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The modulation of various signal transduction pathways in colorectal carcinoma cells by docosahexaenoic acidDu Toit, Joe-Lin 12 1900 (has links)
Thesis (MSc)--University of Stellenbosch, 2006. / ENGLISH ABSTRACT: Introduction: The ability of different polyunsaturated fatty acids (PUFAs),
especially n-3 PUFAs, to prevent the development of cancer has been under
intense investigation the past three decades. Numerous studies have shown
that these fatty acids can kill cancer cells in vitro as well as in vivo whilst normal
cells remain unaffected. Unfortunately, the cellular and molecular mechanisms
responsible for this phenomenon are still poorly understood. This study
investigated the signalling pathways modulated by docosahexaenoic acid
(DHA) in an adenocarcinoma cell line, in order to shed some light on these
unknown mechanisms.
Materials & Methods: NCM460 (normal colon epithelial) and CaCo2 (colon
adenocarcinoma) cells were cultured and treated with low doses of palmitic acid
(PMA), oleic acid (OA), arachidonic acid (AA), and DHA. The effects of these
fatty acids on the proliferation of the cells were measured with the MTT assay.
The composition of membrane phospholipids of CaCo2 cells was determined
after 48h supplementation with different fatty acids by gas chromatography.
Also, CaCo2 cells were treated with DHA (10 μM) only and proteins were
harvested at fixed time points ranging from 2 minutes to 48 hours. The protein
inhibitors wortmannin (PI3 kinase inhibitor), PD 98059 (MEK inhibitor) and SB
203580 (p38 inhibitor) and also RNA interference (RNAi) of the p38 MAPK
protein were used to investigate cross-talk between signalling pathways. ERK,
p38 MAP kinase, Akt, and p53 were then analysed by Western blotting using
phospho-specific and total antibodies. The cleavage of the apoptotic proteins,
caspase-3 and PARP were also analysed.
Results and discussion: MTT assays revealed that none of the fatty acids were
toxic to normal cells. In addition, DHA was shown to be most effective to kill
CaCo2 cells whilst protecting NCM460 cells and a subsequent dose response experiment revealed that lower concentrations are most suitable for this
purpose. DHA was also shown to be readily incorporated into phospholipids,
along with AA. This is associated with increased membrane fluidity, which
could affect the localisation, and downstream effects, of various signalling
proteins within the membrane. Western blot analysis revealed a rapid increase
in activity in most proteins under investigation, especially ERK and Akt
(Ser473). Long-term DHA supplementation suppressed the full activation of
Akt. This down regulation of survival signalling could lead to cell death in
CaCo2 cells. In addition, it was shown that after 48h, DHA induced the
cleavage of caspase-3 and PARP, which is indicative of apoptosis. RNAi
experiments suggested a possible role for p38 MAPK in the phosphorylation of
p53 at Ser15, a site which is associated with DNA damage.
Conclusion: DHA exerts its effects by means of cellular signal transduction
pathways, particularly by suppression of the important survival-related kinase,
Akt. This could have implications for future therapeutic interventions in cancer
patients, as fatty acids are safe to use and do not interfere with the functionality
of normal tissue. / AFRIKAANSE OPSOMMING: Inleiding: Die vermoë van verskillende poli-onversadigde vetsure (POVSe),
veral n-3 POVSe, om die ontstaan van kanker te voorkom, is intens nagevors
die afgelope drie dekades. Menigte studies het aangevoer dat hierdie vetsure
kankerselle in vitro asook in vivo kan doodmaak, terwyl normale selle nie
daardeur beïnvloed word nie. Ongelukkig word die sellulêre and molekulêre
meganismes onderliggend tot hierdie verskynsel nie goed begryp nie. Hierdie
studie het verskeie seintransduksie-paaie wat deur dokosaheksaenoësuur
(DHS) in ‘n adenokarsinoom sellyn gemoduleer word, ondersoek.
