Orientador: Júlio Lopes Sequeira / Resumo: As neoplasias mamárias das gatas frequentemente são malignas e agressivas, sendo os tipos mais comuns classificados como carcinomas tubulopapilíferos, sólidos e cribriformes. O grau histológico tem relação com o comportamento biológico desses tumores. No entanto poucos estudos tem abordado a cinética celular, a expressão de marcadores epiteliais e mioepiteliais, ou mesmo de moléculas de adesão e suas relações com a agressividade tumoral. Assim, o objetivo deste trabalho foi relacionar o tipo histológico dos carcinomas mamários das gatas e seus graus histológicos com os índices proliferativos e apoptóticos, e a expressão imuno-histoquímica de CK14, alfa-SMA, E-caderina e P-caderina. Foram utilizadas 31 amostras de carcinomas mamários de gatas. Submetidas a técnica imuno-histoquímica indireta com os anticorpos Ki-67, caspase-3-clivada, CK14, alfa-SMA, E-caderina e P-caderina. Predominaram as gatas SRD, com média de idade de 12 anos. Em frequência, o percentual dos tipos histológicos foi: 42%, 45,50% e 12,50% para os carcinomas tubulopapilíferos, sólidos e cribriforme; e foi de 9,65%, 41,95% e 48,80%, para os graus I, II e III, respectivamente. Os carcinomas tubulopapilíferos mostraram índice mitótico inferior aos carcinomas sólidos, assim como os carcinomas de grau I em relação aos de grau II e III. A característica basal (CK14 +) foi frequente nesses carcinomas. O subtipo complexo (alfa-SMA + ou alfa-SMA/CK14 +/-) é raro. Houve perda da expressão de E-caderina a medida que se ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Mammary tumors of the cats are often malignant and aggressive, the most common types classified as tubulopapillary, solid and cribriform carcinomas. The histological grade is related to the biological behavior of these tumors. However few studies have addressed the cell kinetics, the expression of epithelial and myoepithelial markers, or adhesion molecules and their relationship with tumor aggressiveness. The objective of this study was to correlate the histologic type of breast carcinomas of the cats and their histological grades with proliferative and apoptotic indices, and immunohistochemical expression of CK14, alpha-SMA, E-cadherin and Pcadherin. 31 samples of breast carcinomas cats were used. Subjected to indirect immunohistochemical technique with antibodies Ki-67, caspase-3, cleaved CK14, alpha-SMA, E-cadherin and P-cadherin. Predominated SRD cats, with a mean age of 12 years. In frequency, the percentage of histological types was 42%, 45.50% and 12.50% for tubulopapillary, solid and cribriform carcinomas; and it was 9.65%, 41.95% and 48.80% for grades I, II and III, respectively. The tubulopapillary carcinomas showed mitotic index lower than the solid carcinomas, as well as grade I carcinomas compared to levels II and III. The basal feature (CK14 +) was common in these carcinomas. The complex subtype (alpha-SMA + or alpha-SMA / CK14 +/-) is rare. There was a loss of Ecadherin expression as it becomes more aggressive. The P-cadherin was expressed high regardless of hi... (Complete abstract click electronic access below) / Mestre

