Adherence to antiretroviral therapy in children in Zimbabwe: a randomized control trial to validate a new self-reported adherence monitoring tool
- Authors: Mugore, Linnetie
- Date: 2014
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/54734 , vital:26607
- Description: Background: Among children taking antiretroviral therapy (ART), self-reports have been widely reported to over-estimate adherence levels. Pill count adherence levels are often lower than self-reported levels, with unannounced home pill count adherence being lower than facility based pill count adherence. There is often poor agreement between pill count adherence levels and those measured using other objective adherence measuring methods such as Medication Event Monitoring Systems (MEMS®), which is widely viewed as the gold standard for adherence measurement. Objectives: The aim of this study was to design and evaluate a new self-reported paediatric adherence monitoring tool, assess the feasibility of using pill count methods in monitoring adherence and identify challenges to reporting adherence among children on ART in rural and urban Zimbabwe. Methods A dual centre, superiority, parallel design RCT was conducted to evaluate the newly-developed visually- and verbally-cued „past 10 days‟ tool for the assessment of adherence in children on ART at two sites in Zimbabwe; Harare Central Children‟s Hospital in an urban setting, and Murambinda Mission Hospital, a rural site. Child-caregiver pairs presenting to one of these facilities for the child‟s review of ART and refill of the medication were recruited, signed informed consent obtained, and were randomised for self-reported adherence monitoring into either the experimental group („new 10-day tool‟) or the control group („PACTG-style‟ self-report tool). Data (demographic, socioeconomic, and reported adherence) were collected in individual interviews with child-caregiver pairs. Additional adherence monitoring methods used for both groups included the Morisky-8-Item Medication Adherence Scale (MMAS-8) and a facility based pill count. FGDs were held with groups of caregivers and groups of children ≥13 years of age to understand reasons for non-adherence as well as issues around reporting non-adherence. Superiority testing was conducted by comparing adherent proportions and their confidence intervals (95% CI). Further concurrent validity test was done using the Mann-Whitney U test to evaluate the relationship between the new tool and the MMAS-8 scores. Agreement between the child and caregiver reports of adherence was used as a test of reliability of the new tool using the kappa statistic. Socio-demographic, clinical and care-related factors associated with adherence were identified using reported adherence in both child and caregiver groups in a logistic regression model. Two pill count methods were assessed for feasibility using the proportions of children with complete data for calculating adherence levels, and their CI and a comparison of the two methods, a routinely-used method and one that incorporated the reported residual quantity (RRQ) of medication at last refill. Results : Analysis included 245 child-caregiver pairs, 123 in the experimental group and 122 in the control group. The median age for children was 9 years. In the experimental group, adherence by caregiver and child reports ranged from 94.3% - 98.4% and 78.4% - 96.1%, and those in the control group ranged from 89.2% - 97.5% and 71.2% - 98.1%, respectively. There was no significant difference between adherence levels in the two groups. Adherence levels measured by both the experimental and control tools were found to be associated with MMAS-8 adherence levels (p <0.05). Agreement between child- and caregiver-reported adherence was moderate though significant (kappa; 0.407, p <0.05). Only about half of the children had adequate data to compute pill counts. Proportions adherent at 95% cut-off were 39% by the „routine pill count‟ and 58% by the „Pill count RRQ‟. Being an orphan was associated with child reported-adherence whereas use of non-human reminders, having a maternal relative as a primary caregiver and knowledge of dose frequency, were all associated with caregiver-reported adherence. Major causes of non-adherence mentioned during the FGDs included interference of medication administration times with scheduling of routine socio-economic activities and lack of support from some non-biological caregivers. Reporting of non-adherence appeared to be hampered by perceptions of negative reactions by healthcare workers to these reports and by caregivers being unaware that the child missed some doses. Conclusions: The „new 10-day tool‟ was not shown to be superior to the „PACTG-style tool‟ in detecting non-adherence, however this new tool was found to be a valid and reliable adherence monitoring tool that included a moderately long recall period of 10 days, can be applied without the need for the respondent to remember names of individual medicines in the
- Full Text:
- Authors: Mugore, Linnetie
- Date: 2014
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/54734 , vital:26607
- Description: Background: Among children taking antiretroviral therapy (ART), self-reports have been widely reported to over-estimate adherence levels. Pill count adherence levels are often lower than self-reported levels, with unannounced home pill count adherence being lower than facility based pill count adherence. There is often poor agreement between pill count adherence levels and those measured using other objective adherence measuring methods such as Medication Event Monitoring Systems (MEMS®), which is widely viewed as the gold standard for adherence measurement. Objectives: The aim of this study was to design and evaluate a new self-reported paediatric adherence monitoring tool, assess the feasibility of using pill count methods in monitoring adherence and identify challenges to reporting adherence among children on ART in rural and urban Zimbabwe. Methods A dual centre, superiority, parallel design RCT was conducted to evaluate the newly-developed visually- and verbally-cued „past 10 days‟ tool for the assessment of adherence in children on ART at two sites in Zimbabwe; Harare Central Children‟s Hospital in an urban setting, and Murambinda Mission Hospital, a rural site. Child-caregiver pairs presenting to one of these facilities for the child‟s review of ART and refill of the medication were recruited, signed informed consent obtained, and were randomised for self-reported adherence monitoring into either the experimental group („new 10-day tool‟) or the control group („PACTG-style‟ self-report tool). Data (demographic, socioeconomic, and reported adherence) were collected in individual interviews with child-caregiver pairs. Additional adherence monitoring methods used for both groups included the Morisky-8-Item Medication Adherence Scale (MMAS-8) and a facility based pill count. FGDs were held with groups of caregivers and groups of children ≥13 years of age to understand reasons for non-adherence as well as issues around reporting non-adherence. Superiority testing was conducted by comparing adherent proportions and their confidence intervals (95% CI). Further concurrent validity test was done using the Mann-Whitney U test to evaluate the relationship between the new tool and the MMAS-8 scores. Agreement between the child and caregiver reports of adherence was used as a test of reliability of the new tool using the kappa statistic. Socio-demographic, clinical and care-related factors associated with adherence were identified using reported adherence in both child and caregiver groups in a logistic regression model. Two pill count methods were assessed for feasibility using the proportions of children with complete data for calculating adherence levels, and their CI and a comparison of the two methods, a routinely-used method and one that incorporated the reported residual quantity (RRQ) of medication at last refill. Results : Analysis included 245 child-caregiver pairs, 123 in the experimental group and 122 in the control group. The median age for children was 9 years. In the experimental group, adherence by caregiver and child reports ranged from 94.3% - 98.4% and 78.4% - 96.1%, and those in the control group ranged from 89.2% - 97.5% and 71.2% - 98.1%, respectively. There was no significant difference between adherence levels in the two groups. Adherence levels measured by both the experimental and control tools were found to be associated with MMAS-8 adherence levels (p <0.05). Agreement between child- and caregiver-reported adherence was moderate though significant (kappa; 0.407, p <0.05). Only about half of the children had adequate data to compute pill counts. Proportions adherent at 95% cut-off were 39% by the „routine pill count‟ and 58% by the „Pill count RRQ‟. Being an orphan was associated with child reported-adherence whereas use of non-human reminders, having a maternal relative as a primary caregiver and knowledge of dose frequency, were all associated with caregiver-reported adherence. Major causes of non-adherence mentioned during the FGDs included interference of medication administration times with scheduling of routine socio-economic activities and lack of support from some non-biological caregivers. Reporting of non-adherence appeared to be hampered by perceptions of negative reactions by healthcare workers to these reports and by caregivers being unaware that the child missed some doses. Conclusions: The „new 10-day tool‟ was not shown to be superior to the „PACTG-style tool‟ in detecting non-adherence, however this new tool was found to be a valid and reliable adherence monitoring tool that included a moderately long recall period of 10 days, can be applied without the need for the respondent to remember names of individual medicines in the
- Full Text:
The plasmodium falciparum exported Hsp40 co-chaperone, PFA0660w
- Authors: Daniyan, Michael Oluwatoyin
- Date: 2014
- Subjects: Molecular chaperones Heat shock proteins Proteins -- Analysis Proteins -- Structure Plasmodium Plasmodium falciparum Malaria -- Prevention -- Research
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4108 , http://hdl.handle.net/10962/d1011780
- Description: Plasmodium falciparum is the pathogen that is responsible for the most virulent, severe and dangerous form of human malaria infection, accounting for nearly a million deaths every year. To survive and develop in the unusual environment of the red blood cells, the parasite causes structural remodelling of the host cell and biochemical changes through the export of virulence factors. Among the exportome are the molecular chaperones of the heat shock protein family, of which Hsp40s and Hsp70s are prominent. PF A0660w, a type II P. falciparum Hsp40, has been shown to be exported in complex with PfHsp70-x into the infected erythrocyte, suggesting possible functional interactions. However, the chaperone properties of PF A0660w and its interactions with proteins of parasite and human origin are yet to be investigated. Using a codon optimised coding region, PF A0660w was successfully expressed in E. coli M 15 [pREP4] cells. However, the expressed protein was largely deposited as insoluble pellet, and analysis of the pellets revealed a high percentage of PF A0660w, characteristic of inclusion body formation. PF A0660w was purified from inclusion bodies using additive enhanced solubilisation and refolding buffers followed by nickel affinity chromatography. SDS-PAGE and western analysis revealed that the purified protein was of high purity. Size exclusion chromatography showed that the protein existed as a monomer in solution and the secondary structure analysis using Fourier transformed infrared spectroscopy (FTIR) confirmed the success of the refolding approach. Its monomeric state suggests that PF A0660w may be functionally different from other Hsp40 that form dimers and that for PF A0660w, dimer formation may not be needed to maintain the stability of the protein in solution, but may occur in response to functional necessities during its interaction with partner Hsp70. PFA0660w was able to significantly stimulate the ATPase activity ofPfl-Isp70-x but not Pfl-Isp70-1 or human Hsp70 (HsHsp70), suggesting a specific functional interaction. Also, PF A0660w produced a dose dependent suppression of rhodanese aggregation and cooperated with Pfl-Isp70-1, PfHsp70-x and HsHsp70 to cause enhanced aggregation suppression. Its ability to independently suppress aggregation may help to maintain substrates in an unfolded conformation for eventual transfer to partner Hsp70s during refolding processes. Also, the in vivo characterisation using a PF A0660w peptide specific antibody confirmed that PF A0660w was exported into the cytosol of infected erythrocytes. Its lack of induction upon heat shock suggests that PF A0660w may not be involved in the response of the parasite to heat stress. Overall, this study has provided the first heterologous over-expression, purification and biochemical evidence for the possible functional role of PF A0660w, and has thereby provided the needed background for further exploration of this protein as a potential target for drug discovery.