Materiale & Metodes: NCM460 (normale kolonepiteel) en CaCo2 (kolon
adenokarsinoom) selle is onderhou in ‘n selkultuur-laboratorium en behandel
met lae dosisse palmitiensuur (PMS), oleïensuur (OS), aragidoonsuur (AS), en
DHS. Die invloed van hierdie vetsure op die proliferasie van die selle is d.m.v.
die MTT toets bepaal. The samestelling van membraan-fosfolipiede van CaCo2
selle is na 48h behandeling met die verskillende vetsure bepaal deur middel
van gaschromatografie. Die CaCo2 selle is ook met DHA (10 μM) alleenlik
behandel en teen vaste tydpunte wat wissel van 2 minute tot 48h, waarna
proteïene geëkstraeer is. Die proteïen-inhibitore wortmannin (PI3 kinase
inhibitor), PD 98059 (MEK inhibitor), en SB 203580 (p38 inhibitor) asook RNAinterferensie
(RNAi) teen die p38 MAPK proteïen is ingespan om oorvleueling
tussen seintransduksie–weë te ondersoek. ERK, p38 MAPK, Akt, en p53 is
geanaliseer deur middel van die Western–klad metode met fosfo–spesifieke en
totale antiliggame. Die kliewing van die apoptotiese proteïene caspase-3 en
PARP is ook bepaal.
Resultate en bespreking: MTT toetse het ontul dat geen vetsure toksies was vir
die normale selle nie. Daar is ook gevind dat DHS die mees effektiewe vetsuur
was om CaCo2 selle te dood, terwyl NCM460 selle beskerm word. Gevolglik het ‘n dosis-respons eksperiment getoon dat laer konsentrasies die beste
geskik is vir hierdie doel. Daar is ook gevind dat DHA maklik in fosfolipiede
geïnkorporeer word, tesame met AS. Dit word geassosieer met verhoogde
membraan-vloeibaarheid, wat die ligging, en ook stroom-af werking, van
verskeie seintransduksie proteïene in die membraan, kan beïnvloed. Westernklad
analises het ‘n vinnige verhoging in die aktiwiteite van die meeste
proteïene onder die soeklig, getoon, veral ERK en Akt (Ser473). Langdurige
DHS behandeling het die maksimale aktiwiteit van Akt onderdruk. Hierdie
afname van oorlewing-gerigte seine kan lei tot seldood in CaCo2 selle. Daar is
boonop geving dat DHS die kliewing van caspase-3 en PARP geïnduseer het
na 48, wat dui op apoptose. Uit die RNAi eksperiment kon daar ook ‘n
moontlike rol vir p38 MAPK in die fosforilering van p53 by Ser15, wat
geassosieer word met DNS-skade, getoon word.
Gevolgtrekking: DHS beoefen sy effekte deur middel van seintransduksie
paaie, veral deur die oorlewing-geassosieerde kinase, Akt, te onderdruk. Dit
kan implikasies hê vir toekomende terapeutiese ingrypings in kankerpasiënte,
aangesien vetsure veilig is om te gebruik en nie skadelik is vir normale weefsel
nie.
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ART-related body composition changes in adult women in a semi-rural South African contextDe Bruto, Petro C. 12 1900 (has links)
Assignment (MPhil)--University of Stellenbosch, 2006. / ENGLISH ABSTRACT: The aim of this study was to investigate practical methods of monitoring AIDS related
wasting and lipodystrophy in a resource-poor clinical setting with HIV infected women as
the population group of interest. Measurement of body composition changes using
anthropometry is both cost- and time-efficient. Various different skinfolds were taken and
two different equations (the equations of Pollock et al. (1975) and Durnin and Womersley
(1974) for calculating body fat were used to determine the most promising method or
methods of monitoring body composition changes in a clinical setting.
Detailed anthropometric measurements were performed, as well as selected measurements
for haematological parameters and quality of life (QoL) for a group of 8 participants on
antiretroviral medication (ART group) and 6 participants who were not on treatment (TN
group). New variables namely, intra-abdominal indicator (IAI) and a percent of ideal body
mass to percent of ideal arm circumference ratio (%IBW:%IAC) were investigated as
possible indicators of lipodystrophy. Although measurements were taken at various timepoints,
three specific time-points were chosen for data-analysis for the ART group and two
time points for the TN group. These three time-points were, baseline (on the day of
recruitment for TN participants and within one month before the initiation of treatment for
ART participants), short-term (2 to 12 weeks after treatment initiation or the baseline
measurement or for the ART and the TN participants) and long-term (within one and a half
year of treatment initiation for the ART group).
