Natural products have been a source of many pharmaceutical drugs and a number of drugs that are currently used in the treatment of cancer are derivatives of compounds originally isolated from natural products. There is evidence that extracts of Tulbaghia violacea can be used to treat cancer. The activation of apoptosis in cancer cells is a target for the development of novel anti-cancer drugs since one of the characteristics of cancer cells is resistance to apoptosis due to the deregulation of biochemical pathways leading to apoptosis. In fact, many current anti-cancer drugs exert their effects through the activation of apoptosis. Previous studies showed that extracts of T.violacea induce apoptosis in cancer cells and one study reported on the isolation of a compound (methyl-ԃ-D-glucopyranoside), which is responsible for the pro-apoptotic activity of the T.violacea extract. Therefore the aim of this study was to investigate the anti-cancer activity of methyl-ԃ-Dglucopyranoside and extracts prepared from T.violacea. In this study the pro-apoptotic activity of methyl-ԃ-D-glucopyranoside and extracts prepared from T.violacea were investigated on a panel of human cancer cell lines, which included HepG2, MCF7, H157, HT29 and the non-cancerous cell line, KMST6. The induction of apoptosis was evaluated by flow cytometry using several bioassays which measures biochemical events (caspase activation, phosphatidylserine externalisation and reactive oxygen species (ROS) production that is associated with the induction of apoptosis. The results demonstrated that the effects of methyl--D-glucopyranoside on cultured cells are transient and that the cells recover from the effects of methyl--D-glucopyranoside. This suggested thatmethyl-ԃ-D-glucopyranoside is not the compound responsible for the pro-apoptotic bioactivity in the T.violacea extract. This study also showed that cytotoxic and pro-apoptotic bioactivity of the leaf-extract was significantly higher in comparison to the tuber-extract. The bioactivity of the organic solvent extracts (dichloromethane, hexane, methanol and 50% methanol/water) of T.violacea leaves was also significantly higher than water extracts of T.violacea leaves. A comparison of the different organic extracts prepared from the T.violacea leaves showed that the highest activity was observed for the dichloromethane and hexane extracts. In an effort to identify the bioactive compound(s) the dichloromethane extract was subjected to Versaflash® column chromatography. However, due to problems experienced with the solubility of the dichloromethane sub-fractions, these compounds could not be tested for their bioactivity. Palmitone (16-hentriacontanone) was identified as one of the major compounds present in the dichloromethane sub-fractions. This compound was previously shown to have anticonvulsant bioactivity but there is no evidence in the literature that it has anti-cancer or pro-apoptotic activities. Fingerprinting of the methanol extract showed the presence of long chain fatty acid derivatives, flavonoids and allicin derivatives in the methanol extract. Although, this study failed to isolate the pro-apoptotic bioactive compound(s) present in the extracts of T.violacea, it confirmed that extracts of this plant induce apoptosis in cultured human cancer cell lines.