- Full Text:
- Authors: Daniyan, Michael Oluwatoyin
- Date: 2014
- Subjects: Molecular chaperones Heat shock proteins Proteins -- Analysis Proteins -- Structure Plasmodium Plasmodium falciparum Malaria -- Prevention -- Research
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4108 , http://hdl.handle.net/10962/d1011780
- Description: Plasmodium falciparum is the pathogen that is responsible for the most virulent, severe and dangerous form of human malaria infection, accounting for nearly a million deaths every year. To survive and develop in the unusual environment of the red blood cells, the parasite causes structural remodelling of the host cell and biochemical changes through the export of virulence factors. Among the exportome are the molecular chaperones of the heat shock protein family, of which Hsp40s and Hsp70s are prominent. PF A0660w, a type II P. falciparum Hsp40, has been shown to be exported in complex with PfHsp70-x into the infected erythrocyte, suggesting possible functional interactions. However, the chaperone properties of PF A0660w and its interactions with proteins of parasite and human origin are yet to be investigated. Using a codon optimised coding region, PF A0660w was successfully expressed in E. coli M 15 [pREP4] cells. However, the expressed protein was largely deposited as insoluble pellet, and analysis of the pellets revealed a high percentage of PF A0660w, characteristic of inclusion body formation. PF A0660w was purified from inclusion bodies using additive enhanced solubilisation and refolding buffers followed by nickel affinity chromatography. SDS-PAGE and western analysis revealed that the purified protein was of high purity. Size exclusion chromatography showed that the protein existed as a monomer in solution and the secondary structure analysis using Fourier transformed infrared spectroscopy (FTIR) confirmed the success of the refolding approach. Its monomeric state suggests that PF A0660w may be functionally different from other Hsp40 that form dimers and that for PF A0660w, dimer formation may not be needed to maintain the stability of the protein in solution, but may occur in response to functional necessities during its interaction with partner Hsp70. PFA0660w was able to significantly stimulate the ATPase activity ofPfl-Isp70-x but not Pfl-Isp70-1 or human Hsp70 (HsHsp70), suggesting a specific functional interaction. Also, PF A0660w produced a dose dependent suppression of rhodanese aggregation and cooperated with Pfl-Isp70-1, PfHsp70-x and HsHsp70 to cause enhanced aggregation suppression. Its ability to independently suppress aggregation may help to maintain substrates in an unfolded conformation for eventual transfer to partner Hsp70s during refolding processes. Also, the in vivo characterisation using a PF A0660w peptide specific antibody confirmed that PF A0660w was exported into the cytosol of infected erythrocytes. Its lack of induction upon heat shock suggests that PF A0660w may not be involved in the response of the parasite to heat stress. Overall, this study has provided the first heterologous over-expression, purification and biochemical evidence for the possible functional role of PF A0660w, and has thereby provided the needed background for further exploration of this protein as a potential target for drug discovery.
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Studies on existing and new isolates of Cryptophlebia leucotreta granulovirus (CrleGV) on Thaumatotibia leucotreta populations from a range of geographic regions in South Africa
- Authors: Opoku-Debrah, John Kwadwo
- Date: 2012
- Subjects: Cryptophlebia leucotreta Cryptophlebia leucotreta -- South Africa Cryptophlebia leucotreta -- Biological control Cryptophlebia leucotreta -- Life cycles Baculoviruses Lepidoptera -- Biological control Tortricidae -- Biological control Microbial insecticides Pests -- Integrated control
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:5778 , http://hdl.handle.net/10962/d1005466
- Description: Baculoviruses are arthropod-specific DNA viruses that are highly virulent to most lepidopteran insects. Their host specificity and compatibility with IPM programmes has enabled their usage as safe microbial insecticides (biopesticides). Two baculovirus-based biopesticides, Cryptogran and Cryptex, which have been formulated with Cryptophlebia leucotreta granulovirus (CrleGV) have been registered for the control of false codling moth (FCM), Thaumatotibia (=Cryptophlebia) leucotreta (Meyrick) (Lepidoptera: Tortricidae) in South Africa and have been successfully incorporated into IPM programmes. However, several studies have indicated that insects can develop resistance to baculovirus-based biopesticide as was shown with field populations of codling moth (CM), Cydia pomonella (L.), which developed resistance to the biopesticide Cydia pomonella granulovirus (CpGV-M) in Europe. Other studies have shown that, under laboratory conditions, FCM populations differ in their susceptibility to Cryptogran and Cryptex. In order to investigate difference in susceptibility as well as protect against any future resistance by FCM to Cryptogran and Cryptex, a search for novel CrleGV-SA isolates from diseased insects from different geographic regions in South Africa was performed. Six geographic populations (Addo, Citrusdal, Marble Hall, Nelspruit, Baths and Mixed colonies) of FCM were established and maintained in the laboratory. Studies on the comparative biological performance based on pupal mass, female fecundity, egg hatch, pupal survival, adult eclosion and duration of life cycle of the Addo, Citrusdal, Marble Hall, Nelspruit and Mixed colonies revealed a low biological performance for the Citrusdal colony. This was attributed to the fact that FCM populations found in the Citrusdal area are not indigenous and may have been introduced from a very limited gene pool from another region. When insects from five colonies, excluding the Baths colony, were subjected to stress by overcrowding , a latent baculovirus resident in the Addo, Nelspruit, Citrusdal, Marble Hall and Mixed colonies was brought into an overt lethal state. Transmission electron micrographs revealed the presence of GV occlusion bodies (OBs) in diseased insects. DNA profiles obtained by single restriction endonuclease analysis of viral genomic DNA using BamH 1, Sa/1, Xba1 , Pst1, Xh01 , Kpn1, Hindlll and EcoR1 revealed five CrleGV-SA isolates latent within the insect populations. The new isolates were named CrleGV-SA Ado, CrleGV-SA Cit, CrleGV-SA Mbl, CrleGVSA Nels and CrleGV-SA Mix isolates. The novelty of the five CrleGV-SA isolates was confirmed by the presence of unique submolar bands, indicating that each isolate was genetically different. PCR amplification and sequencing of the granulin and egt genes from the five isolates revealed several single nucleotide polymorph isms (SNPs) which, in some cases, resulted in amino acid substitutions. DNA profiles from RFLPs, as well as phylogenetic analysis based on granulin and egt sequencing showed the presence of two CrleGV-SA genome types for the CrleGV-SA isolate. Cryptex and CrleGV-SA Ado, CrleGV-SA Cit, CrleGV-SA Mbl and CrleGV-SA Mix were placed as members of Group one CrleGV-SA, and Cryptogran and CrleGV-SA Nels isolate were placed into Group two CrleGV-SA. In droplet feeding bioassays, the median survival time (STso) for neonate larvae inoculated with Group one and two CrleGV-SA were determined to range from 80 - 88 hours (3.33 - 3.67 days), for all five colonies. LDso values for Group one and two CrleGV-SA against neonates from the Addo, Citrusdal, Marble Hall, Nelspruit and Mixed colonies varied between some populations and ranged from 0.80 - 3.12 OBs per larva, indicating some level of variation in host susceptibility. This is the first study reporting the existence of genetically distinct CrleGV baculovirus isolates infecting FCM in different geographical areas of South Africa. The results of this study have broad-ranging implications for our understanding of baculovirus-host interactions and for the application of baculovirus basedbiopesticides.
