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The phenomenon of 'second window of protection' : effect of beta-adrenergic stimulation and melatoninDavids, Ashraf 03 1900 (has links)
Thesis (MSc)--Stellenbosch University, 2004. / ENGLISH ABSTRACT:
Please see fulltext for abstract. / AFRIKAANSE OPSOMMING:
Sien asb volteks vir opsomming.
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The characterisation and expression of HIV-1 subtype C gagSampson, Candice Corene January 2002 (has links)
Thesis (MScMedSc) -- University of Stellenbosch, 2002. / ENGLISH ABSTRACT: The gag gene of HIV-1 encodes for one of the major structural proteins, which
contains several conserved cytotoxic T cell (CTL) epitopes. Gag specific CTL
responses are important in controlling viral load during acute infection and
asymptomatic stages of the infection. Currently, only one complete South African
HIV-1 subtype C gag sequence has been published. The first aim of this study
was to characterise the complete gag gene of 15 HIV-1 subtype C isolates, to be
used as a set of reference sequences in the design of a South African HIV-1
subtype C vaccine.
Fifteen HIV-1 subtype C isolates selected for this study, were isolated during 1998
and 1999 from the HIV-1 positive patients attending the Infectious Disease Clinic at
Tygerberg Hospital. The gag gene of these isolates was amplified by PCR, cloned
into mammalian expression vectors and sequenced. Restriction digest analyses as
well as phylogenetic analyses were performed on the sequencing data. Previously
published mutational analyses and CTL epitopes were compared to the predicted
amino acid sequences of the gag clones.
Sequences of 23 complete gag genes representing the 15 HIV-1 subtype C
isolates as well as one complete sequence of an HIV-1 subtype B isolate were
compiled. Subtyping by restriction fragment length polymorphism (RFLP) would
have correctly identified 14 of the 15 subtype C isolates as subtype C and one as
unidentifiable. The subtype B isolate would have also been correctly identified.
Phylogenetic analyses showed that our subtype C isolates clustered with reference
subtype C strains from various countries, including Botswana, India, Israel,
Tanzania and Zambia. Strains from Ethiopia and Brazil formed a separate subtype
C cluster. The diversity between our isolates was comparable to the diversity seen
between all the HIV-1 subtype C strains. Comparisons of previously published
mutational analyses and CTL epitopes to the predicted amino acid sequences of the gag clones, showed conservation in most of the clones throughout the
sequence.
A second aim was to establish transfection and Western Blot techniques in our
laboratory for use in future studies. An in vitro transcription! translation assay was
performed on the gag clones and the protein producing clones were used to
transfect mammalian cells using electroporation. A Western blot was then used to
screen for Gag protein expression in the transfected cell Iysates.
The in vitro transcription! translation assay showed that seven of the 23 clones
could produce a protein of -55 kDa in size. Four out of the seven of these clones
gave a weak expression of a-55 kDa protein after transfection in a mammalian
cell line. Since the completion of the experimental work of this study, other cloned
HIV-1 genes have successfully been transfected into mammalian cells using the
electroporation technique and the proteins produced were screened for by Western
blot.
To conclude with; the native form of the gag gene does not elicit strong expression
of the protein, but studies have shown that expression can be improved by
sequence-modification of the gag nucleotide sequence. Due to the conservation of
gag, the sequence of any subtype C strain can be used for the development of a
Southern African vaccine. / AFRIKAANSE OPSOMMING: Die HIV-1 gag geen kodeer vir een van die hoof strukturele proterene en bevat
verskeie sitotoksiese T-limfosiet epitope. Gag spesifieke sellulere immuun respons
is belangrik vir die beheer van virale lading tydens akute infeksies en tydens
asimptomatiese fases van die infeksie. Tans is slegs een volledige Suid
Afrikaanse HIV-1 subtipe C nuklerensuur volgorde gepubliseer. Die eerste doel
van hierdie studie was om die volledige gag geen van 15 HIV-1 subtipe C isolate te
karakteriseer, om gebruik te word as In stel verwysings nukleiensuur volgordes, vir
die ontwerp van In Suid Afrikaanse HIV-1 subtipe C entstof.
Die 15 HIV-1 subtipe C isolate wat vir hierdie studie geselekteer is, is tydens 1998
en 1999 ge·lsoleer vanaf HIV-1 positiewe pasiente wat die Infeksiesiekte Kliniek,
Tygerberg Hospitaal bygewoon het. Die gag geen van hierdie isolate is
geamplifiseer deur PKR, gekloneer in soogdier ekspressie vektore en die
nukleiensuur volgorde is bepaal. Die nuklerensuur volgorde is gebruik in restriksie
ensiem analises asook filogenetiese analises. Reeds gepubliseerde mutasie
analises en limfosiet epitope is met die voorspelde aminosuur volgorde van die gag
klone vergelyk.
Die nukleiensuur volgordes van die 23 volledige gag gene wat die 15 HIV-1
subtipe C isolate verteenwoordig, asook een volledige nukleiensuur volgorde van
een HIV-1 subtipe B isolaat, is saamgestel. Subtipering deur middel van restriksie
fragment lengte polimorfisme (RFLP) sou 14 uit die 15 subtipe C isolate korrek
qerdentifiseer het, maar sou een nie kon identifiseer nie. RFLP sou ook die
subtipe B isolaat korrek qerdentifiseer het. Filogenetiese analises het gewys dat
ons subtipe C isolate met die verwysings subtipe C stamme van verskeie lande,
insluitend Botswana, lndie, Israel, Tanzania en Zambie groepeer. Stamme van
Ethiopie en Brasilie het In aparte subtipe C groep gevorm. Die diversiteit tussen
ons isolate was vergelykbaar met die diversiteit tussen al die subtipe C stamme.
Vergelykings van gepubliseerde mutasie analises en limfosiet epitope met die voorspelde aminosuur volgorde van die gag klone, het konservasie in meeste van
die klone, deur die hele nukleiensuur volgorde, getoon.
Die tweede doel was om die metodes van transfeksie en Westerse klad in ons
laboratorium tot stand te bring. In vitro transkripsie/ translasie toetse is gedoen op
die gag klone en die proteten produserende klone is gebruik om soogdierselle te
transfekteer deur gebruik te maak van elektroporasie. In Westerse klad is toe
gebruik om vir Gag proterenuitdrukkinq in die sellisate te toets.
Die in vitro transkripsie/ translasie toets het getoon dat sewe uit 23 klone, In
proteren van -55 kDa kon produseer. Vier uit die sewe van hierdie klone het In
-55 kDa proteren swak uitgedruk na transfektering van soogdier selle. Sedert die
voltooiing van die eksperimentele werk van hierdie stud ie, is ander gekloneerde
HIV-1 gene suksesvol in soogdierselle getransfekteer met die gebruik van
elektroporasie en die proterene is met In Westerse klad aangetoon.
Ten slotte: die natuurlike vorm van die gag geen ontlok nie In sterk ekspressie van
die proteren nie, maar ander studies het wei aangetoon dat die ekspressie verbeter
kan word met modifikasie van die gag nukleiensuur volgorde. As gevolg van die
konservasie van gag, kan die nuklerensuur volgorde van enige subtipe C stam
gebruik word vir die ontwikkeling van In Suider Afrikaanse entstof. / The Poliomyelitis Research Foundation / The South African AIDS Vaccine Initiative / Harry Crossley Foundation
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The role of p38 MAPK activation in preconditioning mediated protection against ischaemia/reperfusion injuryHartley, Shahiem 12 1900 (has links)
Thesis (MSc)--Stellenbosch University, 2002. / ENGLISH ABSTRACT: The ultimate consequence of the interruption of blood flow to the myocardium is
necrosis. In view of the prevalence of coronary artery disease in the general
population, and the deleterious effects of myocardial ischaemia on myocardial
tissue, it is important to develop new strategies to protect the myocardium against
ischaemia. Necrosis of myocardial tissue has for a long time been considered to be
the main component of the damage incurred by myocardial infarction. Recently the
importance of the contribution of apoptotic cell death in the context of myocardial
ischaemia/reperfusion injury has become apparent.
