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Effects of plants-derived oleanolic acid in an in-vitro model hyperglycaemia-induced oxidative stress.Dlamini, Immaculate Nonkululeko. January 2010 (has links)
Diabetes mellitus (DM) has become a global threat in developing and developed countries, where diabetic patients are more prone to cardiovascular complications, a condition called diabetic cardiomyopathy. Studies have shown a direct link between hyperglycaemia and an increase in the production of reactive oxygen species in cardiac cells leading to diabetic cardiomyopathy. This study tests oleanolic acid, a bioactive compound from the plant Syzigium aromaticum as an antioxidant which could have a potential role in management of DM.
Aims
i) To extract Oleanolic acid (OA) from Syzigium aromaticim, ii) Investigate the antioxidant effects of plant derived OA in an in-vitro model of hyperglycaemia induced oxidative stress.
Methods
The flower buds of the Syzigium aromaticim [(Linnaeus) Merrill & Perry] (Myrtaceae) plant (commonly called cloves) were used to isolate OA. The ethyl acetate solubles from the cloves were subjected to chromatographic fractionation to yield OA powder. Spectroscopic analysis was done using 1D and 2D 1H and 13C NMR techniques for the identification of the structure of the compound. This compound was then used in vitro to test for its antioxidative properties. H9C2 cardiac myoblasts were employed which were treated with normoglycaemic (5.5 mM) and hyperglycaemic (33 mM) glucose conditions. The cells were then treated with oleanolic acid to test for its antioxidant properties. We looked at a dose-dependent (0, 20, 50 μM) and time-dependent effects of OA treatment (6 and 24 hrs) following 48 hours glucose exposure. ROS levels were measured using H2DCF-DA fluorescence staining using microscopy and flow cytometry techniques for analysis.
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Results
Recrystallisation of the powder with ethanol and inspection of the 1 and 2- dimensional 1H- and 13C-NMR spectra of the compound with comparison to literature data confirmed OA molecular structure and IUPAC numbering similar to that of literature characterized and confirmed the structure of oleanolic acid.
In cell specific data high glucose treatments on H9C2 cells showed increased ROS production (22 ± 6 % and 20 ± 7 % n= 3 p< 0.01) for 6 and 24 hrs treatments, respectively, compared to their normoglycaemic control groups. The 6 h OA treated group showed a decrease in ROS production with 26.6 ± 17.4 % for the 20 μM while for 50 μM there was a 37.7 ± 14.3% decrease. A ROS reduction trend was observed in the normoglycaemic group, but this was significant at 24 hrs with 46.8 ± 45.3% and 57.3 ± 9 % for both 20 and 50 μM treatments, respectively. The 24 hrs OA treated group showed a dose-dependent decrease in ROS with 50 μM more pronounced (80.7% ± 4.5 %). The 20 μM OA treatments also showed a 15.7 ± 19 % decrease in ROS.
Discussion
In the present study, we have evaluated the antioxidant effects of OA in vitro following extraction of the compound from Syzigium aromaticim. The oxidative stress induced by hyperglycaemia was attenuated by oleanolic acid and this also translated into decreased ROS suggesting its use as an antioxidant in alleviating cardiovascular complications associated with diabetes mellitus.