- Full Text:
- Authors: Opoku-Debrah, John Kwadwo
- Date: 2012
- Subjects: Cryptophlebia leucotreta Cryptophlebia leucotreta -- South Africa Cryptophlebia leucotreta -- Biological control Cryptophlebia leucotreta -- Life cycles Baculoviruses Lepidoptera -- Biological control Tortricidae -- Biological control Microbial insecticides Pests -- Integrated control
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:5778 , http://hdl.handle.net/10962/d1005466
- Description: Baculoviruses are arthropod-specific DNA viruses that are highly virulent to most lepidopteran insects. Their host specificity and compatibility with IPM programmes has enabled their usage as safe microbial insecticides (biopesticides). Two baculovirus-based biopesticides, Cryptogran and Cryptex, which have been formulated with Cryptophlebia leucotreta granulovirus (CrleGV) have been registered for the control of false codling moth (FCM), Thaumatotibia (=Cryptophlebia) leucotreta (Meyrick) (Lepidoptera: Tortricidae) in South Africa and have been successfully incorporated into IPM programmes. However, several studies have indicated that insects can develop resistance to baculovirus-based biopesticide as was shown with field populations of codling moth (CM), Cydia pomonella (L.), which developed resistance to the biopesticide Cydia pomonella granulovirus (CpGV-M) in Europe. Other studies have shown that, under laboratory conditions, FCM populations differ in their susceptibility to Cryptogran and Cryptex. In order to investigate difference in susceptibility as well as protect against any future resistance by FCM to Cryptogran and Cryptex, a search for novel CrleGV-SA isolates from diseased insects from different geographic regions in South Africa was performed. Six geographic populations (Addo, Citrusdal, Marble Hall, Nelspruit, Baths and Mixed colonies) of FCM were established and maintained in the laboratory. Studies on the comparative biological performance based on pupal mass, female fecundity, egg hatch, pupal survival, adult eclosion and duration of life cycle of the Addo, Citrusdal, Marble Hall, Nelspruit and Mixed colonies revealed a low biological performance for the Citrusdal colony. This was attributed to the fact that FCM populations found in the Citrusdal area are not indigenous and may have been introduced from a very limited gene pool from another region. When insects from five colonies, excluding the Baths colony, were subjected to stress by overcrowding , a latent baculovirus resident in the Addo, Nelspruit, Citrusdal, Marble Hall and Mixed colonies was brought into an overt lethal state. Transmission electron micrographs revealed the presence of GV occlusion bodies (OBs) in diseased insects. DNA profiles obtained by single restriction endonuclease analysis of viral genomic DNA using BamH 1, Sa/1, Xba1 , Pst1, Xh01 , Kpn1, Hindlll and EcoR1 revealed five CrleGV-SA isolates latent within the insect populations. The new isolates were named CrleGV-SA Ado, CrleGV-SA Cit, CrleGV-SA Mbl, CrleGVSA Nels and CrleGV-SA Mix isolates. The novelty of the five CrleGV-SA isolates was confirmed by the presence of unique submolar bands, indicating that each isolate was genetically different. PCR amplification and sequencing of the granulin and egt genes from the five isolates revealed several single nucleotide polymorph isms (SNPs) which, in some cases, resulted in amino acid substitutions. DNA profiles from RFLPs, as well as phylogenetic analysis based on granulin and egt sequencing showed the presence of two CrleGV-SA genome types for the CrleGV-SA isolate. Cryptex and CrleGV-SA Ado, CrleGV-SA Cit, CrleGV-SA Mbl and CrleGV-SA Mix were placed as members of Group one CrleGV-SA, and Cryptogran and CrleGV-SA Nels isolate were placed into Group two CrleGV-SA. In droplet feeding bioassays, the median survival time (STso) for neonate larvae inoculated with Group one and two CrleGV-SA were determined to range from 80 - 88 hours (3.33 - 3.67 days), for all five colonies. LDso values for Group one and two CrleGV-SA against neonates from the Addo, Citrusdal, Marble Hall, Nelspruit and Mixed colonies varied between some populations and ranged from 0.80 - 3.12 OBs per larva, indicating some level of variation in host susceptibility. This is the first study reporting the existence of genetically distinct CrleGV baculovirus isolates infecting FCM in different geographical areas of South Africa. The results of this study have broad-ranging implications for our understanding of baculovirus-host interactions and for the application of baculovirus basedbiopesticides.