There is a general agreement that early reperfusion is necessary to salvage
myocardial tissue from cell death. Preconditioning is the phenomenon whereby
brief episodes of ischaemia and reperfusion protect the heart against a subsequent
longer period of ischaemia. This endogenous mechanism is the strongest form of
protection against myocardial infarction that has yet been described. Apart from
ischaemie preconditioning (IPC), protection can also be elicited with pharmacologic
agents, such as activation of the beta-adrenergic receptor with isoproterenol.
Ischaemie preconditioning protects the myocardium against necrosis, arrhythmias
and apoptosis, and increases functional recovery upon reperfusion. Betaadrenergic
receptor stimulated preconditioning (PPC) has been shown to improve
post-ischaemie functional recovery, but it is not known whether it also protects
against myocardial infarction and apoptosis.
The signaling pathways involved in preconditioning have been extensively studied.
A distinction is usually made between factors that act as triggers, or as mediators
of protection. Triggers activate cellular responses before the onset of sustained
ischaemia, and its involvement is demonstrated by showing that inhibitors of the
trigger bracketing the preconditioning protocol can block its protective effect, or that
transient administration with washout before sustained ischaemia can activate a
protective effect. A mediator operates during sustained ischaemia, and its
involvement is demonstrated by showing that infusion of an inhibitor of its action
immediately prior to sustained ischaemia (without washout) can block its protective
effect. Another approach to demonstrate a mediator role is to attempt to activate
signal transduction pathways during sustained ischaemia. As it is not possible to
infuse substances during ischaemia, activators are infused immediately prior to
ischaemia without washout of the agent and subsequently its effect on protection is
observed.
It is clear that the evolutionary conserved stress activated pathways are involved in
preconditioning. There are three pathways i.e., the extracellular receptor activated
pathways (ERK), c-jun terminal activated kinases (JNK) and p38 mitogen-activated
protein kinases (MAPK). The precise role of the p38 MAPK pathway has not been
elucidated. Experimental evidence has suggested a role for the activation of p38
MAPK as a trigger, as well as a mediator of the protective effect of preconditioning.
There is however also strong evidence that the attenuation of p38 MAPK activation
during sustained ischaemia, rather than its activation, is responsible for the protection that is observed. Furthermore, the role of p38 MAPK has only been
investigated in relation to its protection against necrosis, but not apoptosis.
AIMS:
The aim of this study was to:
(I) Establish a model of preconditioning in neonatal cardiomyocyte cell culture.
The reason was that such a model could potentially enable one to rapidly
elucidate the signal transduction pathways in an environment without the
influence of non-cardiac cells.
(II) Investigate whether IPC and ~PC protect against necrosis and apoptosis.
(III) Elucidate the role of the stress-activated kinase, p38 MAPK, in
preconditioning.
METHODS:
1. Neonatal rat cardiomyocyte cell culture model
A viability assay with 3-[4,5- Dimethylthaizol-2-yl]-2,5-diphenyl-tetrazolium bromide
(MTT) was first developed using different concentrations - a concentration of
0.25% was found to be optimal to determine viability. Neonatal cardiomyocyte cell
cultures were subjected to sustained simulated "ischaemia" by using either 5 mM
KCN plus deoxyglucose (DOG) for 5 min or potassium cyanide (KCN) for 45 min.
Some cell cultures were preconditioned with either chemical ischaemia (5 mM KCN
for 5 min) or isoproterenol (10-7 M) for 5 min and 60 min reoxygenation before
being exposed to sustained simulated ischaemia.
2. Isolated adult rat cardiomyocyte model
Isolated cardiac myocytes were exposed to 2 hours of hypoxia, which was induced
by pelletting the cells by centrifugation, and covering them with a thin layer of
mineral oil. Some groups were preconditioned with either hypoxia for 10 min at
37° C or isoproterenol (10-7 M) for 5 min, followed by reoxygenation for 20 minutes.
The trypan blue exclusion method and MTT method developed in the neonatal
cardiomyocytes were used to assess viability.
3. Isolated perfused rat heart model
3.1 Infarct size was determined in a model of regional ischaemia by using
tetrazolium staining and determining the area of necrosis (exclusion of
tetrazolium) as a percentage of area at risk. These hearts were subjected to
35 min global ischaemia and 30 min reperfusion. Some groups were
preconditioned by three cycles of 5 min global ischaemia or addition of
isoproterenol (10-7 M) for 5 min, followed by 5 min reperfusion before the
onset of sustained regional ischaemia.
3.2 p38 MAPK activation and markers of apoptosis: p38 MAPK activation was
determined using antibodies against dual phosphorylated p38 MAPK (i.e.
activated p38 MAPK). Apoptosis was measured by using antibodies against
activated caspase-3, and against a fragment of PARP (PARP cleavage). For
these experiments isolated rat hearts were exposed to global ischaemia for
25 min followed by 30 min reperfusion. Some groups were preconditioned
with three cycles of 5 min global ischaemia. A global ischaemia model was
used in order to have sufficient tissue available for the Western blot
determinations. This necessitated a shorter period of sustained ischaemia,
as the globally ischaemie heart does not recover sufficiently after a longer
period of ischaemia such as is necessary in regional ischaemia
experiments.
3.3 The role of p38 MAPK in ischaemie preconditioning was investigated by
administration of SB 203580 (1IJM),a selective inhibitor of p38 MAPK, either
bracketing the preconditioning (i.e. to determine its role as a trigger) or for
10 min immediately prior to sustained ischaemia (i.e. to determine its role as
a mediator). The second approach was to use anisomycin, an activator of
p38 MAPK, as a trigger (infusion for 10 min followed by wash out) or as a
mediator (10 min immediately prior to sustained ischaemia) in the same
model as used for determination of p38 MAPK activity. The infusion of
anisomycin for 10 min has been shown to elicit activation of p38 MAPK to a
similar extent as has been observed with an ischaemie preconditioning
protocol. The endpoints used were infarct size and markers of apoptosis.
RESULTS:
1. Neonatal rat cardiomyocyte cell culture model
It was not possible to establish a model of preconditioning of neonatal
cardiomyocytes that was consistently successful. It was therefore decided to
abandon the attempts and to use a different cell model.
2. Isolated adult rat cardiomyocyte model
Isolated adult cardiomyocytes were preconditioned successfully, but produced too
little material to perform simultaneous determinations of cell viability and Western
blots (p38 MAPK activation and markers of apoptosis). It was therefore decided to
use the isolated perfused adult rat heart.
3. Isolated perfused adult rat heart model
3.1 Both IPC and PPCprotect against infarction and apoptosis:
Using two models of preconditioning i.e., IPC and PPC, the protective effects of
preconditioning were demonstrated convincingly against infarction (necrosis). IPC
and PPC both caused a significant reduction in infarct size (12.2±1.4 and
15.2±2.6%) versus Non-PC hearts (29.6±2.9%) (p < 0.001). Both forms of
preconditioning also protected against apoptosis, by significantly reducing the
markers of apoptosis, caspase-3 activation and PARP cleavage. The protection
afforded by both forms of preconditioning was accompanied by a marked decrease
in activation of p38 MAPK upon reperfusion. The relationship between p38 MAPK
and the protection that was elicited by preconditioning was then investigated, namely whether p38 MAPK acted as a trigger, or as a mediator of protection. To
investigate the role of p38 MAPK as a mediator or a trigger in preconditioning, use
was made of (i) a specific inhibitor of p38 MAPK activation i.e., SB 203580 and (ii)
a known activator of p38 MAPK i.e., anisomycin.