ART and TN participants did not differ for many variables at baseline. The major
differences between ART and TN were in measured and derived variables of the arm,
especially percent of ideal arm circumference (%IAC) and upper arm fat area (UAFA),
which were significantly lower in the ART group.
CD4+ and QoL improved significantly for the ART participants from baseline to long-term.
This was not associated with changes in muscle mass, but rather some fat mass variables.
Participants on antiretroviral medication exhibited changes relating to abdominal obesity.
It was concluded that antiretroviral therapy contributed greatly to the QoL of the participants and it probably aided in the recovery from wasting for at least one participant
in this study. Measures of the arm can be used in a rural clinical setting to effectively
monitor patients with regard to AIDS related wasting. The new variables IAI and
%IBW:%IAC could be helpful in the monitoring of lipodystrophy and should be
investigated in future research. / AFRIKAANSE OPSOMMING: Die doelwit van hierdie studie is om praktiese metodes te ondersoek om VIGS-verwante
uittering en lipodistrofie te meet in ‘n plattelandse kliniese omgewing (waar hulpbronne
dikwels beperk is) met MIV ge-infekteerde vroue as populasiegroep. Die gebruik van
antropometrie om veranderinge in liggaamssamestelling te meet is beide koste- en
tydeffektief. Verskeie velvoumetings is geneem en twee verskillende vergelykings (die
vergelykings van Pollock et al. (1975) en Durnin en Womersley (1974)) is gebruik om
liggaamsvetinhoud te bereken, met die doel om ‘n belowende metode te vind om
veranderinge in liggaamssamestelling te meet in ‘n kliniese omgewing.
Verskeie antropometriese metings is geneem, sowel as uitgesoekte hematologiese en
lewenskwaliteitmetings (QoL) vir ‘n groep van agt deelnemers wat antiretrovirale
medikasie ontvang het (ART groep) en ses deelnemers wat nie hierdie behandeling ontvang
het nie (TN groep). Nuwe veranderlikes (binnebuikindikator (IAI) en die verhouding van
persentasie van ideale liggaamsmassa tot persentasie van ideale armomtrek
(%IBW:%IAC)) is ondersoek as moontlike aanwysers van lipodistrofie. Drie spesifieke
tydpunte vir die ART groep en twee tydpunte vir die TN groep is gekies uit die verskeie
tydpunte waarby metings geneem is, nl. basislyn (gedefinieer as die dag wat TN deelnemers
in die studie opgeneem is en 0 tot 4 weke voor die begin van behandeling vir die ART
deelnemers), korttermyn (2 tot 12 weke nadat behandeling begin is of na die basislyn
meting) en lang-termyn (binne een en ‘n half jaar nadat behandeling begin is vir die ART
groep).
By die basislyn tydpunt het min van die ART en TN deelnemers se gemete veranderlikes
verskil. Die ART en TN groepe het hoofsaaklik verskil ten opsigte van veranderlikes wat
betrekking het op die arm, veral persentasie van ideale armomtrek (%IAC) en bo-arm vetarea
(UAFA). Hierdie twee veranderlikes was beduidend laer in die ART groep as in die
TN groep.
CD4+ seltelling en lewenskwaliteit tellings het beduidend verbeter vir die ART deelnemers
van die basislyn tot die lang-termyn tydpunt. Hierdie veranderinge is nie samehangend met veranderinge in spiermassa nie, maar eerder met sommige vetmassa veranderlikes.