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Genetic regulation of vascular and floral patterning in Arabidopsis thalianaDeyholos, Michael K. January 2000 (has links)
The mechanisms that genes use to direct patterns of development are of fundamental interest. Using Arabidopsis thaliana as a model, I have investigated aspects of these mechanisms in the separate processes of vascular and floral development. Specifically, I conducted a screen for vascular-defective mutants, and analyzed a region of the genome that regulates the expression of the floral homeotic gene, AGAMOUS ( AG). / In this report, I describe the identification of over forty mutants that are abnormal in tracheary element development or vein patterning. The spectrum of mutant phenotypes that I observed indicates that the mechanisms that pattern primary and secondary veins of leaves or cotyledons are at least partially separable; that among the genes that affect vascular development, a significant proportion are repressors of vascular differentiation; and that the majority of vascular mutants that can be identified in this type of screen have pleiotropic phenotypes. / I characterized two of the mutants, varicose ( vcs) and scarface (sfc), in more detail. vcs mutants are temperature sensitive, and at the non-permissive temperature, accumulate distended tracheary elements around veins. VCS is also required at an early stage of leaf development for normal vein patterning, and interacts with the AUXIN RESISTANT 1 gene in this process. sfc mutants fail to develop normal, contiguous vein networks in cotyledons, leaves, sepals, and petals. It is specifically the secondary and higher order veins in these organs that are affected by the mutation. sfc mutants have exaggerated responses to exogenous auxin, and the SFC gene overlaps in primary and secondary vein patterning functions with an auxin-response factor gene MONOPTEROUS. / This report also includes an analysis of the cis-regulatory regions that control expression of AGAMOUS, a gene that when properly expressed in two central domains of the developing flower, directs the formation of carpels and stamens. My dissection of an AG intragenic region demonstrated that AG expression in stamens can be activated independently of carpels. Moreover, the stamen-specific expression pattern was found to be independent of APETALA2, a known negative regulator of AG, while the carpel-specific expression pattern was shown to be independent of LEUNIG, another negative regulator of AG.
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Characterisation of the CD161+ CD4+ T cell population in HIV and MTB infected individuals.Govender, Pamla. January 2012 (has links)
Human Immunodeficiency Virus (HIV) infection is characterized by immune dysfunction
that predisposes infected individuals to opportunistic infections such as Mycobacterium
Tuberculosis (MTB). The result of this is an exacerbation of HIV-TB related deaths
annually. Therefore there is an imperative need for HIV-TB focused research that aims to
identify immunological factors that are involved in the control of MTB and HIV in both
mono- and co-infected individuals. The CD161+ CD4+ T cell subset is linked to a distinct
phenotypic and functional profile. Importantly, these CD161+ T cells may act as an
important component of immunological defense and provide protection in infected tissues.
CD161+ CD4+ T cells have also been identified as the precursor population of Th17 cells
and it has been previously reported that reduction of CD161+ CD4+ T cells during HIV
infection may limit Th17 reconstitution (Prendergast et al., 2010). This may ultimately
contribute to impairment of mucosal immunity leading to the acquisition of opportunistic
infections such as MTB and disease progression in HIV infected individuals. Our study
aimed to comprehensively characterise the impact of HIV and MTB infection on the
CD161+ CD4+ T cell subset and to assess the frequency, phenotype and function of these
cells. The study also aimed to correlate the longitudinal variation in frequency, phenotype
and function with markers of HIV disease progression.
Methods
The frequency, phenotype and function of the CD161+ CD4+ T cell subset was measured
by flow cytometry. For the frequency and phenotypic assessment, whole blood was
collected from HIV negative and HIV/MTB mono and co-infected subjects (n = 17 per
patient group). Whole blood was surface stained with antibodies specific to CD3, CD4,
CD8, CD161 and chemokine receptors CD103, CCR6, CXCR4, CCR5 and CXCR6. The
percentage positive expression of CD161 on CD4+ T cells and chemokine receptor
expression was measured. The functional assessment of CD161+ CD4+ T cells involved
PBMC stimulation with antigenic stimulant, phorbol 12-myristate 13-acetate (PMA) and
ionomycin or ESAT-6/CFP-10, GAG, TB10.4 and Ag85a followed by intracellular
cytokine staining for IFN-γ, IL-17A, IL-22 and TNF-α. A subgroup of HIV negative
(frequency and phenotype, n = 10, function n = 7) and HIV mono-infected subjects
(frequency and phenotype, n = 10, function n = 7) were longitudinally followed to assess
variations in the frequency, phenotype and function of CD161+ CD4+ T cells over time.