3.2 p38 MAPK as a trigger of protection:
Administration of SB 203580 during the IPC protocol and washed out before
sustained ischaemia did not abolish the protective effect of ischaemie
preconditioning, and resulted in a small, but significant increase in caspase-3
activation and PARP cleavage. On the other hand, activation of p38 MAPK with
anisomycin for 10 min followed by washout also resulted in a significant reduction
in necrosis (infarct size 14.9±2.2 versus 29.6±2.9% in Non-PC hearts) (p < 0.001)
and both markers of apoptosis. The latter results suggested that p38 MAPK was a
trigger of preconditioning. If this was the case, why didn't SB 203580 abolish the
protection of IPC? The most likely explanation was that multiple protective
mechanisms were activated during a multi-cycle protocol of ischaemic
preconditioning, of which activation of p38 MAPK was only one. Inhibition of p38
MAPK with SB 203580 would therefore not be expected to block the activation of
those mechanisms that were independent of p38 MAPK, but were still capable of
protecting against necrosis or apoptosis. It is very interesting that a small increase
in apoptosis was observed when SB 203580 was used in this situation, as it may
indicate that the protection against apoptosis was more dependent on the
activation of p38 MAPK than the protection against necrosis, as no effect was seen on infarct size. Another explanation could be that infarct size determination was not
sensitive enough to detect such small effects.
3.3 p38 MAPK as a mediator of protection:
Inhibition of p38 MAPK activation with SB 203580 administered 10 min before
sustained ischaemia caused a significant decrease in infarct size compared to
Non-PC hearts (12.6±1.9 vs 29.6±2.9%) (p < 0.001) equivalent to that of hearts
preconditioned with ischaemia. This was accompanied by a similar pattern of
protection against apoptosis, with significantly reduced activation of caspase-3
activation and PARP cleavage.
These results strongly supported a role for the attenuation of p38 MAPK activation
as a mediator of preconditioning against ischaemia/reperfusion-mediated necrosis
and apoptosis. However, the results of the experiments with anisomycin were at
first glance not compatible with such a conclusion. The administration of the
activator of p38 MAPK, anisomycin, for 10 min immediately prior to sustained
ischaemia resulted in significant protection against necrosis (infarct size 16.6±2.4%
vs 29.6±2.9% in Non-PC hearts) (p < 0.01) and reduced caspase-3 activation and
PARP cleavage indicating less apoptosis. The reason for these findings were
probably that this method of administration of anisomycin did in fact not activate
p38 MAPK during sustained ischaemia, but actually served as a trigger to protect
against ischaemia - similarly as if it had been infused with washout of the drug.
Support for this notion was found in the fact that p38 MAPK activation was decreased upon reperfusion. These results suggested that the logistical problem of
not being able to infuse a drug into the myocardium during ischaemia could not be
overcome by immediate prior infusion, and that the administration of anisomycin in
this way had activated downstream effectors of the p38 MAPK signal transduction
pathway. An important contender for such an effector would be heat shock protein
27 (HSP27), which has been shown to play an important role in protection against
apoptosis, and stabilisation of actin, and thus the cytoskeleton. Another possibility
was that anisomycin had activated the JNK stress activated kinases. The
elucidation of a role of this signal transduction pathway would necessitate the use
of anisomycin in the presence of an agent such as curcumin, an inhibitor of JNK.
Final conclusion:
The work in this thesis showed that the stress activated kinase, p38 MAPK, was
involved in the protective effect of ischaemie preconditioning. The results
suggested a role for the activation of p38 MAPK as a trigger of protection, and the
attenuation of p38 MAPK as a mediator of protection, which was observed in the
reduction of both necrosis (infarct size) and apoptosis as determined with caspase-
3 activation and PARP cleavage. / AFRIKAANSE OPSOMMING: Die afsluiting van bloedvloei na die miokardium gee aanleiding tot nekrose. In die
lig van die voorkoms van koronêre bloedvatsiekte onder die algemene populasie,
en die nadelige effekte van miokardiale isgemie op miokardiale weefsel, is dit
belangrik om nuwe strategieë te ontwikkel wat die miokardium teen isgemie
beskerm. Nekrose van miokardiale weefsel word tradisioneel as die belangrikste
komponent van die skade aangerig deur miokardiale infarksie beskou. Die belang
van apoptotiese seldood in die konteks van miokardiale isgemie/herperfusie (I/R)
het onlangs na vore getree.
Dit word algeneem aanvaar dat vroeë vroegtydige herperfusie noodsaaklik is om
miokardiale weefsel te beskerm teen seldood. Prekondisionering is 'n verskynsel
waartydens kort episodes van IIR die hart teen 'n daaropvolgende langer periode
van isgemie beskerm. Hierdie endogene meganisme is die kragtigste vorm van
beskerming teen miokardiale infarksie tot dusver beskryf. Afgesien van isgemiese
prekondisionering (IPC), kan beskerming ook deur farmakologiese middels, soos
byvoorbeeld die aktivering van die beta-adrenerge reseptore met isoproterenol,
ontlok word. IPC beskerm die miokardium teen nekrose, arritmieë en apoptose, en
verhoog funksionele herstel na herperfusie. Daar is reeds aangetoon dat betaadrenerge
prekonsionering (~PC) post-isgemiese funksionele herstel verbeter,
maar dit is nog onbekend of beskerming ook teen miokardiale infarksie en
apoptose verleen word.
Die seintransduksie paaie betrokke tydens prekondisionering is reeds in detail
bestudeer. Daar word gewoonlik tussen faktore wat optree as snellers, of as
mediators van beskerming, onderskei. Snellers aktiveer sellulêre response voor die
aanvang van volgehoue isgemie, en hul betrokkenheid word aangetoon deurdat
inhibisie van snellers tydens die prekondisionering protokol, beskerming ophef.
Snellers se effekete kan ook ontlok word deur hulle tydelike toe te dien en dan net
voor volgehoue isgemie weer uit te was. Mediators oefen hulle effek tydens
volgehoue isgemie uit, en hulle betrokkenheid word gedemonstreer deurdat
toediening van inhibitors net voor volgehoue isgemie (sonder uitwas) hulle
beskermende effekte ophef. Mediators se rol kan ook aangetoon word deur te
poog om seintransduksie paaie tydens volgehoue isgemie te aktiveer. Aangesien
dit ontmoontlik is om middels tydens isgemie te infuseer, word aktiveerders
onmiddelik voor die aanvang van isgemie toegedien sonder om hulle uit te was,
sodat hulle effekte op beskerming vervolgens bestudeer kan word.
Dit is duidelik dat die evolusionêr-behoue stres geaktiveerde paaie tydens
prekondisionering betrokke is. Daar is drie paaie nl. die ekstrasellulêre reseptor
geaktiveerde pad (ERK), c-jun terminaal geaktiveerde kinases (JNK) en p38
mitogeen geaktiveerde proteïen kinases (MAPK). Die spesifieke rol van die p38
MAPK pad is nog nie ontrafel nie. Eksperimentele bewyse stel 'n rol vir die
aktivering van p38 MAPK as 'n sneller, sowel as 'n mediator van die beskermende
effek van prekondisionering, voor. Daar is egter ook sterk bewyse dat 'n afname in
p38 MAPK aktivering tydens volgehoue isgemie, eerder as sy aktivering, verantwoordelik is vir die waargenome beskermende effek. Verder is die rol van
p38 MAPK slegs in die konteks van beskerming teen nekrose, maar nie teen
apoptose nie, bestudeer.