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Metal interactions with neural substrates and their role in neurodegeneration
- Authors: Lack, Barbara Anne
- Date: 2003
- Subjects: Nervous system -- Degeneration Neurotoxicology Chemical reactions Metals -- Physiological effect Melatonin -- Physiological effect
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4391 , http://hdl.handle.net/10962/d1005709
- Description: "Life" may be characterized as a controlled stationary flow equilibrium, maintained by energy consuming chemical reactions. The physiological functioning of these life systems include at least 28 of the elements isolated on the periodic table thus far, most of which are metals. However, as with Paracelsus Principle: "The dose makes the poison", there exists a definite link between metal levels, essential and toxic, and the onset of neurodegenerative diseases. The economic costs of brain dysfunction are enormous, but this pales in comparison to the staggering emotional toll on the victims themselves and their families. In an attempt to improve the understanding of the causes of neurodegeneration, this study focuses on one potential aspect: the possible link between metals and neurotransmitter homeostasis utilising a variety of electronanalytical techniques. Adsorptive cathodic stripping voltammetry was employed to investigate the binding affinities and complex formation of melatonin and its precursor serotonin with calcium, potassium, sodium, lithium and aluminium. The results showed that all the metals studied formed complexes with both pineal indoleamines. However, the stability and affmity of the ligands toward the various metals varied greatly. The study suggests a further role for melatonin, that of metalloregulator and possible metal detoxifier in the brain, the in vivo studies which followed will further substantiate this notion. This research additionally focused on the cholinergic system, in particular acetylcholine complex formation studies with mercury, lead, cadmium, copper and zinc using the adsorptive cathodic stripping voltammetry method. The formation and characterisation of a solid mercury-acetylcholine complex lent further strength to the in situ electrochemical complex formation observed. The results showed the preference of acetylcholine for environmentally toxic heavy metals (such as Cd²⁺) over those divalent cations that occur naturally in the body. The possible metalloregulatory role melatonin played in the three brain regIOns: cerebellum, cortex and corpus striatum of male Wistar rats was studied as an in vivo extension of the earlier in vitro studies. Anodic stripping voltammetry was employed to detect metal levels present. The results showed that daily injections of melatonin was responsible for significantly decreasing copper(I), cadmium(II) and lead(II) levels in various regions of the rat brain of those animals that had undergone a pinealectomy in comparison to the saline injected group having undergone the same treatment. Histological and electrochemical stripping techniques were applied to investigate the implications of high A1³⁺ levels in the brain regions, particularly the hippocampus. Melatonin showed signs of promise in indirect symptom alleviation and by significantly decreasing A1³⁺ levels in rats that had been dosed with melatonin prior to A1³⁺ treatments in comparison with the control groups. Finally a preliminary study outlining a method for the production of a calcium selective microelectrode was undertaken. Further work is still needed to optimise the microelectrode production as well as its possible applications. However, whilst the overall conclusions of this entire multidisciplinary study may indeed only be in effect one piece of a very large puzzle on neurodegenerative diseases, this piece will no doubt serve as a building block for further ideas and work in this field.
- Full Text:
- Authors: Lack, Barbara Anne
- Date: 2003
- Subjects: Nervous system -- Degeneration Neurotoxicology Chemical reactions Metals -- Physiological effect Melatonin -- Physiological effect
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4391 , http://hdl.handle.net/10962/d1005709
- Description: "Life" may be characterized as a controlled stationary flow equilibrium, maintained by energy consuming chemical reactions. The physiological functioning of these life systems include at least 28 of the elements isolated on the periodic table thus far, most of which are metals. However, as with Paracelsus Principle: "The dose makes the poison", there exists a definite link between metal levels, essential and toxic, and the onset of neurodegenerative diseases. The economic costs of brain dysfunction are enormous, but this pales in comparison to the staggering emotional toll on the victims themselves and their families. In an attempt to improve the understanding of the causes of neurodegeneration, this study focuses on one potential aspect: the possible link between metals and neurotransmitter homeostasis utilising a variety of electronanalytical techniques. Adsorptive cathodic stripping voltammetry was employed to investigate the binding affinities and complex formation of melatonin and its precursor serotonin with calcium, potassium, sodium, lithium and aluminium. The results showed that all the metals studied formed complexes with both pineal indoleamines. However, the stability and affmity of the ligands toward the various metals varied greatly. The study suggests a further role for melatonin, that of metalloregulator and possible metal detoxifier in the brain, the in vivo studies which followed will further substantiate this notion. This research additionally focused on the cholinergic system, in particular acetylcholine complex formation studies with mercury, lead, cadmium, copper and zinc using the adsorptive cathodic stripping voltammetry method. The formation and characterisation of a solid mercury-acetylcholine complex lent further strength to the in situ electrochemical complex formation observed. The results showed the preference of acetylcholine for environmentally toxic heavy metals (such as Cd²⁺) over those divalent cations that occur naturally in the body. The possible metalloregulatory role melatonin played in the three brain regIOns: cerebellum, cortex and corpus striatum of male Wistar rats was studied as an in vivo extension of the earlier in vitro studies. Anodic stripping voltammetry was employed to detect metal levels present. The results showed that daily injections of melatonin was responsible for significantly decreasing copper(I), cadmium(II) and lead(II) levels in various regions of the rat brain of those animals that had undergone a pinealectomy in comparison to the saline injected group having undergone the same treatment. Histological and electrochemical stripping techniques were applied to investigate the implications of high A1³⁺ levels in the brain regions, particularly the hippocampus. Melatonin showed signs of promise in indirect symptom alleviation and by significantly decreasing A1³⁺ levels in rats that had been dosed with melatonin prior to A1³⁺ treatments in comparison with the control groups. Finally a preliminary study outlining a method for the production of a calcium selective microelectrode was undertaken. Further work is still needed to optimise the microelectrode production as well as its possible applications. However, whilst the overall conclusions of this entire multidisciplinary study may indeed only be in effect one piece of a very large puzzle on neurodegenerative diseases, this piece will no doubt serve as a building block for further ideas and work in this field.