Results
The CD161+ CD4+ T cell subset demonstrated high-level expression of chemokine
receptors CCR5, CCR6, CXCR4 and low-level expression of CD103 and CXCR6. The
subset also demonstrated the ability to produce cytokines IFN-γ, IL17A, IL-22 and TNF-α
in healthy subjects. Analysis of HIV infected samples revealed a significant reduction in
the frequency of the CD161+ CD4+ subset (median = 06.86%, p < 0.0001) compared to
that of healthy individuals (median = 14.75%). Correlation of the subset frequency to
markers of disease progression revealed a positive trend to CD4 count (r = 0.2590,
p = 0.0787) and a significant negative correlation to viral load (r = -0.3522, p = 0.0152).
Unlike with HIV infection, no significant changes in CD161+ CD4+ T cell frequency was
observed in individuals with LTBI (mono- or HIV co-infected) or active TB disease
compared to that of the healthy patient group. However, the exception to this was HIV
infected individuals with active TB disease (co-infected) (median = 03.80%, p < 0.0001).
Decreased CCR6 expression on CD161+ CD4+ T cells was observed in HIV monoinfected
(p = 0.0065) and HIV infected individuals with active TB disease (p = 0.007). No
functional changes were observed in both the HIV and MTB mono- and co-infected
cohorts following non-specific stimulation. An interesting positive trend in correlation
between IFN-γ production and CD4 count (r = 0.2727, p = 0.0733) was demonstrated with
a significant negative correlation between IFN-γ production and viral load observed
following non-specific antigenic stimulation (r = -0.3705, p = 0.0133). CD161+ CD4+ T
cells demonstrated antigen-specific T cell responses to peptides ESAT-6/CFP-10, TB10.4,
Ag85a and GAG in a small proportion of 69 study participants with variable ranges in
magnitude of the responses observed. The longitudinal assessment of CD161+ CD4+ T
cell frequency and phenotype demonstrated low-level proportion of CD4+ T cells
expressing CD161 and CCR6 expression longitudinally maintained in HIV mono-infected
compared to that of healthy individuals.
Conclusion
The phenotypic and functional profile of the CD161+ CD4+ T cell population indicates
that it may be an important component of immunological defense that may provide
mucosal defense and protection at epithelial sites and tissues e.g. expression of tissue
homing markers like CCR6 and the production of cytokines such as IL-17A and IL-22
(important in mucosal immunity). HIV infection is associated with a reduced frequency of
CD161+ CD4+ T cells. The correlation between CD161+ CD4+ T cell frequency and
markers of disease progression suggests that the observed low-level frequency in HIV
infected individuals may in part be a result of non-specific HIV-mediated depletion of
CD4+ T cells. However, lower levels of CD161+ CD4+ T cells in HIV infected individuals
could also be a result of naturally lower levels being present in individuals prior to
infection, thereby making these individuals more susceptible to HIV infection. The
significantly reduced levels of CCR6 expression on CD161+ CD4+ T cells in HIV monoinfected
individuals may also be an indication of cell subset migration to gut associated
lymphoid tissue (GALT, target site of HIV replication) during HIV infection. Given their
potential role in mediating signals that are essential for immune responses to microbes and
microbial products, migration of CCR6+ CD161+ CD4+ T cells to target sites of HIV
infection could serve as a protective measure in the fight against HIV infection. Although
there were no observable changes in the functional capacity of the CD161+ CD4+ T subset
in HIV infection, we believe that the reduction in frequency may contribute to HIV disease
progression and susceptibility to opportunistic infections such as MTB or active TB
disease. Unlike with HIV infection, infection with MTB appeared to have no significant
impact on CD161+ CD4+ T cells as there were no observable differences in frequency or
the functional capacity of the cell subset following PMA stimulation. However, MTB and
HIV antigen-specific responses were observed in a small proportion of the total 69 subjects
tested. This therefore indicates that a subset of CD161+ CD4+ T cells may act in an HIV
and MTB-specific manner. Additional MTB and HIV-specific responses may be present in
this CD161+ CD4+ population and may only be identified through stimulation with
additional antigenic targets.