DOELWITTE:
Die doelwit van hierdie studie was:
(I) Die vestiging van 'n prekondisionering model in neonatale kardiomiosiet in
selkultuur. Hierdie model sou potensieel 'n spoedige ontrafeling van die
seintransduksie paaie sonder die invloed van nie-kardiale selle bewerkstellig.
(II Om ondersoek in te stelof IPC en PPCteen nekrose en apoptose beskerm.
(III) Die ontrafeling van die rol van die stres geaktiveerde kinase, p38 MAPK,
tydens prekondisionering.
METODES:
1. Neonatale rot kardiomiosiet weefselkultuur model
'n Lewensvatbaarheids essai is ontwikkel deur van verskillende konsentrasies van
3-[4,5-dimetielthiazol-2-yl]-2,5-difeniel-tetrazolium bromied (MTT) gebruik te maak
- 'n konsentrasie van 0.25% was optimaalom lewensvatbaarheid te bepaal.
Neonatale kardiomiosiet weefselkulture is onderwerp aan volgehoue gesimuleerde
"isgemie" deur gebruik te maak van 5 mM KCN plus deoksiglukose (DOG) vir 5
minute of 45 min KCN. Sommige weefselkulture is geprekondisioneer deur middel
van chemiese isgemie (5 mM KCN vir 5 min) of van isoproterenol (10-7 M) vir 5 minute en 60 minute reoksigenasie alvorens dit bloot gestel is aan volgehoue
gesimuleerde isgemie.
2. Geïsoleerde volwasse rot kardiomiosiet model
Geïsoleerde kardiomiosiete is aan twee uur hipoksie blootgestel deur selle in 'n
pellet te sentrifugeer en met 'n dun lagie mineraalolie te bedek. Sommige groepe is
geprekondisioneer deur middel van 10 minute hipoksie by 37°C, of toediening van
isoproterenol (10-7 M) vir 5 minute gevolg deur 20 minute reoksigenasie. Die
tripaanblou uitsluitings metode en MTT metode soos ontwikkel in die neonatale
kardiomiosiet model is gebruik om lewensvatbaarheid te bepaal.
3. Geïsoleerde geperfuseerde volwasse rot hart model
3.1 Infarkgrootte is bepaal met 'n model van streeks isgemie deur van
tetrazolium kleuring gebruik te maak, waarna die area van nekrose (uitsluiting van
tetrazolium) as 'n presentasie van die risiko area bepaal is. Hierdie harte was
onderwerp aan 35 minute globale isgemie en 30 minute herperfusie. Sommige
groepe is geprekondisioneer met 3 siklusse van 5 minute globale isgemie, of die
toevoeging van isoproterenol (10-7 M) vir 5 minute, gevolg deur 5 minute
herperfusie voor die aanvang van volgehoue streeks isgemie.
3.2 p38 MAPK aktivering en merkers van apoptose: p38 MAPK aktivering is
bepaal deur gebruik te maak van anti-liggame teen tweeledige gefosforileerde p38
MAPK (d.w.s. geaktiveerde p38 MAPK). Apoptose is bepaal deur gebruik te maak van anti-liggame teen geaktiveerde kaspase-3, en teen 'n fragment van PARP
(PARP kliewing). Tydens hierdie eksperimente is geïsoleerde rotharte bloot gestel
aan 25 minute globale isgemie gevolg deur 30 minute herperfusie. Sommige
groepe is geprekondisioneer met drie siklusse van 5 minute globale isgemie. Om
voldoende weefsel vir Westerse klad tegnieke te verkry, is gebruik gemaak van 'n
globale isgemie model. As gevolg hiervan was 'n kort periode van volgehoue
isgemie genoodsaak, aangesien die globale isgemiese hart nie voldoende herstel
na 'n langer periode van isgemie nie, soos wat benodig word in streeks isgemiese
eksperimente.
3.3 Die rol van p38 MAPK tydens IPC is bepaal deur die toediening van 'n 1IJM
konsentrasie van SB 203580, 'n selektiewe inhibitor van p38 MAPK, hetsy tydens
prekondisionering (d.w.s. om die rol as 'n sneller te bepaal), óf vir 10 minute direk
voor die aanvang van volgehoue isgemie (d.w.s. om dus sy rol as mediator te
bepaal). Die tweede benadering was om anisomisien, 'n aktiveerder van p38
MAPK, as sneller (toediening vir 10 minute gevolg deur uitwassing) of as mediator
(10 minute direk voor aanvang van volgehoue isgemie) in dieselfde model as in die
geval van p38 MAPK aktiviteit bepaling, te gebuik. Die toediening van anisomisien
vir 10 minute het aangetoon dat dit p38 MAPK aktivering kan ontlok tot dieselfde
maate as die IPC protokol. Die eindpunte was infarkgrootte en merkers van
apoptose.
RESULTATE:
1. Neonatale rot kardiomiosiet weefselkultuur model
Dit was nie moontlik om 'n suksesvolle model met konsekwente resultate vir die
prekondisionering van neonatale kardiomiosiete te vestig nie. Daar is dus besluit
om af te sien van hierdie pogings en eerder 'n alternatiewe selmodel te gebruik.
2. Geïsoleerde volwasse rot kardiomiosiet model
Geïsoleerde volwasse kardiomiosiete is suksesvol geprekondisioneer, maar het te
min materiaalopgelewer vir die gelyktydige bepaling van sellewensvatbaarheid,
p38 MAPK aktivering en merkers vir apoptose. Daar is dus besluit om die
geïsoleerde geperfuseerde volwasse rothart te gebruik.
3. Geïsoleerde geperfuseerde volwasse rothart model
3.1 Beide IPC en PPCbeskerm teen infarksie en apoptose:
Deur gebruik te maak van twee prekondisionering modelle d.w.s. IPC en PPC, is
die beskermende effekte van prekondisionering teen infraksie (nekrose) oortuigend
gedemonstreer. Beide IPC en PPC het In betekenisvolle afname in infarkgrootle
veroorsaak (12.2 ± 1.4 en 15.2 ± 2.6% respektiewelik), vs Nie-PC harte (29.6 ±
2.9%)(p < 0.001). Beide vorme van prekondisionering het ook teen apoptose
beskerm deur die apoptose merkers, kaspase-3 aktivering en PARP kliewing te
verlaag. Die beskerming verkry deur beide vorms van prekondisionering is
geassosieer met In merkbare afname in die aktivering van p38 MAPK na
herperfusie. Die verband tussen p38 MAPK en die beskerming ontlok deur prekondisionering is gevolglik ondersoek, naamlik of p38 MAPK optree as 'n
sneller of as 'n mediator van beskerming. Om die rol van p38 MAPK as 'n mediator
of sneller tydens prekondisionering te ondersoek is daar gebruik gemaak van (I) 'n
spesifieke inhibitor van p38 MAPK aktivering nl. SB 203580 en (II) 'n bekende
aktiveerder van p38 MAPK nl. anisomisien.