Deelnemers wat antiretrovirale medikasie ontvang het, het veranderinge getoon wat gedui
het op ‘n verhoogde neerlegging van vet in die buikarea. Ten slotte is bevind dat
antiretrovirale medikasie bygedra het tot die verbeterde lewenskwaliteit van die deelnemers
en dat dit waarskynlik ook die omkeer van uittering van ten minste een deelnemer
aangehelp het. Daar is ook bevind dat armverwante metinge gebruik kan word in die
plattelandse kliniese omgewing om pasiënte suksesvol te monitor ten opsigte van VIGSverwante
uittering. Die nuwe veranderlikes, IAI en %IBW:%IAC kan moontlik gebruik
word om lipodistrofie-verwante veranderings te meet en die gebruik van hierdie
veranderlikes behoort ondersoek te word in verdere navorsing.
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The effect of the TGF-β isoforms on progenitor cell recruitment and differentiation into cardiac and skeletal muscleSchabort, Elske Jeanne 12 1900 (has links)
Thesis (PhD (Physiology (Human and animal))-- University of Stellenbosch, 2007. / Definition: Stem cells are unspecialised cells with the capacity for long-term self-renewal and
the ability to differentiate into multiple cell-lineages.
The potential for the application of stem cells in clinical settings has had a profound effect on
the future of regenerative medicine. However, to be of greater therapeutic use, selection of
the most appropriate cell type, as well as optimisation of stem cell incorporation into the
damaged tissue is required. In adult skeletal muscle, satellite cells are the primary stem cell
population which mediate postnatal muscle growth. Following injury or in diseased
conditions, these cells are activated and recruited for new muscle formation. In contrast, the
potential of resident adult stem cell incorporation into the myocardium has been challenged
and the response of cardiac tissue, especially to ischaemic injury, is scar formation.
Following muscle damage, various growth factors and cytokines are released in the afflicted
area which influences the recruitment and incorporation of stem cells into the injured tissue.
Transforming Growth Factor-β (TGF-β) is a member of the TGF-β-superfamily of cytokines and
has at least three isoforms, TGF-β1, -β2, and -β3, which play essential roles in the regulation
of cell growth and regeneration following activation and stimulation of receptor-signalling
pathways. By improving the understanding of how TGF-β affects these processes, it is
possible to gain insight into how the intercellular environment can be manipulated to improve
stem cell-mediated repair following muscle injury. Therefore, the main aims of this thesis
were to determine the effect of the three TGF-β isoforms on proliferation, differentiation,
migration and fusion of muscle progenitor cells (skeletal and cardiac) and relate this to
possible improved mechanisms for muscle repair.
The effect of short- and long-term treatment with all three TGF-β isoforms were investigated
on muscle progenitor cell proliferation and differentiation using the C2C12 skeletal muscle
satellite and P19 multipotent embryonal carcinoma cell-lineages as in vitro model systems.
Cells were treated with 5 ng/mℓ TGF-β isoforms unless where stated otherwise. In C2C12
cells, proliferating cell nuclear antigen (PCNA) expression and localisation were analysed, and
together with total nuclear counts, used to assess the effect of TGF-β on myoblast
proliferation (Chapter 5). The myogenic regulatory factors MyoD and myogenin, and structural
protein myosin heavy chain (MHC) were used as protein markers to assess early and terminal
differentiation, respectively. To establish possible mechanisms by which TGF-β isoforms
regulate differentiation, further analysis included determination of MyoD localisation and the
rate of MyoD degradation in C2C12 cells. To assess the effect of TGF-β isoforms on P19 cell differentiation, protein expression levels of
connexin-43 and MHC were analysed, together with the determination of embryoid body
numbers in differentiating P19 cells (Chapter 6). Furthermore, assays were developed to
analyse the effect of TGF-β isoforms on both C2C12 and P19 cell migration (Chapter 7), as
well as fusion of C2C12 cells (Chapter 8).
Whereas all three isoforms of TGF-β significantly increased proliferation of C2C12 cells,
differentiation results, however, indicated that especially following long-term incubation,
TGF-β isoforms delayed both early and terminal differentiation of C2C12 cells into myotubes.