Further investigation of CD161+ CD4+ T cells should be performed at the actual sites of
infection to investigate if CD161+ CD4+ T cells are concentrated at sites of disease. Also
it may be important to investigate the polyfunctionality of CD161+ CD4+ T cells to
understand the multifunctional capacity of the cell subset in providing immunological
defense to pathogens such as HIV and MTB. / Thesis (M.Med.)-University of KwaZulu-Natal, Durban, 2012.
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Studies on a multicatalytic, protease complex from Trypanosoma brucei brucei.Lomo, Peter Onyimbo. 20 December 2013 (has links)
Subcellular fractionation (together with immunocytochemical localisation studies) showed that
the parasite Trypanosoma brucei brucei possesses a multicatalytic protease complex (MCPTb).
This complex is predominantly cytosolic but some activity is also present in the nuclear
fraction. MCP-Tb was isolated from T. b. brucei and compared to the properties of other
proteasomes reported in the literature and to the 20S MCP isolated from bovine red blood cells
(MCP-rbc). The isolation procedure employed four-steps: anion exchange chromatography on
Q-Sepharose, adsorption chromatography on HA-Ultrogel, molecular exclusion
chromatography on Sephacryl S-300 and glycerol density gradient sedimentation.
The molecular mass of intact MCP-Tb was shown to be smaller than that of MCP-rbc.
Separation of the different proteasome subunits by 2D-PAGE showed that MCP-Tb has 12
different polypeptide components compared to the 28 different polypeptide components of
MCP-rbc. The N-terminal sequence of an MCP-Tb subunit showed that this subunit did not
have any obvious sequence homology with the subunits of proteasomes from other cells.
Furthermore, anti-MCP-Tb antibodies (which exhibited the in vitro inhibitory activity of
MCP-Tb) did not cross-react with MCP-rbc showing that MCP-Tb and MCP-rbc are antigenically distinct.
The basic enzymatic properties of MCP-Tb were fairly typical of other 20S proteasomes.
MCP-Tb had multiple peptidase activities (identified as chymotrypsin-like, trypsin-like and
peptidyl glutamylpeptide hydrolase activities) that are characteristic of proteasomes.
Furthermore, the characteristics of inhibition by a variety of inhibitors were similar to those of
other proteasomes, including MCP-rbc. The activities of 20S proteasomes from most cell
types are activated by endogenous high molecular mass complexes such as the bovine 19S
complex called PA700. These complexes form end-on associations with the 20S proteasome.
However, no endogenous MCP-activator was found in T. b. brucei. Nevertheless, MCP-Tb
was activated in an ATP-dependent manner by bovine PA700. Inhibition of the intrinsic
phosphatase activity of PA700 inhibited the protease enhancing effect of PA700. Electron microscopic examination of negatively stained MCP-Tb and MCP-rbc showed
particles that were morphologically indistinguishable. However, the MCP-Tb also exhibited
unique end-on associations between individual units forming long (up to 200 nm) ribbon-like
chains. Since access to the active sites of proteasomes occurs through the pores at the end of the complexes, this end-on association, when coupled to our observation of an apparent lack of an endogenous activator, suggests that T. b. brucei may have evolved an alternate mechanism
for controlling their proteasome activity. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1999.