- Full Text:
Identification of Cowdria ruminantium proteins that induce specific cellular immune responses
- Authors: Van Kleef, Mirinda
- Date: 2002
- Subjects: Ruminants--Pathogens Heartwater
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4061 , http://hdl.handle.net/10962/d1004373
- Description: Cowdria ruminantium (Cowdria) is an obligate intracellular pathogen that causes heartwater in ruminants. Cellular immunity and the type I cytokine IFN-γ have been implicated in protective immunity to heartwater. The aim of this thesis was to identify proteins of the Welgevonden isolate of Cowdria that induce lymphocyte proliferation and IFN-γ production. Differential centrifugation was found to be the simplest and most efficient method of Cowdria purification. Cowdria organisms were fractionated into their constituent proteins of between 11 and 168 kDa by continuous flow electrophoresis. The resulting fractions were tested for their ability to stimulate lymphocyte proliferation in vitro. In an attempt to simulate the natural infective process, peripheral blood mononuclear cells (PBMC) were obtained from two cattle rendered immune by infection and treatment and assayed in proliferation assays with the proteins fractions. In a parallel study, four cattle were immunised with inactivated Cowdria to determine whether their lymphocytes responded similarly. Cowdria-specific proliferation was detected for only a brief period after immunisation by infection with live organisms. This response was only detected again two to three years later. In contrast, PBMC from animals immunised with inactivated organisms were continuously responsive for at least three years. Only Cowdria proteins with molecular masses of 11, 12, 14 to 17 and 19 to 23 kDa induced proliferative responses in PBMC obtained from all six animals. Cell surface phenotypic analysis of Cowdria specific T-cell lines indicated that CD4⁺ lymphocytes were enriched over time with a concomitant increase in antigen-specific proliferation and IFN-γ production. Proteins of molecular masses 13 to 18 kDa induced CD4⁺ lymphocyte proliferation and IFN-γ production by T-cell lines from all the animals tested. Antibodies raised in a chicken and in rabbits to these low molecular weight proteins had low titres and specificity. Two-dimensional electrophoresis indicated that proteins within a single molecular weight range comprised several components with different pIs, revealing the complexity of the Cowdria proteome. This complicates the search for potentially protective antigens. Nevertheless, since they cause proliferation and IFN-γ production by lymphocytes from immunised cattle, these low molecular weight proteins merit further investigation as potential vaccine antigens. , Author: Mirinda van Kleef neé Rossouw
- Full Text:
- Authors: Van Kleef, Mirinda
- Date: 2002
- Subjects: Ruminants--Pathogens Heartwater
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4061 , http://hdl.handle.net/10962/d1004373
- Description: Cowdria ruminantium (Cowdria) is an obligate intracellular pathogen that causes heartwater in ruminants. Cellular immunity and the type I cytokine IFN-γ have been implicated in protective immunity to heartwater. The aim of this thesis was to identify proteins of the Welgevonden isolate of Cowdria that induce lymphocyte proliferation and IFN-γ production. Differential centrifugation was found to be the simplest and most efficient method of Cowdria purification. Cowdria organisms were fractionated into their constituent proteins of between 11 and 168 kDa by continuous flow electrophoresis. The resulting fractions were tested for their ability to stimulate lymphocyte proliferation in vitro. In an attempt to simulate the natural infective process, peripheral blood mononuclear cells (PBMC) were obtained from two cattle rendered immune by infection and treatment and assayed in proliferation assays with the proteins fractions. In a parallel study, four cattle were immunised with inactivated Cowdria to determine whether their lymphocytes responded similarly. Cowdria-specific proliferation was detected for only a brief period after immunisation by infection with live organisms. This response was only detected again two to three years later. In contrast, PBMC from animals immunised with inactivated organisms were continuously responsive for at least three years. Only Cowdria proteins with molecular masses of 11, 12, 14 to 17 and 19 to 23 kDa induced proliferative responses in PBMC obtained from all six animals. Cell surface phenotypic analysis of Cowdria specific T-cell lines indicated that CD4⁺ lymphocytes were enriched over time with a concomitant increase in antigen-specific proliferation and IFN-γ production. Proteins of molecular masses 13 to 18 kDa induced CD4⁺ lymphocyte proliferation and IFN-γ production by T-cell lines from all the animals tested. Antibodies raised in a chicken and in rabbits to these low molecular weight proteins had low titres and specificity. Two-dimensional electrophoresis indicated that proteins within a single molecular weight range comprised several components with different pIs, revealing the complexity of the Cowdria proteome. This complicates the search for potentially protective antigens. Nevertheless, since they cause proliferation and IFN-γ production by lymphocytes from immunised cattle, these low molecular weight proteins merit further investigation as potential vaccine antigens. , Author: Mirinda van Kleef neé Rossouw
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A framework for interpreting noisy, two-dimensional images, based on a fuzzification of programmed, attributed graph grammars
- Authors: Watkins, Gregory Shroll
- Date: 1998
- Subjects: Music -- Data processing Computer sound processing Artificial intelligence -- Musical applications Fuzzy systems
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4604 , http://hdl.handle.net/10962/d1004862
- Description: This thesis investigates a fuzzy syntactic approach to the interpretation of noisy two-dimensional images. This approach is based on a modification of the attributed graph grammar formalism to utilise fuzzy membership functions in the applicability predicates. As far as we are aware, this represents the first such modification of graph grammars. Furthermore, we develop a method for programming the resultant fuzzy attributed graph grammars through the use of non-deterministic control diagrams. To do this, we modify the standard programming mechanism to allow it to cope with the fuzzy certainty values associated with productions in our grammar. Our objective was to develop a flexible framework which can be used for the recognition of a wide variety of image classes, and which is adept at dealing with noise in these images. Programmed graph grammars are specifically chosen for the ease with which they allow one to specify a new two-dimensional image class. We implement a prototype system for Optical Music Recognition using our framework. This system allows us to test the capabilities of the framework for coping with noise in the context of handwritten music score recognition. Preliminary results from the prototype system show that the framework copes well with noisy images.