Similarly, myocyte migration and fusion were also negatively regulated following TGF-β
treatment. In the P19 cell-lineage, results demonstrated that isoform-specific treatment with
TGF-β1 could potentially enhance differentiation. Further research is however required in this
area, especially since migration was greatly reduced in these cells.
Taken together, results demonstrated variable effects following TGF-β treatment depending
on the cell type and the duration of TGF-β application. Circulating and/or treatment
concentrations of this growth factor could therefore be manipulated depending on the area of
injury to improve regenerative processes. Alternatively, when selecting appropriate stem or
progenitor cells for therapeutic application, the effect of the immediate environment and
subsequent interaction between the two should be taken into consideration for optimal
beneficial results.
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Dietary red palm oil-supplementation offers cardioprotection against Ischaemia/Reperfusion injury : possible cellular mechanisms involvedEsterhuyse, Adriaan Johannes 12 1900 (has links)
Dissertation (PhD)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: Activation of the NO-cGMP pathway is associated with myocardial protection
against ischaemia/reperfusion injury. However, high-cholesterol diets alter
function of this pathway and these alterations have been implicated in both
ischaemic/reperfusion injury and the development of ischaemic heart disease.
Little is known about the effects of supplements such as Red Palm Oil (RPO)
on the myocardial NO-cGMP-signalling pathway. RPO consists of saturated,
mono-unsaturated and poly-unsaturated fatty acids and is rich in antioxidants
such as β-carotene and Vitamin E (tocopherols and tocotrienols). The aims of
this study were: 1) to determine whether dietary RPO-supplemention protects
against ischaemia/reperfusion injury in rats fed a standard rat chow (control)
and cholesterol-enriched diets and 2) if so, to investigate possible mechanisms
for this protection.
Male Long-Evans rats were fed a standard rat chow or a standard rat chow
plus cholesterol and/or RPO-supplementation for 6 weeks. Myocardial
functional recovery was measured and hearts were freeze-clamped for
determination of myocardial phospholipid, cAMP/cGMP concentrations, total
myocardial nitric oxide concentrations, lipid hydroperoxide production and
superoxide dismutase- and nitric oxide synthase activity in isolated rat hearts
subjected to 25 minutes of normothermic total global ischaemia. In addition,
the degree of phosphorylation of extracellular signal-regulated kinase (ERK),
p38, c-Jun N-terminal protein kinase (JNK) and protein kinase B (PKB/Akt)
was investigated. Furthermore, the effect of RPO-supplementation on
caspase-3 activation and poly (ADP-ribose) polymerase (PARP)-cleavage in
hearts subjected to ischaemia and reperfusion was also investigated. Our data show that dietary RPO-supplementation protects the hearts of rats on
a standard rat chow (control) and hypercholesterolaemic diet against
ischaemia/reperfusion injury as reflected by improved aortic output recovery.
Increased intracellular cardiomyocyte NO concentrations as observed in
control hearts supplemented with RPO after 120 minutes hypoxia may
contribute to the elevated cGMP concentration and may confer some of the
cardioprotection to the ischaemic/reperfused heart. Although improved
functional recovery with RPO-supplementation of a high-cholesterol diet was
also associated with an increase in intracellular cardiomyocyte NO production
after hypoxia compared to the non-hypoxic conditions, it could not be linked to
increased NO-cGMP signalling. These data are in agreement with other
studies, which showed that high-cholesterol diet impairs NO-cGMP signalling
and confirms our hypothesis that elevated cGMP concentrations may not be
the only mechanism of protection. We have also shown that RPOsupplementation
caused increased phosphorylation of p38 and PKB, reduced
phosphorylation of JNK and attenuation of PARP cleavage, which may
contribute to the protection of the cell against apoptosis.
Based on our results we propose that the myocardial protection offered by
RPO-supplementation of rats on a normal and hypercholesterolaemic diet may
be associated with either its antioxidant characteristics and/or changes in the
fatty acid composition of the myocardium during ischaemia/reperfusion.