>Magister Scientiae - MSc / Apoptosis or programmed cell death is vital to the development of organisms as they maintain the balance between cell death and cell growth. Failure to activate apoptosis has been implicated in carcinogenesis and often results from the over expression of anti–cancer proteins such as the X–linked inhibitor of apoptosis protein (XIAP). XIAP is over expresses in certain cancers and is a potent inhibitor of the initiator caspase 9 and effector caspases 3 and 7. The increased expression of XIAP in cancer cells result in the resistance to apoptosis. The control of XIAP is therefore considered as a target for anti–cancer drug development. Embelin or 2,5–dihydroxy–3–undecyl–1,4–benzoquinoine is a dihydroxyquinone compound that was previously shown to inhibit XIAP. This drug was discovered by structure based computational screening. The binding of embelin to XIAP displaces XIAP from caspases, consequently eliminating the inhibitory effect of XIAP on apoptosis. The objective of this study was to develop a gold nanoparticle that can be used for the targeted delivery of embelin to cancer cells thereby enhancing pro–apoptotic effects of the pro–apoptotic drug, ceramide. XIAP expression levels were investigated by Western blot analysis in a panel of human cancer cell lines available in the laboratory to identify two cell lines that can be used as low and high XIAP expression controls. Gold nanoparticles were synthesized and conjugated with embelin and a cancer targeting peptide with the amino acid sequence LTVSPWY. The biconjugated nanoparticles were used to co–treat MCF7 and HepG2 cells with ceramide. Apoptosis was quantified using flow cytometry. The uptake of gold nanoparticles was investigated using HR–TEM and ICP–OES. This study showed that gold nanoparticles conjugated with the LTVSPWY peptide is specifically targeted to and taken up by cancer cells. Gold nanoparticles conjugated with embelin promoted ceramide induced apoptotic cell death of cancer cells. However, it was observed that gold nanoparticles biconjugated with the LTVSPWY peptide and embelin failed to enhance the pro–apoptotic effects of ceramide. iii This study successfully demonstrated that gold nanoparticles conjugated with embelin could be used to enhance the effects of anti–cancer drugs using ceramide as an example.

Thesis (M.Sc.(Chemistry)) -- University of Limpopo, 2020 / Breast cancer is a malignant tumour that starts in the cells of the breast. Many studies revealed aromatase (CYP19A1) and cyclin-dependent kinase 2 (CDK2) as possible therapeutic targets regarding breast cancer treatment, because they play crucial roles in anti-apoptotic processes during cell proliferation. Quinoxaline derivatives have attracted a great deal of attention due to their biological activities against fungi, virus, bacteria and cancer. Computer modelling was employed in order to reduce time and cost by searching the library of molecules and identifying those which are likely to bind to the drug target. A library of new one hundred (100) nitro and amino quinoxaline alkyne derivatives were successfully designed and screened against target proteins (CYP19A1 and CDK2) using virtual screening technique and thirteen (13) molecules were identified to be hit compounds against both targets with the docking score ranging from -6.143 to -8.372 kcal/mol as a measure of binding affinity. The hit compounds were subjected to IFD in order to identify tight binding through intermolecular interactions with active site residues of the binding pocket of the target proteins. All identified nitro and amino quinoxaline alkyne derivatives were successfully synthesised in a multi-step reaction sequence and their spectroscopic analysis (NMR, FTIR and MS) were in good agreement with the proposed structures in a good to moderate yield. The newly synthesised novel amino and nitro-quinoxaline derivatives were evaluated for anti-proliferative activity against breast cancer (MCF-7). Compound 59 showed to possess good inhibition against MCF-7 with an IC50 of 9.102 μM, whereas compounds 34, 54, 56 and 61 showed promising activity against MCF-7 with an IC50 value of < 50 μM. However, the MTT assay results showed that 59 was found to be toxic with an IC50 value of 0.205 μM against Raw 264.7 cell line. The dose response investigations showed that 31 and 34 have the promising anti-cancer activity against CYP19A and the correlation between molecular modelling (in-silico) and CYP19A inhibition activities (in- vitro), was established as compounds 31 and 34 were identified to bind to the drug target (CYP19A) with the docking score of -8.372 and 7.630 kcal/mol respectively. All the synthesized compounds were evaluated for the antitubercular activity against Mtb H37Rv strain as a secondary study. Compounds 57-62 with nitro-quinoxaline derivatives exhibited stronger inhibitory effects on Mtb H37Rv strain. In addition, compounds 60 and 62 were found to be most active against Mtb H37Rv with the high activity at MIC90 of <0.65 and <0.64 μM respectively. All active compounds are currently investigated for their cytotoxicity which have not been investigated before / National Research Foundation (NRF) and SASOL Inzalo Foundation