- Full Text:
- Authors: Watkins, Gregory Shroll
- Date: 1998
- Subjects: Music -- Data processing Computer sound processing Artificial intelligence -- Musical applications Fuzzy systems
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4604 , http://hdl.handle.net/10962/d1004862
- Description: This thesis investigates a fuzzy syntactic approach to the interpretation of noisy two-dimensional images. This approach is based on a modification of the attributed graph grammar formalism to utilise fuzzy membership functions in the applicability predicates. As far as we are aware, this represents the first such modification of graph grammars. Furthermore, we develop a method for programming the resultant fuzzy attributed graph grammars through the use of non-deterministic control diagrams. To do this, we modify the standard programming mechanism to allow it to cope with the fuzzy certainty values associated with productions in our grammar. Our objective was to develop a flexible framework which can be used for the recognition of a wide variety of image classes, and which is adept at dealing with noise in these images. Programmed graph grammars are specifically chosen for the ease with which they allow one to specify a new two-dimensional image class. We implement a prototype system for Optical Music Recognition using our framework. This system allows us to test the capabilities of the framework for coping with noise in the context of handwritten music score recognition. Preliminary results from the prototype system show that the framework copes well with noisy images.
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Aspects of the biology of the infaunal bivalve Mollusc Solen cylindraceus (Hanley) in the Kariega estuary
- De Villiers, Casper Johannes
- Authors: De Villiers, Casper Johannes
- Date: 1990
- Subjects: Mollusks -- South Africa -- Eastern Cape Bivalves -- South Africa -- Eastern Cape
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:5639 , http://hdl.handle.net/10962/d1005321
- Description: Solen cylindraceus is an infaunal filter-feeding bivalve inhabiting the intertidal mud banks of many southern African estuaries. It is particularly abundant in the Kariega estuary (33°41'S; 26°42'E) where it reaches densities of 400m⁻² (192g shell-free dry wt. m⁻²). The Kariega is a permanently open, marine dominated estuary about 18km in length, and S. cylindraceus is most abundant in its middle and upper reaches. Some physical characteristics of the estuary (temperature, salinity, sediment and water turbidity) are described, and the possible role of these factors in determining the density and distribution of S. cylindraceus within the Kariega estuary, is discussed. The structure of the alimentary system, gills and labial palps of S. cylintfraceus is described, all of which showed no major variation from the "typical" eulamellibranchiate form. Solen cylintfraceus was found to be a euryhaline osmoconformer with a salinity tolerance range of 15-65%. When animals were removed from their burrows, osmotic equilibration of the haemolymph was rapid (1-2 hours). By contrast, in animals left undisturbed in their burrows, osmotic equilibration was retarded (72-204 hours). It is suggested that the observed decrease in the rate of change of haemolymph osmolarity for animals in their burrows is linked to the stability of the interstitial salinity. A temperature tolerance range of 5-44°C was determined for S. cylintfraceus (in situ), in which prolonged exposure to 5°C and 40-45°C (12-36 hours respectively) resulted in a decreased burrowing ability, coma and death. Animal burrowing responses were not affected by temperatures in the range 15-35°C. Field experiments were carried out over several tidal cycles, in which the measurement of crystalline style volume was used as a means of assessing extracellular digestive activity. No major variation in style volume was recorded and it appeared that S. cylindraceus did not exhibit any cyclical pattern of style dissolution and regeneration. It is suggested that S. cylindraceus feeds continuously from the water column during high tide and possibly within its burrow, at or below the water table, during low tide. At a suspensoid concentration of 5Omg l⁻¹, S. cylindraceus was found to filter water almost continuously (90-95% of the time). Time spent filtering dropped to 68% at 100mg l⁻¹ and 32% at 500mg l⁻¹. Filtration rates for summer collected animals (25°C) were 22.86 ± 4.36ml min.⁻¹, some 3ml min.⁻¹ greater than that recorded for winter (16°C) collected animals. Filtration rate may be expressed as a function of shell length by the equations: y=0.247x¹̇⁰⁶⁶ (winter) and y=0.758x⁰̇⁸²⁶ (summer). Solen cylindraceus was capable of acclimating its filtration rate to both high and low temperatures under laboratory conditions. Filtration rate exhibited a thermal optimum in the range 15-35°C, declining at higher and lower temperatures. Q₁₀ values of filtration decreased rapidly from greater than 4 to less than 2, when the thermal optimum was reached. Maximum rates generally occurred at approximately 5°C above the temperature to which the animal had been acclimated. Optimal filtration rates (19-23ml min.⁻¹) were recorded in the salinity range 15-45%. When subjected to abrupt changes in salinity, filtration rates were immediately depressed. The extent and duration of these decreased filtration rates were dependent upon the magnitude and direction of salinity change, and were always less in animals exposed to hyper- than hyposaline conditions. Animals exposed to increased temperature and simultaneous elevated or unchanged salinity, showed a slight increase in filtration rate followed by rapid acclimation. A decrease in both temperature and salinity resulted in an initial decrease in filtration rate and a longer acclimation period. The ability of S. cylindraceus to acclimate fully within a wide temperature and salinity range, and to filter maximally in hypersaline conditions may, in part, explain its unusually high abundance in the Kariega estuary, despite it being close to the southernmost limit of the animal's geographical distribution. No significant difference in flItration rate was recorded at suspensoid concentrations of 5-100mg 1⁻¹. However, at 250 and 500mg l⁻¹ filtration rates decreased significantly, and coincided with increased levels of pseudofaecal production. Solen cylindraceus retained particles down to 2.5-3.0µm with great efficiency (ca. 60-90% efficiency). Below this particle size, retention efficiency decreased rapidly and a net production of particles was recorded below 1.51µm. Particle retention was independent of temperature (15 and 25°C) and salinity (15 and 35%). Use was made of stable carbon isotope analyses (¹³C/¹²C ratios) in an attempt to determine the important food sources of S. cylindraceus within the Kariega estuary. The results obtained demonstrated an enrichment in δ¹³ values for S. cylindraceus from the upper (-27.9%) to the middle (-25%c) and lower (-21.6%o ) reaches of the estuary, with no seasonal variation apparent. The bivalve was substantially more depleted in ¹³C relative to the dominant aquatic macrophytes Zostera capensis (-9.1 to -15.6%o) and Spartina maritima (-12.5%o). The use of δ¹³ alone, however, to unequivocally "pin point" specific food sources of a filter feeder in a predominantly detritus based food web, is limited. It is suggested that in the Kariega estuary, riparian litter and other terrestrially derived vegetation contribute to the carbon pool. A possible contribution of ¹³C depleted food sources via chemoautotrophic and/or anaerobic pathways, to the diet of S. cylindraceus, is suggested.