Šioje disertacijoje aprašyti tyrimai, atlikti siekiant įvertinti plazminių ląstelių imunotipavimo, taikant tėkmės citometriją, prognostinį potencialą. Patikrinome hipotezę, jog cirkuliuojančių plazminių ląstelių kinetika gydymo metu gali, anksčiau, nei standartiniai metodai įvertinti atsaką į gydymą. Tyrime naudojome iki tol neaprašytą plazminių ląstelių imunofenotipavimo metodiką. Mėginiai dažyti dviem skirtingais žymenų deriniais: CD56/CD138/CD45/CD19/CD38/CD20 ir cLambda/cKappa/CD138/CD19/CD38/CD56. Nustatėme, kad sveikų donorų kaulų čiulpuose aptinkama ~30% plazminių ląstelių, turinčių atipine CD56 ir/ar CD19 žymenų raiška. Optimizavome tėkmės citometrijos metodiką normalių ir piktybinų plazminų ląstelių aptikimui. Nustatėme, kad piktybinių cirkuliuojančių plazminių ląstelių proporcijos nesumažėjimas po pirmojo chemoterapijos kurso, su 91,7% jautrumu bei 93,3% specifiškumu prognozuoja ankstyvą progresiją. Pacientų, kuriems cirkuliuojančių plazminių ląstelių proporcija nesumažėjo, laikas iki progresijos ir bendras išgyvenamumas buvo statistiškai patikimai trumpesnis, nei pacientų, kuriems piktybinių cirkuliuojančių plazminių ląstelių proporcija sumažėjo ar šios ląstelės buvo neaptinkamos. Ištyrėme normalių plazminių ląstelių populiacijos klinikinę vertę pacientams, sergantiems mielomine liga. Apibendrinant, plazminių ląstelių imunofenotipavimas taikant tėkmės citometrijos metodą suteikia prognostiškai reikšmingos informacijos. Svarbiausias radinys, jog cirkuliuojančių... [toliau žr. visą tekstą] / The investigations presented in this dissertation were initiated with the intention to evaluate the prognostic value of plasma cells immunophenotypic analysis in multiple myeloma patients. We tested the hypothesis that kinetics of peripheral blood circulating plasma cells in response to first chemotherapy cycle could identify patients refractory to given treatment. We employed novel original methodology for plasma cells immunophenotyping: cells were stained in two tubes with antibody combinations CD56/CD138/CD45/CD19/CD38/CD20 and cLambda/cKappa/CD138/CD19/CD38/CD56. We found that ~30% of all plasma cells in bone marrow of healthy donors are immunophenotypically aberrant by CD56 and/or CD19 marker expression. We optimized immunophenotypic differentiation between malignant and normal plasma cells. Non reduction of malignant circulating plasma cells in response to first chemotherapy cycle predicted early progression with sensitivity and specificity of 91.7% and 93.3%, respectively. Time to progression and overall survival were significantly shorter in these patients as compared to patients with undetectable or reduced malignant circulating plasma cells. We also evaluated the clinical value of normal plasma cell subpopulation detection in peripheral blood and bone marrow of multiple myeloma patients. In summary, we demonstrated that immunophenotyping of plasma cells using multiparameter flow cytometry provides important prognostic information. The major finding was that the... [to full text]

The investigations presented in this dissertation were initiated with the intention to evaluate the prognostic value of plasma cells immunophenotypic analysis in multiple myeloma patients. We tested the hypothesis that kinetics of peripheral blood circulating plasma cells in response to first chemotherapy cycle could identify patients refractory to given treatment. We employed novel original methodology for plasma cells immunophenotyping: cells were stained in two tubes with antibody combinations CD56/CD138/CD45/CD19/CD38/CD20 and cLambda/cKappa/CD138/CD19/CD38/CD56. We found that ~30% of all plasma cells in bone marrow of healthy donors are immunophenotypically aberrant by CD56 and/or CD19 marker expression. We optimized immunophenotypic differentiation between malignant and normal plasma cells. Non reduction of malignant circulating plasma cells in response to first chemotherapy cycle predicted early progression with sensitivity and specificity of 91.7% and 93.3%, respectively. Time to progression and overall survival were significantly shorter in these patients as compared to patients with undetectable or reduced malignant circulating plasma cells. We also evaluated the clinical value of normal plasma cell subpopulation detection in peripheral blood and bone marrow of multiple myeloma patients. In summary, we demonstrated that immunophenotyping of plasma cells using multiparameter flow cytometry provides important prognostic information. The major finding was that the... [to full text] / Šioje