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A microsatellite evaluation of the genetic status of the p27Kip1 and p21Cip1/WAF1 genes in oesophageal cancer.Gaffoor, Zakir. January 2008 (has links)
p21 C/P 1/"El and p 2 7K/P 1 are cyclin-dependant kinase inhibitors that fonn an integral part of the cell cycle process. These proteins function as cell-cycle inhibitors, and are able to induce cell cycle arrest by binding to cyclin complexes at key stages. p21 and p27 have been found to be down-regulated in various cancers. This study investigated aberrations at microsatellite markers linked to the p21 and p27 cell cycle genes, in a large cohort of oesophageal squamous cell carcinomas in South Africa. Fluorescent-based PCR were performed on markers linked to both the p21 and p27. The products were run with a 50-500hp marker on 6% denaturing polyacrylamide gels, on the ALFexpresstm' DNA sequencer. The detection and analysis of PCR products was achieved using the AL F e xp res sT M and Fragment M an a aerTm software programmes. Our findings indicate that markers linked to p27 display infrequent aberrations, with loss of heterozygosity ranging from 19% to 37%, and microsatellite instability at 3% to 7%. However, significant relationships between decreased survival time, and aberrations in markers DI2S391 and Dl2S364, were found to exist. Marker D6S1575 linked to p21 displayed frequent allelic loss at 47%, and was comparable to similar studies on the 6p region Further, LOH-Al in this marker was found to be significantly associated with poorly differentiated tumours. The findings from our study indicate that microsatellite aberrations occur infrequently at the p21 and p27 loci in oesophageal cancer. with the exception of marker D6S1575. In addition,this study clearly demonstrates the accuracy and sensitivity of the technology employed. This is the first microsatellite-based investigation of the p21/p27 gene loci in oesophageal cancer in South Africa, using a fluorescent-based PCR assay. / Thesis (M.Med.Sc.)-University of KwaZulu-Natal, Durban, 2008.
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Myotonic dystrophy : clinical and molecular spectrum in KwaZulu-Natal.Motala, Ayesha. January 2006 (has links)
Myotonic dystrophy is the commonest form of adult muscular dystrophy. Myotonic dystrophy 1 and 2 (DM 1 and DM 2) are autosomal dominant inherited disorders with unusual multisystem clinical features characterized by myotonia, progressive muscle weakness and wasting, cataracts, hypogonadism, frontal balding, cardiac conduction defects and diabetes. Severity varies from asymptomatic to severely affected phenotypes. DM1 presents with predominantly distal weakness whereas DM2 have predominantly proximal weakness.98% of patients identified worldwide present with DM1. DM 1 is caused by the expansion of an unstable CTG trinucleotide repeat in the 3' untranslated region of the myotonic dystrophy protein kinase gene on chromosome 19ql3.3. DM 2 is linked to the long arm of chromosome 3q21. It is caused by a tranucleotide, CCTG expansion in intron 1 of the zinc finger protein 9(ZNF9) gene that interferes with processing of a variety of RNAs. All DM mutations can be detected using a combination of the Southern Blot and Polymerase Chain reaction (PCR) techniques. Aim: This study aims to characterize the clinical spectrum and molecular features of myotonic dystrophy patients in KwaZulu - Natal between 1989 and 2005. Methodology: Patients included in this study were obtained from the database of patients diagnosed with Myotonic Dystrophy at the Department of Neurology in KwaZulu-Natal from 1989 to 2005. Patients were subjected to clinical, radiological and neurophysiological assessment. Molecular testing was performed using PCR and Southern blot. Results: Thirty-seven patients with Myotonic Dystrophy were identified. Twenty patients consented and were included into the study. Eighty-five percent of patients were of Indian descent and the remaining fifteen percent were White. No African patients were identified. Sixty-five percent were male and thirty-five percent female. Myotonia was clinically present in all patients. Ninety-five percent of patients presented with predominantly distal weakness of which 40% demonstrated mild weakness, 35% moderate weakness and 25 % severe weakness. No patients were identified with predominantly proximal wasting or weakness. Southern blotting demonstrated expanded CTG repeats (DM1) in all 20 samples analysed. The PCR analysis was unable to demonstrate expanded alleles. Conclusion: This study identified patients presenting with Myotonic dystrophy to the Department of Neurology in KwaZulu-Natal and demonstrated that Myotonic Dystrophy Type 1 remains the commonest clinical and molecular presentation. In addition it substantiated previous research findings wherein no South African of African descent was found to be affected by the disease. There have been no reported cases of Myotonic Dystrophy in African Black patients presenting to the Department of Neurology in Durban, no African Black patients have been diagnosed with Myotonic Dystrophy over the past 20 years. However ,the predominance of Indians in this study is more likely a reflection of referral bias than differing incidence amongst sections of the population. PCR analysis cannot detect trinucleotide repeat expansions beyond 200 repeats and as a result Southern Blotting remains the gold standard in obtaining a molecular diagnosis. A clinical diagnosis is sufficient and molecular confirmation is not an absolute requirement. / Thesis (M.Med)-University of KwaZulu-Natal, Durban, 2006.