3.2 p38 MAPK as 'n sneller vir beskerming:
Toediening van SB 203580 tydens die IPC protokol en uitwassing daarvan voor die
aanvang van volgehoue isgemie het nie die beskermende effek van IPC opgehef
nie, en het gelei tot 'n klein maar betekenisvolle verhoging in kaspase-3 aktivering
en PARP kliewing. Andersins het die aktivering van p38 MAPK met anisomisien vir
10 minute gevolg deur In uitwas ook tot In betekenisvolle afname in nekrose
(infarkgrootte 14.9 ± 2.2 vs 29.6 ± 2.9% in Nie-PC harte) (p < 0.001) in beide
merkers van apoptose gelei. Laasgenoemde resultate dui daarop dat p38 MAPK
inderdaad 'n mediator van prekondisionering is. Indien dit die geval is, waarom het
SB 203580 nie die beskermende effek van IPC opgehef nie? Die mees
waarskynlike verklaring is dat veelvuldige beskermingsmeganismes tydens 'n
multi-siklus protokol van IPC geaktiveer word, waarvan p38 MAPK aktivering slegs
een is. Dit is dus onwaarskynlik dat die inhibisie van p38 MAPK met SB 203580 die
aktivering van daardie meganismes onafhanklik van p38 MAPK sal blokkeer en
steeds in staat sal wees tot beskerming teen nekrose en apoptose. Dit is
interessant dat In klein verhoging in apoptose waargeneem is toe SB 203580
gebruik is onder hierdie toestande, aangesien dit daarop kan dui dat die beskerming teen apoptose meer afhanklik was van die aktivering van p38 MAPK
as die beskerming teen nekrose, siende dat geen effek op infarkgrootte
waargeneem is nie. 'n Verdere verklaring kan wees dat die bepaling van
infarkgrootte nie sensitief genoeg is om sulke klein effekte waar te neem nie.
3.3 p38 MAPK as 'n mediator vir beskerming:
Inhibisie van p38 MAPK aktivering deur SB 203580 toediening 10 minute voor
volgehoue isgemie het 'n betekenisvolle verlaging in infarkgrootte in vergelyking
met Nie-PC harte veroorsaak (12.6 ± 1.9 vs 29.6 ± 2.9%) (p < 0.001) soortgelyk
aan dié van harte geprekondisioneer met isgemie. Dit is geassosieer met In
soortgelyke patroon van beskerming teen apoptose, met betekenisvolle verlaagde
kaspase-3 aktivering en PARP kliewing.
Hierdie resultate ondersteun die rol van die afname van p38 MAPK aktivering as 'n
mediator van prekondisionering teen I/R-gemedieerde nekrose en apoptose. Die
resultate van die anisomisien eksperimente was met die eerste oogopslag nie in
oorstemming met hierdie gevolgtrekking nie. Die toedienning van die p38 MAPK
aktiveerder, anisomisien, vir 10 minute voor volgehoue isgemie het tot 'n
betekenisvolle beskerming teen nekrose aanleiding gegee (infarkgrootte 16.6 ± 2.4
vs 29.6 ± 2.9% in Nie-PC harte) (p < 0.01) en verlaagde kaspase-3 aktivering en
PARP kliewing wat dui op verlaagde apoptose. Die rede vir hierdie bevindings is
moontlik dat die metode van anisomisien toediening nie p38 MAPK geaktiveer het
tydens volgehoue isgemie nie, maar eintlik gedien het as 'n sneller vir beskerming teen isgemie - amper asof dit toegedien sou word sonder om uitgewas te word.
Ondersteuning vir hierdie aanname word gevind in die feit dat p38 MAPK
aktivering verlaag is na herperfusie. Hierdie resultate stel voor dat die logistiese
probleem dat In middel nie tydens isgemie toegedien kan word nie, nie oorkom kan
word deur onmiddelike voortydige infusie nie, en dat die toediening van
anisomisien op hierdie manier gelei het tot die aktivering van stroom-af effektors
van die p38 MAPK seintransduksie pad. 'n Belangrike kandidaat vir so 'n effektor is
"heat shock protein 27" (HSP27), wat reeds aangetoon is om 'n belangrike rol in
die beskerming teen apoptose en destabilisering, en dus die sitoskelet, te speel. 'n
Ander moontlikheid is dat anisomisien die JNK stres geaktiveerde kinases
geaktiveer het. Die ontrafeling van die rol van hierdie seintransduksie pad
noodsaak die gebruik van anisomisien in die teenwoordigheid van 'n agent soos
curcumin, 'n JNK inhibitor.
Finale gevolgtrekking:
Die werk soos vervat in hierdie tesis toon aan dat die stres geaktiveerde kinase,
p38 MAPK, betrokke is in die beskermings effek van isgemiese prekondisionering.
Die resultate dui op 'n rol vir die aktivering van p38 MAPK as 'n sneller vir
beskerming, en die afname in p38 MAPK as 'n mediator vir beskerming, soos
waargeneem in die vermindering van veranderlikes van beide nekrose
(infarkgrootte) en apoptose soos bepaal deur kaspase-3 aktivering en PARP
kliewing.
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Characterisation of the HIV-1 subtype C Env gene and the expression of the Env protein from selected isolates in mammalian cellsDe Villiers, Tania 03 1900 (has links)
Thesis (MSc)--Stellenbosch University, 2000. / ENGLISH ABSTRACT: At the end of 2002, human immunodeficiency virus (HIV) had infected 42 million people
worldwide. The morbidity and mortality rate, as well as the epidemic proportions of the
disease have led to concentrated scientific efforts to reveal the disease's pathogenesis
and develop effective preventative and treatment measures. Advances have been made
to inhibit viral replication by suppressing the virus' ability to replicate by developing antiretroviral
treatments, although development of a save and effective vaccine is the only
way to stem the pandemic. Advances in vaccine design, animal models and clinical
research have led to the creation of promising candidate vaccines to counter this
rampage, but most of these vaccines entering phase I-III clinical trials are based mainly
only subtype B genomes. HIV-1 subtype C is the most commonly transmitted subtype
worldwide, and is the predominant subtype in India, China, East and Southern Africa. A
subtype C vaccine is critical for the developing nations such as South Africa, where antiretroviral
therapies are largely unaffordable. The envelope gene (env) is an attractive
target as immunogen to be included in a HIV vaccine. The envelope protein (Env) elicits
neutralising antibodies and cytotoxic T-Iymphocyte (CTl) responses. This protein will
therefore be useful in creating a humoral and cellular immune response in the host. A
shortage in characterised subtype C env gene sequences from South Africa was
recognised, and this study focussed on the characterisation of generated sequences, as
well as the expression of selected env genes. These immunogens were created for
possible use in a prime-boost vaccine modality. The env genes from recent circulating
strains in South Africa were amplified by polymerase chain reaction (PCR). The genes
were then cloned for sequencing and expression purposes. Phylogenetic relationships
were determined by comparing the sequences to reference subtype strains and subtype
C strains. Expression of the genes was assessed by Western Blot in 293 cells with HIV-
1 positive patient sera.
Sequence analysis showed a more conserved third variable (V3) loop in South African
subtype C sequences, with a more variable region downstream from the loop. The
crown sequence (GPGQ) and positions of uncharged or negatively charged residues in the V3 loop indicated a non-syncytium-inducing (NSI) phenotype for the isolates.
Phylogenetic analysis showed the sequences to all belong to the C subtype, and further
that the sequences were not recombinant, which was confirmed by recombination
analysis. The intersample diversity observed for strains from South Africa was
significantly higher than distances observed to the subtype C consensus sequence. The
South African sequences were distributed across several subclusters in a subtype C
phylogenetic tree, highlighting the concept that these infections represent a more
longstanding epidemic with multiple introductions from different geographic areas.
Western Blot with HIV-1 positive patient sera showed the expression of uncleaved
gp160 Env proteins, which were Rev dependent.
This study has generated much needed subtype C South African env gene sequences
that can be used as basis for modification for use as immunogens in a South African
vaccine. / AFRIKAANSE OPSOMMING: Teen die einde van 2002 was 42 miljoen mense wêreldwyd geïnfekteer met die
menslike immuniteitsgebrekvirus (MIV). Die dode- en sterfte syfers, asook die skaal van
die epidemie, het gelei tot 'n wetenskaplike poging om die siekte se patogenese te
openbaar en om effektiewe voorkomende en terapeutiese middels te ontwikkel.