- Full Text:
- Authors: De Villiers, Casper Johannes
- Date: 1990
- Subjects: Mollusks -- South Africa -- Eastern Cape Bivalves -- South Africa -- Eastern Cape
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:5639 , http://hdl.handle.net/10962/d1005321
- Description: Solen cylindraceus is an infaunal filter-feeding bivalve inhabiting the intertidal mud banks of many southern African estuaries. It is particularly abundant in the Kariega estuary (33°41'S; 26°42'E) where it reaches densities of 400m⁻² (192g shell-free dry wt. m⁻²). The Kariega is a permanently open, marine dominated estuary about 18km in length, and S. cylindraceus is most abundant in its middle and upper reaches. Some physical characteristics of the estuary (temperature, salinity, sediment and water turbidity) are described, and the possible role of these factors in determining the density and distribution of S. cylindraceus within the Kariega estuary, is discussed. The structure of the alimentary system, gills and labial palps of S. cylintfraceus is described, all of which showed no major variation from the "typical" eulamellibranchiate form. Solen cylintfraceus was found to be a euryhaline osmoconformer with a salinity tolerance range of 15-65%. When animals were removed from their burrows, osmotic equilibration of the haemolymph was rapid (1-2 hours). By contrast, in animals left undisturbed in their burrows, osmotic equilibration was retarded (72-204 hours). It is suggested that the observed decrease in the rate of change of haemolymph osmolarity for animals in their burrows is linked to the stability of the interstitial salinity. A temperature tolerance range of 5-44°C was determined for S. cylintfraceus (in situ), in which prolonged exposure to 5°C and 40-45°C (12-36 hours respectively) resulted in a decreased burrowing ability, coma and death. Animal burrowing responses were not affected by temperatures in the range 15-35°C. Field experiments were carried out over several tidal cycles, in which the measurement of crystalline style volume was used as a means of assessing extracellular digestive activity. No major variation in style volume was recorded and it appeared that S. cylindraceus did not exhibit any cyclical pattern of style dissolution and regeneration. It is suggested that S. cylindraceus feeds continuously from the water column during high tide and possibly within its burrow, at or below the water table, during low tide. At a suspensoid concentration of 5Omg l⁻¹, S. cylindraceus was found to filter water almost continuously (90-95% of the time). Time spent filtering dropped to 68% at 100mg l⁻¹ and 32% at 500mg l⁻¹. Filtration rates for summer collected animals (25°C) were 22.86 ± 4.36ml min.⁻¹, some 3ml min.⁻¹ greater than that recorded for winter (16°C) collected animals. Filtration rate may be expressed as a function of shell length by the equations: y=0.247x¹̇⁰⁶⁶ (winter) and y=0.758x⁰̇⁸²⁶ (summer). Solen cylindraceus was capable of acclimating its filtration rate to both high and low temperatures under laboratory conditions. Filtration rate exhibited a thermal optimum in the range 15-35°C, declining at higher and lower temperatures. Q₁₀ values of filtration decreased rapidly from greater than 4 to less than 2, when the thermal optimum was reached. Maximum rates generally occurred at approximately 5°C above the temperature to which the animal had been acclimated. Optimal filtration rates (19-23ml min.⁻¹) were recorded in the salinity range 15-45%. When subjected to abrupt changes in salinity, filtration rates were immediately depressed. The extent and duration of these decreased filtration rates were dependent upon the magnitude and direction of salinity change, and were always less in animals exposed to hyper- than hyposaline conditions. Animals exposed to increased temperature and simultaneous elevated or unchanged salinity, showed a slight increase in filtration rate followed by rapid acclimation. A decrease in both temperature and salinity resulted in an initial decrease in filtration rate and a longer acclimation period. The ability of S. cylindraceus to acclimate fully within a wide temperature and salinity range, and to filter maximally in hypersaline conditions may, in part, explain its unusually high abundance in the Kariega estuary, despite it being close to the southernmost limit of the animal's geographical distribution. No significant difference in flItration rate was recorded at suspensoid concentrations of 5-100mg 1⁻¹. However, at 250 and 500mg l⁻¹ filtration rates decreased significantly, and coincided with increased levels of pseudofaecal production. Solen cylindraceus retained particles down to 2.5-3.0µm with great efficiency (ca. 60-90% efficiency). Below this particle size, retention efficiency decreased rapidly and a net production of particles was recorded below 1.51µm. Particle retention was independent of temperature (15 and 25°C) and salinity (15 and 35%). Use was made of stable carbon isotope analyses (¹³C/¹²C ratios) in an attempt to determine the important food sources of S. cylindraceus within the Kariega estuary. The results obtained demonstrated an enrichment in δ¹³ values for S. cylindraceus from the upper (-27.9%) to the middle (-25%c) and lower (-21.6%o ) reaches of the estuary, with no seasonal variation apparent. The bivalve was substantially more depleted in ¹³C relative to the dominant aquatic macrophytes Zostera capensis (-9.1 to -15.6%o) and Spartina maritima (-12.5%o). The use of δ¹³ alone, however, to unequivocally "pin point" specific food sources of a filter feeder in a predominantly detritus based food web, is limited. It is suggested that in the Kariega estuary, riparian litter and other terrestrially derived vegetation contribute to the carbon pool. A possible contribution of ¹³C depleted food sources via chemoautotrophic and/or anaerobic pathways, to the diet of S. cylindraceus, is suggested.
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