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Gene expression analysis of squamous cell carcinoma of the oesophagus using a novel real time PCR probe systemMalik, Neelam. January 2010 (has links)
Squamous cell carcinoma of the oesophagus (OSCC) is a common malignancy that occurs with high frequency in certain parts of the world, including South Africa. The aetiology of OSCC has remained unclear although many studies suggest that it is caused by a combination of variable risk factors. Recent reports implicate a variety of genetic factors in the carcinogenesis of OSCC but their involvement is yet to be defined. / Thesis (M.Med)-University of KwaZulu-Natal, Durban, 2010.
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Dissecting contributions of structural elements of PSGL-1 to its interaction with P-selectin using AFMSánchez, René Javier 05 1900 (has links)
No description available.
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Membrane permeability of HIV-1 protease inhibitors.Ramlucken, Uraisha. 29 October 2014 (has links)
According to the 2012 UNAIDS global report, sub-Saharan Africa hosts 69% of the world’s total population living with HIV, South Africa being the most affected with a reported 24% incidence rate. To date, extensive research is being conducted globally, particularly involving anti-HIV treatment that targets the retroviral enzymes: reverse transcriptase, integrase and protease. The discovery of inhibitors to HIV protease which disrupts virion protein assembly has made this enzyme a prime target of anti-retroviral therapies, thus there exists a concerted research initiative to identify compounds with HIV protease inactivation potential. This study employs HIV protease that is isolated and purified from a genetically modified HIV protease overexpressing Escherichia coli strain to monitor the inhibitory capacity of new lead compounds. Optimized growth conditions for HIV protease production displayed that the use of chemically defined media resulted in higher yields of the enzyme. Recent research studies have shown that peptide-based cage and glycosylated compounds displayed HIV protease inhibitor activity in cell free enzymatic reactions that are comparable to commercially available HIV protease inhibitors. However, in contrast it has also been reported that these inhibitors are inactive in whole T-cell assays, when employing HIV infected CD4 cells.
It is a well-known fact that potential new chemical entities that do not possess oral bioavailability, in terms of their absorption properties, are not successful candidates within the drug discovery industry. Following this, the current study was designed to determine if inefficient membrane permeability of these promising anti-HIV protease lead compounds could result in their inactivity in whole T-cell assays. Two different methods were considered, a cell-based method using the Madin Darby Canine Kidney strain I (MDCKI) cell line and a non-cell based method, the parallel artificial membrane permeability assay (PAMPA). MDCKI cells have been extensively used to form monolayers that mimic human intestinal membranes whilst the PAMPA utilizes an artificial lipid membrane composition on a filter support. Data from permeability assays using the novel chemically synthesized inhibitors have been compared to commercially available drugs, antipyrine, metoprolol and caffeine, which displayed efficient membrane permeability characteristics, thereby validating the assay. The results indicated that novel cage-derived and glycosylated peptide inhibitors do not possess sufficient passive diffusion properties which may explain their inactivity in whole T-cell assays. / M.Sc. University of KwaZulu-Natal, Durban 2014.
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Effects of short-term intensified training on molecular factors related to myofiber regulationHinkley, James M. 05 August 2011 (has links)
Access to abstract permanently restricted to Ball State community only / Access to thesis permanently restricted to Ball State community only / School of Physical Education, Sport, and Exercise Science
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