Vordering is reeds gemaak om die virus se replikasie te hinder deur die ontwerp van
antivirale middels, alhoewel die ontwikkeling van 'n doeltreffende en veilige entstof die
enigste manier is om die pandemie te stuit. As gevolg van die vordering in entstof
ontwerp, diere modelle en kliniese navorsing is belowende kandidaat entstowwe wat die
infeksie kan teenwerk ontwikkel, maar die meeste van hierdie enstowwe wat vir fase I-III
kliniese proewe gebruik word is gebaseer op subtipe B genome. MIV-subtipe C is
wêreldwide die algemeenste subtipe wat oorgedra word en is die oorheersende subtipe
in lande soos Indië, China, oostelike en suidelike Afrika. 'n Subtipe C entstof word
dringend benodig in ontwikkelende lande soos Suid-Afrika waar antivirale middels
onbekostigbaar is. Die membraangeen is 'n aanloklike teiken om as immunogeen in 'n
MIV entstof te dien. Die membraanproteïen lok neutraliserende teenliggame en
sitotoksiese T-limfosiet reaksies uit. Die proteïen sal dus 'n humorale en sellulêre
immuunrespons in die gasheer ontlok. 'n Tekort aan gekarakteriseerde subtipe C
membraangeen volgordes van Suid-Afrika is opgemerk, en dus fokus hierdie studie op
die karakterisering van gegenereerde volgordes, asook die uitdrukking van
geselekteerde membraangene. Die immunogene is geskep om moontlik gebruik te word
in 'n stimuleer-versterkingsenstof toedieningstrategie. Die membraangene van onlangs
sirkulerende virusstamme in Suid-Afrika was geamplifiseer deur polimerase
kettingreaksie (PKR). Die gene is daarna gekloneer vir beide volgordebepalings en
uitdrukkingdoeleindes. Filogenetiese verhoudings is uitgewerk deur die volgordes met
verwysingsstamme en subtipe C stamme te vergelyk. Uitdrukking van die gene is
waargeneem in 293 selle deur die Westerse kladtegniek te gebruik met MIV-1 positiewe
pasiëntsera as teenliggaam.
Volgorde-analise het aangetoon dat die derde varieerbare (V3) lus meer gekonserveer
is, en dat die gedeelte wat op die lus volg meer varieerbaar is. Die kroonvolgorde
(GPGQ) asook posisies van ongelaaide of negatief gelaaide aminosure in die V3 lus het
aangedui dat die isolate 'n nie-syncytia induserende fenotipe het. Filogenetiese analise
het aangedui dat al die volgordes subtipe C is en dat die volgordes nie rekombinant is
nie. Dit is ook deur rekombinasie analise bewys. Die inter-monster diversiteit van die
Suid-Afrikaanse volgordes was hoër as die waargenome afstand vanaf die subtipe C
konsensus volgorde. Die Suid-Afrikaanse volgordes is versprei oor verskeie subgroepe
in 'n subtipe C boom, wat die konsep dat hierdie infeksies 'n meer gevestigde epidemie
voorstel waar veelvuldige infeksies met verskillende geografiese oorspronge
plaasgevind het beklemtoon. Die Westerse klad het ongeprosesseerde gp160
membraanproteïne aangetoon wat Rev afhanklik was.
Hierdie studie het hoogs benodigde subtipe C Suid-Afrikaanse volgordes van
membraangene geproduseer. Die volgordes kan as basis dien om die gene te
modifiseer sodat dit gebruik kan word as immunogene in 'n entstof vir Suid-Afrika.
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Violence against women : impact on reproductive health and pregnancy outcomeSchoeman, Jeanne 03 1900 (has links)
Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Introduction
Worldwide, up to 25% of women are assaulted during pregnancy, with estimates
varying between populations. Violence has been associated with adverse
pregnancy outcome, including preterm birth, abruptio placentae and low birth
weight. Among the Coloured population of the Western Cape the incidence of
spontaneous preterm birth is 20%, compared to the global figure of 10%.
Overall, the rate of preterm labour has not dropped over the past 40 years and
no clearer answer as to a specific cause has been found.
The objective of this study was to determine whether patients who deliver
preterm experience more domestic violence than those who deliver at term.
Methods
Two groups of patients were assessed. Firstly, patients who spontaneously
delivered between 24 and 33 weeks (24wOd - 33w6d), who were admitted for
suppression of active labour after 24 weeks, or who experienced placental
abruption before 34 weeks, were screened for domestic violence using the
"Abuse Assessment Screen". A second group of women, attending a local
Midwife Obstetric Unit with uncomplicated pregnancies, completed the same
questionnaire. The questionnaires were all administered by the same person
(J.S.) after written informed consent was given.
Results
A total of 229 patients were interviewed, 99 in the low risk (LR) and 130 in the
preterm labour (PTL) group, which included 23 women with abruptio placentae.
The PTL group experienced significantly more violence throughout their lives
than the LR group (59.7% vs. 40.4%, p = 0.038). Experiences of violence within the last year or during the pregnancy did not reach statistical significance
between the two groups, although the numbers were higher for the PTL group.
The PTL group smoked significantly more cigarettes per day (p = 0.009), used
more alcohol (p < 0.001) and had a higher incidence of syphilis than the LR
group (p = 0.005). These differences remained the same when the abruptio's
were analyzed as a separate group.
Conclusions: Women who delivered preterm did experience more violence at
some point in their lives and were also more likely to engage in high-risk
behaviour. Violence alone does not seem to cause PTL directly, but is part of a
low socioeconomic lifestyle. The fact that the alcohol use is so high among these
women is a problem that needs to be addressed, but once again, it is possibly
the result of deeper social problems. The need for education on values and
respect, family planning use and low risk sexual behaviour is once again
challenged. / AFRIKAANSE OPSOMMING: GEWELD TEEN VROUE -IMPAK OP REPRODUKTIEWE GESONDHEID EN
UITKOMS VAN SWANGERSKAP
Inleiding
Daar word beraam dat tot 25% van alle swanger vroue aangerand word, maar
die insidensie wissel tussen verskillende populasies. Ervarings van geweld kan
'n direkte of indirekte oorsaak wees van swak verloskundige uitkoms wat
voortydse kraam, abruptio placentae en lae geboortegewig insluit. In die Wes-
Kaap, onder die Kleurlingbevolking, is die insidensie van voortydse kraam 20%,
wat swak vergelyk met die wêreldwye insidensie van 10%. Gedurende die laaste
40 jaar het die voorkoms van voortydse kraam nie verminder nie en geen
deurbrake is gemaak t.o.v die oorsaak van die probleem nie. Die doel van
hierdie studie was om te bepaal of vroue wat prematuur verlos moontlik meer
geweld ervaar as vroue wat op normale swangerskapsduur verlos.
Metodes
Twee groepe vroue is bestudeer. Die eerste groep het vroue ingesluit wat
spontaan verlos het tussen 24 en 33 weke (24wOd - 33w6d) of vroue wat na 24
weke swangerskapsduur toegelaat is vir onderdrukking van kraam. Vroue met
plasentale loslating (abruptio placentae) voor 34 weke, sonder onderliggende
hipertensiewe toestande, was ook ingesluit in die groep. Daar is m.b.v. 'n
vraelys ("Abuse Assessment Screen") bepaal watter van die vroue gesinsgeweld
ervaar het. Die tweede groep het vroue ingesluit met ongekompliseerde
swangerskappe en wat by 'n nabygeleë kliniek voorgeboortesorg ontvang het.