disertacijoje aprašyti tyrimai, atlikti siekiant įvertinti plazminių ląstelių imunotipavimo, taikant tėkmės citometriją, prognostinį potencialą. Patikrinome hipotezę, jog cirkuliuojančių plazminių ląstelių kinetika gydymo metu gali, anksčiau, nei standartiniai metodai įvertinti atsaką į gydymą. Tyrime naudojome iki tol neaprašytą plazminių ląstelių imunofenotipavimo metodiką. Mėginiai dažyti dviem skirtingais žymenų deriniais: CD56/CD138/CD45/CD19/CD38/CD20 ir cLambda/cKappa/CD138/CD19/CD38/CD56. Nustatėme, kad sveikų donorų kaulų čiulpuose aptinkama ~30% plazminių ląstelių, turinčių atipine CD56 ir/ar CD19 žymenų raiška. Optimizavome tėkmės citometrijos metodiką normalių ir piktybinų plazminų ląstelių aptikimui. Nustatėme, kad piktybinių cirkuliuojančių plazminių ląstelių proporcijos nesumažėjimas po pirmojo chemoterapijos kurso, su 91,7% jautrumu bei 93,3% specifiškumu prognozuoja ankstyvą progresiją. Pacientų, kuriems cirkuliuojančių plazminių ląstelių proporcija nesumažėjo, laikas iki progresijos ir bendras išgyvenamumas buvo statistiškai patikimai trumpesnis, nei pacientų, kuriems piktybinių cirkuliuojančių plazminių ląstelių proporcija sumažėjo ar šios ląstelės buvo neaptinkamos. Ištyrėme normalių plazminių ląstelių populiacijos klinikinę vertę pacientams, sergantiems mielomine liga. Apibendrinant, plazminių ląstelių imunofenotipavimas taikant tėkmės citometrijos metodą suteikia prognostiškai reikšmingos informacijos. Svarbiausias radinys, jog cirkuliuojančių... [toliau žr. visą tekstą]

No description available.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Reovirus is internalized after interaction of the outer proteins μ1, σ1 and σ3 with the host cell. Proteolysis of σ3 and cleavage of μ1 (into δ and φ) eventually leads to the formation of a more infectious subviral particle named “ISVP”. The infectious entry of viruses, but not of ISVPs, can be blocked using various entry inhibitors and therefore, suggests that there is a threshold of σ3 digestion required to allow particle to bypass entry blockers. By combining protease and detergent to the digestion of virions, data from this work showed distinct particles generated along the transition pathway. In addition, studies involving flow cytometry and specific antibodies (anti-μ1) showed that between virus and ISVP there is a gradual yet heterogeneous particle proteolysis that is directly related to the virus infectivity. The findings and approaches taken for this thesis work can possibly be extended for studying other non-enveloped viruses. Moreover, it may help to shed some light on the development of safe and effective oncolytic agents.

This study was aimed at investigating adverse and beneficial long-term effects of jejunoileal bypass (JIB) sugery in obese patients. The JIB was the first widly used surgical procedure for treatment of morbid obesity. The weight loss was remarkable, but the procedure was declared not appropiate for obesity surgery in the late 1970's. Serious late adverse effects such as liver cirrhosis and malignancies, have been postulated. Unexpectedly few studies have adressed these problems. In the long-term follow-up of 87 uniformly operated patients, several persisting beneficial effects were found. The mean Body Mass Index was 41.5 kg/m2 at the time of operation and 29.7 kg/m2 sixteen years after the operation. Diabetes type II and hyperlipidemia, common in an obese population, was not found in this group. Reversals were performed in 3% of the patients in contrast to 20-30% in many earlier studies. Revisions performed in 8% of the patients due to excessive weight loss could have contributed to the good long-term outcome. Percutaneous liver biopsies from 44 patients taken 14-20 (mean 17) years after JIB revealed normal or fatty liver, a lower degree of histological abnormalities than in 11 biopsies taken at the time of operations 1-14 (mean 6) years postoperatively. Liver cirrhosis seen early in one patient could not be found in the late biopsies. Reduced activity of the fibrinolytic system has been shown to be a new cardiovacular risk factor. In 45 patients studied 14-20 years after JIB, the levels of both plasminogen activator inhibitor type 1 (PAI-1) and tissue plasminogen activator (tPA) were significantly lower than in a control group of 10 morbidly obese patients ( PAI-1: 8.4 vs 32 U/mL, tPA: 7.2 vs 12 pg/L). Bile acids are regarded as cofactors in the carcinogenesis in the colon and experimentally an increased frequency of malignant tumors has been demonstated after JIB in carcinogen-induced rats. In 