Thesis (M.Sc. (Biochemistry)) -- University of Limpopo, 2021 / Lung cancer is the most diagnosed cancer with an estimated 3 million deaths expected by 2035. Bioactive phytochemicals present in plants are preferred as anticancer therapeutic agents, due to their ability to differentiate between cancerous and normal cells. One such plant, Senna italica, is traditionally used to treat diabetes, malaria, constipation, jaundice, fever and sexually transmitted diseases. Several studies have reported on its anti-proliferative potential against different types of cancers. However, there is scanty information regarding its molecular mechanism of action against different types of cancers, more especially lung cancer. This study, therefore, aims to determine the differential expression profiles of apoptotic genes in lung A549 cancer cells induced by treatment with S. italica leaf and root extracts in an attempt to understand its purported anticancer molecular mechanism of action. The leaves and roots of S. italica were dried in the dark and extracted with ethyl acetate and methanol. Screening for the presence of secondary metabolites was performed using thin layer chromatography and various standard chemical-based tests. The total phenolic and flavonoid compounds were evaluated using gallic acid and quercetin equivalence assays. The antioxidant activity of S. italica extracts was determined using DPPH free radical scavenging and ferric ion reducing power assays. The cytotoxicity of both leaf and root extracts on lung A549 cancer cells was evaluated using 3-(4,5- dimethylthiazol-2-yl)-2-5-diphenyl tetrazolium bromide (MTT) and further confirmed by Muse cell count and cell viability assays. The proliferation of cells, after treatment with different concentrations of the extracts, was examined using the Ki67 proliferation assay. Genotoxicity was determined to assess the potential damage caused by the extracts on the DNA using a MUSETM multicolour DNA damage kit following manufacturer’s protocol. The morphological change of cells treated with different concentrations of S. italica ethyl acetate root extract was analysed using acridine orange/ ethidium bromide (AO/EB) dual staining assay and examined under fluorescent light. The total number of cells undergoing apoptosis was also determined using the Annexin V assay. The expression of 84 key genes, involved in programmed cell death or apoptosis, was determined using the Human Apoptosis RT² Profiler PCR array kit. Senna italica methanol extract had a high content of plant materials in both leaves and roots compared to the ethyl acetate extract. A higher phenolic content was observedxii mainly in the leaf extract and a higher flavonoid content was observed in the root extract. Phytochemicals, such as phenols, tannins, flavonoids, terpenoids and steroids, which are known to exhibit anti-cancer activity against cancerous cells were abundant in the ethyl acetate leaf and root extracts as compared to the methanol leaf and root extracts. Additionally, the ethyl acetate root extract exhibited more antioxidants and radical scavenging activity in comparison to the methanol root extract. The IC50 of ethyl acetate root extract was determined to be 200µg/ml. Both methanol and ethyl acetate root extracts had little to no effect on the viability of lung A549 lung cancer cells. The results were confirmed by cell count and viability assay results. The cytotoxicity of ethyl acetate root extract was also evaluated against the normal kidney HEK-293 cells, which displayed little cytotoxic effect. The proliferation results indicated that S. italica ethyl acetate root extract has the potential to reduce the proliferation of lung A549 cancer cells. The ethyl acetate root extract was found to induce late apoptosis in A549 cells, but the genotoxicity data indicated that the DNA double strand breaks (DSBs) were repairable. The results further showed an expression of different genes that inhibit apoptosis, such as XIAP in lung A549 cells, following treatment with S. italica ethyl acetate root extract. In conclusion, the ethyl acetate root extract displayed a promising anti-cancer therapeutic potential, and thus warrants further investigation to elucidate the identity of the inherent chemical components that are responsible for the observed biological activity. / University of Limpopo and SAMRC