Hulle is ook gevra om die vraelys te voltooi en is opgevolg om die uitkoms van
hulle swangerskappe te noteer. Die vraelyste is almal deur een persoon (J.S.)
aan die vroue voorgelê nadat hulle ingeligte, skriftelike toestemming gegee het. Resultate
'n Totaal van 229 vroue was ingesluit, 99 in die lae risiko (LR) groep en 130 in
die voortydse kraam (VK) groep, waarvan 23 abruptio placentae gehad het. In
vergelyking met die LR groep, het die VK groep het betekenisvol meer geweld in
hulle leeftyd ervaar (59.7% teenoor 40.4%, p = 0.038). Geweld wat tydens die
afgelope jaar of tydens die swangerskap ervaar is, het nie betekenisvol verskil
tussen die twee groepe nie, alhoewel die getalle hoër was vir die VK groep. Die
VK groep het betekenisvol meer sigarette per dag gerook (p = 0.009), meer
alkohol gebruik (p < 0.001) en het 'n hoër insidensie van sifilis gehad as die LR
groep (p = 0.005). Hierdie verskille was steeds beduidend nadat dié met
abruptio placentae as 'n aparte groep geanaliseer is.
Gevolgtrekking
Die vroue wat prematuur verlos het, het meer emosionele en fisiese geweld in
hulle leeftyd ervaar en is meer geneig om 'n ongesonde leefstyl te handhaaf.
Geweld blyk nie 'n direkte oorsaak van voortydse kraam te wees nie, maar gaan
gepaard met 'n lae sosio-ekonomiese lewensstyl. Die hoë insidensie van
alkoholgebruik onder swanger vroue is 'n probleem wat aangespreek moet word,
maar dit is waarskynlik die manifestasie van dieper emosionele probleme.
Opvoeding in terme van waardes en respek, gesinsbeplanning en veilige
seksuele gedrag is gevolglik 'n noodsaaklikheid.
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Gender selection: separation techniques for X- and Y-chromosome bearing human spermatozoaVan Der Linde, Michelle 12 1900 (has links)
Thesis (MScMedSc)-- Stellenbosch University, 2013. / ENGLISH ABSTRACT: Preconceptual sex selection is an ethically justifiable process whereby X- and Y-chromosome bearing spermatozoa are isolated prior to fertilization of the oocyte in order to generate either a male or a female offspring. Although various separation techniques are available, none can guarantee 100% accuracy. There are various physiological differences between X- and Y-chromosome bearing spermatozoa which can be used to separate these two populations of sperm.
For the purpose of this study, X- and Y-chromosome bearing spermatozoa were separated based on (1) their respective abilities to remain viable when subjected to adverse environments, including extreme pH values, increased temperatures and various hydrogen peroxide (H2O2) concentrations; (2) the ability of Y-chromosome bearing spermatozoa to swim faster and/or more progressively than X-chromosome bearing spermatozoa; and (3) the X-chromosome bearing spermatozoa’s increased size and weight when compared to the Y-chromosome bearing spermatozoa.
The efficacy of live and dead cell separation through (i) Magnetic Antibody Cell Separation (MACS) and (ii) a modified swim-up technique was also assessed and compared. Changes in the sex-chromosome ratio of samples were established by double-label fluorescent in situ hybridization (FISH) before and after processing. Sperm motility (CASA) and viability (eosin/nigrosin) was assessed before and after each intervention. Ethical clearance for this study was granted by the Health Research Ethics Committee 1 (Ethics #: S13/04/068).
The results indicated successful enrichment of X-chromosome bearing spermatozoa upon incubation in acidic media, increased temperatures, and H2O2. In contrast, Y-chromosome bearing spermatozoa were successfully enriched through a direct swim-up method as well as discontinuous gradient centrifugation.
In conclusion, this study demonstrated the potential role for physiological differences between X- and Y-chromosome bearing spermatozoa in the development of preconceptual gender selection through sperm sorting. / AFRIKAANSE OPSOMMING: Prekonsepsie geslagselektering is 'n eties regverdigbare proses waardeur X- en Y- chromosoom draende spermatosoë geïsoleer word voordat bevrugting van die oösiet plaasvind, om óf 'n manlike óf 'n vroulike nageslag te genereer. Alhoewel verskeie skeidingstegnieke beskikbaar is, kan geeneen 100% akkuraatheid waarborg nie. Daar bestaan verskeie fisiologiese verskille tussen X- en Y- chromosoom draende spermatosoë wat skeiding van hierdie twee groepe spermatosoë moontlik kan maak.
Vir die doel van hierdie studie is skeidingsmetodes vir die X- en Y- chromosoom draede spermatosoë gebaseer op (1) hul onderskeie vermoëns om lewensvatbaar te bly tydens blootstelling aan ‘n ongunstige milieu, insluitend ekstreme pH waardes, verhoogde temperature en verskeie waterstofperoksied (H2O2) konsentrasies; (2) die vermoë van die Y-chromosoom draende spermatosoon om vinniger en/of meer progressief as X-chromosoom draende spermatosoë te swem; en (3 ) die X-chromosoom draende spermatosoon se verhoogde grootte en gewig in vergelyking met die Y- chromosoom draende spermatosoon.
Die effektiwiteit van die (i) Magnetiese Anti-liggaam Sel Skeidingstegniek (MACS) en (ii) 'n aangepaste weergawe van die op-swem tegniek om lewendige en dooie selle te skei is ook bepaal en vergelyk. Veranderinge in die geslagschromosoom verhouding van die monsters is bepaal deur dubbel-etiket fluoresensie in situ hibridisering (FISH) voor en na verwerking. Spermmotiliteit (CASA) en lewensvatbaarheid (eosien/nigrosin) is bepaal voor en na elke intervensie. Etiese goedkeuring vir hierdie studie is verleen deur die Gesondheids-Navorsingsetiekkomitee 1 (Etiese # : S13/04/068).
Die resultate dui suksesvolle verryking van X-chromosoom draende spermatosoë deur inkubasie in suur media, verhoogde temperature, en H2O2. Y-chromosoom draende spermatosoë is verryk deur middel van 'n direkte op-swem metode sowel as diskontinue gradiënt sentrifugering .
Ten slotte, hierdie studie toon die potensiële rol vir fisiologiese verskille tussen X- en Y- chromosoom draende spermatosoë in die ontwikkeling van prekonsepsie geslagselektering metodes deur skeiding van X- en Y-chromosoom draende sperme.
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An investigation into the scope of practice of a registered critical care nurse in a private hospitalBell, Janet 10 1900 (has links)
Thesis (MCur)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: The critical care nurse works in an environment where patient need often
shifts the parameters within which she or he practices. It is expected of a
skilled critical care nurse to be able to make independent decisions and take
action regarding patient care based on her or his knowledge and skills without
discounting the parameters of her or his scope of practice. Practice
experience has indicated that the critical care nurse is often uncertain about
whether her or his clinical activities are protected by the regulations provided
by the Nursing Council. This is more specifically true in the private hospital
industry where medical advice or assistance is not always easily available.
This situation led to the following research question:
Do the available professional and legal guidelines provide an appropriate
foundation to guide the practice of the registered critical care nurse in the
private hospital sector critical care environment?
A non-experimental descriptive study with a qualitative orientation was
conducted in 19 private hospitals in the Western Cape. Through nonprobability,
random sampling, 71 registered critical care nurses were included
in the study. A questionnaire was designed and validated to collect the data.
Quantitative data was analysed through Excel® while qualitative data was
analysed thematically.
It was found that the legal and professional guidelines in place at present do
provide a foundation for the clinical activities of critical care nursing in the
private hospital sector. It is suggested that it is rather the critical care nurses’
interpretation of the Scope of Practice (No.R.2598 of 30/11/1984 as amended)
that limits their practice as opposed to the wording of the regulations.
It is recommended that critical care nurses must determine nursing care
parameters based on patient need, using the regulations as a foundation for
critical, analytical and reflective practice rather than as a set of rules to be
followed.