30 of the operated patients, colonoscopy with biopsy was performed 11-17 yeras after the operation. No evidence for malignant transformation was found as reflected by an abscense of polyp formation, histologic dysplasia or aneuploidia in flow cytometric DNA analysis. Eight hundred and thirty patients from 10 hospitals subjected to JIB were compared to 1660 controls with respect to malignant diagnosis over a 20 years period. No significantly increased risk for colorectal carcinoma could be demonstrated. However the overall risk for malignant disease was increased in the operated patients. The frequency of endometrial carcinoma was significantly elevated up to five years after the operation but was normal after that time. In conclusion the postulated progress of serious adverse effects of JIB such as liver cirrhosis and malignant disease has not been possible to demonstrate. Several beneficial effects such as weight loss and reduction of cardiovascular risk factors have been found a long time after the operation. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 1995, härtill 5 uppsatser.</p> / digitalisering@umu

For the past two decades, flow cytometry has been widely used as a powerful analysis tool for the diagnosis of many diseases due to its ability to count, characterize and sort cells. However, conventional flow cytometers are often bulky, expensive and complicated because sophisticated fluidic, electronic and optical systems are required to realize the functions of flow cytometry. The high cost and the complexity in operation and maintenance associated with flow cytometers as well as the large size have limited its use. In recent years, the rapid development of microfluidics-based lab-on-a-chip technology has created a new pathway for flow cytometry. Microfluidic devices allow for the integration of multiple liquid handling processes required in the diagnostic assays, such as pumping, metering, sampling, dispensing, sequential loading and washing. These lab-on-a-chip solutions have been recognized as an opportunity to bring portable, accurate and sensitive diagnostic tests to the flow cytometry. However, most current microfluidic flow cytometry devices are micro- only in the microfluidic chip, the rest of most apparatuses are still large and costly, usually involving tubes, microscopes, lasers and mechanical pumps. Therefore, the objective of this study is to develop a novel lab-on-a-chip system based on the electrokinetically-induced pressure-driven flow and dual-wavelength fluorescent detection, which lights a promising pathway for making a real portable, compact, low-cost microfluidic flow cytometry device. In this study, the core of this microfluidic system is the custom-designed PDMS (polydimethylsiloxane) microchip. A novel method was applied to generate the electrokinetically-induced pressure-driven flow in a T-shaped microchannel using parameters settings that had been optimized by numerical study. This method combined both the electrokinetic pumping force and the pressure pumping force to eliminate their shortcomings associated with the use of each force alone. This is the fundamental of my study. By using this microchip, the size of the fluidic control subsystem is reduced significantly. Furthermore, the dual-wavelength fluorescent detection strategy is proposed in this thesis. On the optical detection side, excitation lights of two different wavelengths are provided by a single LED (light-emitting diode) from one side of the microchannel. Then the two emission lights are captured individually by two photo-detectors placed on the top and the bottom of the microchip. Compared with other microfluidic detection devices reported in the literatures that use lasers or PMTs (Photomultiplier tubes), this design allows for a significant reduction of 90% in the volume and cost. As another important part of my thesis research, a novel flow focusing method that allows the hydrodynamic focusing in a T-shaped microchannel with two sheath flows is developed. This method solves the biggest obstacle which exists in current microfluidic flow cytometry devices. In this method, no external pumps, valves and tubing are involved in the system. Although substantial progress has been made in current microfluidic flow cytometry, there is still a need for a low-cost, compact, portable microfluidic devices, especially in low-resource settings as well as the developing world for POC (point-of-care) diagnosis and analysis. This thesis work has made a great achievement towards the final goal.

Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2009.

Malaria is currently one of the most imperative parasitic diseases in developing countries. Artesunate has a short half-life, low aqueous solubility and resultant poor and erratic absorption upon oral administration, which translate to low bioavailability. Mefloquine is eliminated slowly with a terminal elimination half-life of approximately 20 days and has neuropsychiatric side effects. Novel drug delivery systems have been utilised to optimise chemotherapy with currently available antimalarial drugs. Pheroid™ technology is a patented drug delivery system which has the ability to capture, transport and deliver pharmaceutical compounds. Pheroid™ technology may play a key role in ensuring effective delivery and enhanced bioavailability of novel antimalarial drugs. The aim of this study was to evaluate the possible efficacy and bioavailability enhancement of the selected antimalarial drugs, artesunate and mefloquine, in combination with Pheroid™ vesicles. The in vitro efficacy of artesunate and mefloquine co-formulated in the oil phase of Pheroid™ vesicles and entrapped in Pheroid™ vesicles 24 hours after manufacturing were investigated against a 3D7 chloroquine-sensitive strain of Plasmodium falciparum. Parasitemia (%) was quantified with flow cytometry after incubation periods of 48 and 72 hours. Drug sensitivity was expressed as 50% inhibitory concentration (IC50) values. An in vivo bioavailability study with artesunate and mefloquine was also conducted in combination with Pheroid™ vesicles, using a mouse model. A sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to analyse the drug levels. C57 BL6 mice were used during this study. The selected antimalarial drugs were administered at a dose of 20 mg/kg with an oral gavage tube. Blood samples were collected by means of tail bleeding. The in vitro drug sensitivity assays revealed that artesunate, co-formulated in the oil phase of Pheroid™ vesicles and evaluated after a 48 hour incubation period, decreased the IC50 concentration significantly by 90%. Extending the incubation period to 72 hours decreased the IC50 concentration of artesunate, also co-formulated in the oil phase of Pheroid ™ vesicles significantly by 72%. No statistically significant differences between the reference and Pheroid™ vesicle groups were achieved when artesunate was entrapped 24 hours after manufacturing of Pheroid™ vesicles. Mefloquine co-formulated in the oil phase of Pheroid™ vesicles and evaluated after a 48 hour incubation period decreased the IC50 concentration by 36%. Extending the incubation period to 72 hours increased the efficacy of the Pheroid™ vesicles and the IC50 concentration was significantly decreased by 51%. In contrast with the results obtained with artesunate, entrapment of mefloquine in Pheroid™ vesicles 24 hours after manufacturing decreased the IC50 concentration significantly by 66%. The LC-MS/MS method was found to be sensitive, selective and accurate for the determination of artesunate and its active metabolite, dihydroartemisinin (DHA) in mouse plasma and mefloquine in mouse whole blood. Most of the artesunate plasma concentrations were below the limit of quantification in the reference group and relatively high outliers were observed in some of the samples. The mean artesunate levels of the Pheroid™ vesicle group were lower compared to the reference group, but the variation within the Pheroid™ vesicle group lessened significantly. The mean DHA concentrations of the Pheroid™ vesicle group were significantly higher. DHA obtained a higher peak plasma drug concentration with the Pheroid™ vesicle group (173.0 ng/ml) in relation to the reference group (105.0 ng/ml) and at a much faster time (10 minutes in Pheroid™ vesicles in contrast to 30 minutes of the reference group). Pharmacokinetic models could not be constructed due to blood sampling per animal limitation. The incorporation of mefloquine in Pheroid™ vesicles did not seem to have improved results in relation to the reference group. No statistical significant differences were observed in the pharmacokinetic parameters between the two groups. The relative bioavailability (%) of the Pheroid™ vesicle incorporated mefloquine was 7% less bioavailable than the reference group. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2010.