Furthermore, we demonstrated for the first time that RPO-supplementation
protects the isolated perfused working rat heart during reperfusion from
ischaemia/reperfusion-induced injury through a MAPK-dependent pathway. / AFRIKAANSE OPSOMMING: Aktivering van die NO-cGMP sein transduksie pad word geassosieer met
miokardiale beskerming teen isgemie/herperfusie skade. Hoë cholesterol diëte
verander egter die funksie van die pad en hierdie veranderings speel ‘n rol in
beide isgemie/herperfusie besering en die ontwikkeling van isgemiese
hartsiekte.
Daar is egter min inligting beskikbaar oor die uitwerking van aanvullings soos
rooi palm olie (RPO) op die miokardiale NO-cGMP sein transduksie pad. RPO
bevat versadigde, mono-onversadigde en poli-onversadigde vetsure en is ryk
aan anti-oksidante nl. β-karotene en vitamien E (tokoferole en tokotriënole).
Die doelwitte van hierdie studie was: 1) om vas te stel of ‘n RPO-aanvulling
beskerming bied teen isgemie/herperfusie besering in rotte wat gevoed is met
‘n standaard rotmengsel (kontrole) en cholesterol-verrykte dieet en 2) indien
wel, om moontlike meganismes van beskerming te ondersoek.
Long-Evans manlike rotte is vir 6 weke gevoer met ‘n standaard rotmengsel of
‘n standaard rotmengsel plus cholesterol en/of RPO-aanvulling. Miokardiale
funksionele herstel is gemeet en harte is gevriesklamp vir die bepaling van
miokardiale fosfolipied, cAMP/cGMP, totale stikstofoksied, lipied
hidroperoksied, superoksied dismutase en stikstofoksied sintase in
geïsoleerde rotharte wat vir 25 minute onderwerp was aan normotermiese
totale globale isgemie. Hiermee saam is die graad van fosforilering van
ekstrasellulêre sein gereguleerde kinase (ERK), p38 mitogeen-geaktiveerde
proteïen kinase (p38 MAPK), c-Jun-N-terminale proteïenkinase (JNK) en proteïen kinase B (PKB/Akt) ondersoek, asook kaspase-3 aktivering en poli
(ADP-ribose) polimerase (PARP) kliewing in harte blootgestel aan isgemie en
herperfusie.
Ons resultate toon dat RPO-aanvulling van rotte op ‘n normale en
hipercholesterolemiese dieet die hart beskerm soos getoon deur verbeterde
herstel van aortiese uitset. Verhoogde intrasellulêre miokardiale NO vlakke in
kontrole harte met ‘n RPO-aanvulling wat blootgestel was aan 120 minute
hipoksie, mag bygedra het tot die verhoogde cGMP vlakke en beskerming van
die hart tydens isgemie en herperfusie. Alhoewel verbeterde funksionele
herstel met RPO-aanvulling van ‘n hoë cholesterol dieet ook geassosieer is
met ‘n toename in intrasellulêre miokardiale NO produksie ná hipoksiese
toestande, kon dit nie verbind word met verhoogde aktivering van die NOcGMP
sein transduksie pad nie. Hierdie resultate stem ooreen met ander
studies wat aangetoon het dat hoë-cholesterol diëte die NO-cGMP seinpad
onderdruk. Hierdie bevinding bevestig ons hipotese dat verhoogde cGMP
vlakke moontlik nie die enigste beskermingsmeganisme is nie. Ons resultate
het ook gewys dat RPO-aanvulling fosforilering van p38 en PKB/Akt verhoog,
fosforilering van JNK verminder en PARP kliewing onderdruk. Dit dui op
beskerming van die sel teen apoptose.
Ons resultate dui aan dat die miokardiale beskerming wat RPO-dieet
aanvulling bied moontlik geassosieer kan word met sy anti-oksidant eienskap
en/of veranderinge in die vetsuur samestelling van die miokardium tydens
isgemie/herperfusie. Ons het ook vir die eerste keer bewys dat RPO-aanvulling die geïsoleerde geperfuseerde werkende rothart gedurende herperfusie
beskerm teen isgemie/herperfusie besering deur die aktivering en/of
deaktivering van die MAPK afhanklike pad.
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