Key words: Scope of practice, critical care practice, ICU nursing care, private
hospital nursing practice. / AFRIKAANSE OPSOMMING: Die kritiekesorgverpleegkundige werk in ‘n omgewing waar pasiëntebehoeftes
gereeld die parameters waarin sy of hy praktiseer, verskuif. Dit word van ’n
bekwame kritiekesorgverpleegkundige verwag dat sy of hy onafhanklike
besluite en aksies met betrekking tot pasiëntesorg, gebaseer op haar of sy
kennis en vaardighede, sal neem sonder om die parameters van haar of sy
bestek van praktyk te oorskry. Praktykondervinding het getoon dat die
kritiekesorgverpleegkundige dikwels onseker is oor watter van haar of sy
optredes deur die Regulasies, soos deur die Raad op Verpleging
gespesifiseer word, beskerm word. Dit is nog meer spesifiek van toepassing
in die privaathospitaal-industrie waar geneeskundige advies en bystand nie
altyd maklik beskikbaar is nie. Die situasie het tot die volgende
navorsingsvraag aanleiding gegee:
Voorsien die beskikbare professionele en wetlike riglyne ’n geskikte grondslag
om die praktyk van ’n geregistreerde kritiekesorgverpleegkundige in die
privaatsektor- kritiekesorgomgewing te rig?
’n Nie-eksperimentele, beskrywende studie met ’n kwalitatiewe oriëntasie is in
19 hospitale in die Wes-Kaap onderneem. Deur nie-waarskynlikheids-,
toevallige steekproefneming is 71 geregistreerde kritiekesorgverpleegkundiges
in die studie ingesluit. ’n Vraelys is ontwerp en gevalideer
om inligting in te samel. Kwantitatiewe data is deur middel van Excel ontleed
terwyl kwalitatiewe data tematies ontleed is.
Daar is gevind dat die wetlike en professionele riglyne wat tans beskikbaar is,
‘n grondslag bied vir die kliniese aktiwiteite van kritiekesorgverpleegkundiges
in die privaathospitaal.. Dit word voorgestel dat dit die kritiekesorgverpleegkundige
se interpretasie van die Bestek van Praktyk (No.R.2598 of
30/11/1984 soos aangepas) is wat hulle praktyk beperk, eerder as die
bewoording van die regulasie self.
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The role of renin-angiotensin-aldosterone system (RAAS) genes in the development of hypertrophy in hypertrophic cardiomyopathy (HCM)Carstens, N. 03 1900 (has links)
Thesis (MScMedSc (Biomedical Sciences. Molecular Biology and Human Genetics))--University of Stellenbosch, 2009. / Hypertrophic cardiomyopathy (HCM), an inherited primary cardiac disorder mostly
caused by defective sarcomeric proteins, is considered a model for studying left
ventricular hypertrophy (LVH) in the absence of increased external loading conditions.
The disease manifests extreme variability in the degree and pattern of LVH, even in
HCM patients with the same causal mutation. The clinical phenotype of HCM can
therefore be viewed as a product of the effect of sarcomere dysfunction and of additional
genetic modifiers. Components of the renin-angiotensin-aldosterone system (RAAS) are
plausible candidate modifiers because of their effect on blood pressure and their direct
hypertrophic effect on cardiomyocytes.
The present study investigated genes encoding components of the RAAS for association
with cardiac hypertrophy traits, in 353 individuals comprised of genetically and
echocardiographically affected and unaffected family members, belonging to 22 HCM
families with HCM founder mutations by employing a multi-SNP approach with TaqMan
allelic discrimination technology. Gene-gene interaction analysis was also performed to
investigate the effect of epistasis on hypertrophy. Candidate genes for analysis included
the angiotensin II type 2 receptor (AT2 receptor), renin, renin-binding protein (RnBP), the
(pro)renin receptor, the mineralocorticoid receptor as well as genes encoding subunits of
the epithelial sodium channels (ENaC) and Na+/K+-ATPase that showed evidence for
cardiac expression.
The present study demonstrates for the first time that variations in the renin and RnBP
genes play a role in modulating hypertrophy in HCM, independent of blood pressure and
confirms the involvement of the AT2 receptor in hypertrophy in HCM. Additionally we
report an association between Na+/K+-ATPase α1- and β1-subunits as well as the ENaC
α- and β-subunits and hypertrophy. Significant evidence for epistasis was found between
renin and downstream RAAS effectors, suggesting a complex interplay between these
RAAS variants and the hypertrophic phenotype in HCM. The identification of such
modifiers for HCM may offer novel targets for hypertrophy research and ultimately antihypertrophic
therapy.
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Identification of novel ligands of WDR47, using yeast two-hybrid analysisMcGillewie, L. 12 1900 (has links)
Thesis (MScMedSc (Biomedical Sciences. Molecular Biology and Human Genetics. Medical Biochemistry))--University of Stellenbosch, 2009. / The mammalian neocortex contributes to the increasing functional complexity of the mammalian brain,
partly because of its striking organisation into distinct neuronal layers. The development of the neocortex
has been well studied because disrupted neurodevelopment results in several human diseases.
The basic principles of neocortical development have been well established for some time; however the
molecular mechanisms have only recently been identified. One major advance in our understanding of
these molecular mechanisms was the discovery of Reelin, an extracellular matrix protein that directs the
migration of neurons to their final positions in the developing neocortex.
Reelin is a large multi-domain protein that exerts its functions by binding to its ligands on the cell surface
and initiating a signal transduction cascade that ultimately results in cytoskeletal rearrangements. Several
investigations have been undertaken to elucidate the functions of each of these domains to gain a better
understanding reelin’s functions.
We have previously identified the WR40 repeat protein 47 (WDR47), a protein of unknown function, as a
novel putative ligand for the N-terminal reeler domain of reelin. To gain better understanding into the
functional significance of this interaction, the present study sought to identify novel WDR47- interacting
proteins. In order to achieve this, a cDNA encoding a polypeptide that contains the two N-terminal
domains of WDR47, i.e. the Lis homology and the C-terminal Lis homology domain (CTLH) was used as
bait in a Y2H screen of a foetal brain cDNA library. Putative WDR47 ligands were subsequently verified
using 3D in vivo co-localisation.
Results of these analyses showed that SCG10, a microtubule destabilizing protein belonging to the
stathmin family of proteins, interacted with the N-terminal of WDR47. The identification of SCG10 as a
novel WDR47 interacting protein not only sheds some light on the role and function of WDR47 but also
aids in a better understanding of the reelin pathway and cortical lamination. Moreover, the data presented
here, may also provide researchers with new avenues of research into molecular mechanisms involved in
neuronal migration disorders.
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The influence of sex chromosomes on the outcome of human embryo developmentRaja, Kimenthra 12 1900 (has links)
Thesis (MScMedSc (Obstetrics and Gynaecology))--University of Stellenbosch, 2005. / CHAPTER 1 presents comprehensive background information regarding all aspects
addressed in this thesis. Special attention was given to literature on paternal influences
on embryonic development, the role of sperm RNA, sperm chromatin and sperm
functional aspects i.e. morphology and acrosomal status and size. The experimental
design and all relevant methods used during the study as well as the material that were
used are presented in CHAPTER 2. The results of the different techniques and
evaluations are provided in CHAPTER 3. It was found that 70% of the embryos that
showed no developmental potential were Y-chromosome bearing embryos. The sperm
selection process for ICSI based on the approach of choosing the “best looking“
spermatozoon in the ejaculate seem to provide cells that can be classified as normal
based on the length width ratio set by the WHO for normal cells. The chromatin
packaging quality of the sperm correlated significantly and negatively with the
percentage normal cells in the ejaculates. CHAPTER 4 comprises of a general
discussion of the results and short summary of the major findings during the project.
The discussion section focused on the paternal influence on the embryonic
development and provided a suggestion for future research that can possibly lead to
the use of X-chromosome bearing sperm in case of severe male factor cases.
CHAPTER 5 contains the bibliographical information of the study.
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