Cell death is a fundamental aspect of development and homeostasis; its dysregulation is commonly associated with disease. Historically, apoptosis has been the most heavily studied type of cell death, but there are many other non-apoptotic forms of cell death. The Drosophila ovary provides a powerful in vivo model to study non-apoptotic cell death. Each egg chamber in the ovary contains 15 nurse cells that support an oocyte throughout development, and at the end of oogenesis the nurse cells are surrounded by stretch follicle cells and undergo non-apoptotic cell death. The work in this dissertation investigated the role of stretch follicle cells in nurse cell death. Genetic ablation of the stretch follicle cells revealed that they are required for multiple nurse cell death events including the transport of cytoplasm to the oocyte and DNA fragmentation. We found that phagocytic machinery is required in the stretch follicle cells for the acidification and elimination of nurse cells, suggesting nurse cells die by phagoptosis. Furthermore, live imaging and a transgenic engulfment detector demonstrated that nurse cells are not engulfed piece-wise despite the requirement of phagocytosis machinery, but are instead surrounded and acidified extracellularly. To determine the mechanism driving nurse cell acidification, we performed a targeted RNAi screen against lysosome-associated genes. Using tissue-specific RNAi, we demonstrated that the V-ATPase proton pump is required in the stretch follicle cells for nurse cell acidification. GFP fusion proteins and antibody staining revealed that V-ATPases become enriched and localize to the stretch follicle cell plasma membranes to acidify the nurse cells that they surround. Following acidification, the stretch follicle cells were found to release cathepsins, lysosomal proteases, to break down and degrade the nurse cell. To uncover novel pro-death proteins that mediate signaling between the stretch follicle cells and nurse cells, we utilized proximity-dependent protein labeling and identified proteins enriched in the stretch follicle cells. Altogether this work uncovers a new role for lysosomal machinery acting at the plasma membrane of stretch follicle cells to drive nurse cell death, and identifies pro-death proteins in the stretch follicle cells that promote nurse cell death.

No description available.

No description available.

No description available.

<p> Bax is a pro-apoptotic protein believed to permeabilize mitochondria during apoptosis. The mechanism Bax uses is not well understood. In this work, we use fluorescence techniques to shed light on how tBid activates Bax and we examine the topology of the pore-forming domain of Bax. </p> <p> The manner in which tBid promotes apoptosis via Bax activation is not known. Study of tBid and Bax interaction using a new FRET pair showed that the proteins only interacted in the presence ofmembranes. The Bax pore was shown to have a variable size distribution. A fluorescence technique of simultaneously measuring pore formation, Bax insertion and FRET showed that tBid interaction with Bax occurred before all the Bax inserted or formed pores in the liposomes. A chronological order is proposed for Bax pore formation. tBid first binds to liposomes. tBid proceeds to interact with Bax, and Bax inserts into the membrane. After insertion, Bax oligomerizes and forms small pores. More Bax is recruited and the pores become larger. </p> <p> The two central hairpin helices of Bax, helices 5 and 6, are known as the pore-forming domain. We used cysteine scanning with the environment sensitive fluoroprobe NBD to gain insight into the topology of these helices. Fluorescence intensity changes and emission blue shifts showed that residues in these helices undergo conformational reorganization during pore formation. In the activated oligomeric conformation, fluorescence lifetimes showed that helix 5 was more inaccessible to water than helix 6. Cobalt, a cationic NBD quencher, effectively quenched residues in the pore-forming domain, consistent with a pore that is lined with anionic lipid head groups. Quenching with nitroxide groups at various lipid depths showed that residues on helix 6 were most quenched by a shallow quencher, while residues on helix 5 were quenched by deeper quenchers. Compared to beta sheet pore-forming proteins, the data obtained suggests that Bax and possibly other alpha helical pore-forming proteins form a lipidic pore in a dynamic environment. Combined together, the data suggest a model for Bax in which helix 5 spans the bilayer, and helix 6 is buried just below the lipid headgroups of a toroidal pore. </p> / Thesis / Master of Science (MSc)

No description available.