Currently plant-derived drugs are perceived to be more effective as they display both cytotoxic activity and are less harmful to overall human health. Thus the aim of the study was to determine the cytotoxic effects of the plant Lobostemon fruticosus on A549 cells. The IC50 of the methanol and butanol extracts of L. fruticosus were obtained at 40 μg/ml and 50 μg/ml, respectively. DNA fragmentation was observed after 48 hour exposure to treatments, indicating that the plant extracts induced apoptosis. Cell cycle analysis indicated that the plant extracts inhibited cell cycle progression at the sub-G0 phase, which indicated that the cells had undergone apoptosis. RT-PCR showed that the expression of p53 was down-regulated; however, p21 and Bax were up-regulated in all treatments. LC-MS identified that the compounds from the plant extracts are known apoptotic inducers. The results lead to the conclusion that the extracts of L. fruticosus, induce cell death in A549 cells. The plant extracts induced a p53-independent apoptotic mechanism, which was mediated by Bax and p21. Key words: Lobostemon fruticosus, camptothecin, taxol, Non-small cell lung cancer (NSCLC)

Thesis (Ph. D.)--University of Missouri--Columbia, 1998. / Typescript. Vita. Includes bibliographical references (leaves: 138-170). Also available on the Internet.

Thesis (Ph.D.)--Memorial University of Newfoundland, Faculty of Medicine, 1999. / Includes bibliographical references.