Dendritic spines are small protrusions that receive 90% of excitatory cortical synapses and are critically important to neural function. Each dendritic spine is supported by a dynamic actin cytoskeleton that responds to internal and external cues to allow spine development, elongation, retraction and movement. Multiple proteins have roles in spinogenesis, but until now, a regulatory role for actin itself has not been established. Here, we show that, in the acute slice preparation, actin expression increases during a period of rapid spinogenesis. Furthermore, actin overexpression in organotypic hippocampal cultures leads to a significant increase in spine density on CA1 pyramidal cells. Specifically, the number of filopodia (long, thin protrusions without heads) increases by 38% on secondary apical dendrites and 88% on basal dendrites and the number of elongated spines with heads increases by 162% on secondary apical dendrites and 113% on basal dendrites. Synapsin-I immunostaining demonstrated that the majority of filopodia and elongated spines are apposed by axon terminals. Additionally, we show that overexpressed actin enters both new and established spines within 24 h. These data demonstrate that neurons undertaking spinogenesis upregulate actin expression, that actin overexpression per se increases spine density, and that both new and established spines incorporate exogenous actin.

No description available.

Inflammation caused by oxidative stress (ROS) demonstrates an essential mechanism in the pathogenesis of acute pancreatitis (AP). Important sources for ROS comprise the reactive compound methylglyoxal (MGO) itself and the MGO-derived formation of advanced glycation end-products (AGEs). AGEs bind to the transmembrane receptor RAGE and activate NF-κB, and lead to the production of pro-inflammatory cytokines. MGO is detoxified by glyoxalase-I (Glo-I). The importance of Glo-I was shown in different models of inflammation and carcinogenesis. Nevertheless, the role of Glo-I and MGO in AP has not been evaluated so far. This study analyzed Glo-I in cerulein-(CN)-induced AP and determined the effects of Glo-I knockdown, overexpression and pharmacological modulation. Methods: AP was induced in C57BL6/J mice by i.p. injection of CN. Glo-I was analyzed in explanted pancreata by Western Blot, qRT-PCR and immunohistochemistry. AR42J cells were differentiated by dexamethasone and stimulated with 100 nM of CN. Cells were simultaneously treated with ethyl pyruvate (EP) or S-p-bromobenzylglutathione-cyclopentyl-diester (BrBz), two Glo-I modulators. Knockdown and overexpression of Glo-I was achieved by transient transfection with Glo-I siRNA and pEGFP-N1-Glo-I-Vector. Amylase secretion, TNF-α production (ELISA) and expression of Glo-I, RAGE and NF-κB were measured. Results: Glo-I was significantly upregulated on protein and mRNA levels in CN-treated mice and AR42J cells. Dexamethasone-induced differentiation of AR42J cells increased the expression of Glo-I and RAGE. Treatment of AR42J cells with CN and EP or BrBz resulted in a significant reduction of CN-induced amylase secretion, NF-κB, RAGE and TNF-α. Overexpression of Glo-I led to a significant reduction of CN-induced amylase levels, NF-κB expression and TNF-α, whereas Glo-I knockdown revealed only slight alterations. Measurements of specific Glo-I activity and MGO levels indicated a complex regulation in the model of CN-induced AP. Conclusion: Glo-I is overexpressed in a model of CN-induced AP. Pharmacological modulation and overexpression of Glo-I reduced amylase secretion and the release of pro-inflammatory cytokines in AP in vitro. Targeting Glo-I in AP seems to be an interesting approach for future in vivo studies of AP.

No description available.

Brucella abortus (BA) are gram-negative, facultative intracellular bacteria that cause abortions in cattle and debilitating illness in humans. The US is now virtually free of bovine brucellosis, but the disease is endemic in wildlife. The official brucellosis vaccine in the US is strain RB51 (RB51). It elicits protective cell-mediated immunity (CMI) against BA infections. Mycobacterium avium subspecies paratuberculosis (MAP) causes paratuberculosis in ruminants. It is a slow growing intracellular parasite that requires CMI for its control, belongs to the genus Mycobacterium, and is closely related to M. avium avium (MA). Using RB51 as a vector that induces strong protective CMI may be useful to protect against MAP if it expresses MAP protective antigens. Therefore, MAP 85A and 35kDa proteins were expressed at low levels in RB51, and the immune responses elicited by these vaccines in BALB/c mice were evaluated. Strong anti-Brucella immunity was generated, but the anti-mycobacterial response was low. To evaluate protective efficacy, a BALB/c model using MA was developed. When mice were challenged with MA, protection was obtained in some experiments but was inconsistent. This may be due to the low expression of MAP antigens in RB51. Another objective was to evaluate the effect of an ongoing Brucella-infection on the efficacy of RB51 vaccination, and whether vaccination of already infected animals could have a curative effect. Mice acutely or chronically infected with virulent BA, rapidly cleared the RB51 vaccine organisms, but there was no significant decrease in the number of virulent BA. Brucella spp. have been developed as biological weapons, but there are no vaccines to protect humans. The development of a very attenuated protective vaccine is necessary to prevent human infections, as well as to protect wildlife. To generate such a vaccine, RB51 based vaccines were irradiated to render them non-replicative, but metabolically active. We demonstrated that in general, irradiated and non-irradiated RB51 vaccines remain protective at levels similar to those elicited by the live vaccines. Therefore, irradiation of strain RB51 is an effective means of attenuating the strain without affecting its protective characteristics, and could eventually be used as a vaccine for wildlife and humans. / Ph. D.

Although androgens are known to exert anabolic effects in skeletal muscle, overexpression of androgen receptor (AR) selectively in this tissue causes androgen dependent motor deficits and muscular atrophy. The cellular and subcellular changes underlying this phenotype are unknown. Therefore, this study aimed to elucidate the ultrastructural and histologic changes accompanying myopathy and to determine the importance of androgens and overexpression level for myopathic features. Transmission electron microscopy revealed augmented mitochondrial content and reduced myofibril width in androgen exposed transgenics. Additionally, male transgenics demonstrated increased glycogen content. Histochemical analyses confirmed sex-specific changes in glycogen content and revealed a surprising loss in the proportion of oxidative fibers in symptomatic animals. However, increased mitochondrial content was confirmed by the presence of ragged red fibers. Overexpression of AR in muscle fiber results in mitochondrial pathology and dysregulation of glycogen metabolism, possibly reflecting normal but exaggerated function of androgens in skeletal muscle fibers.

Although androgens are known to exert anabolic effects in skeletal muscle, overexpression of androgen receptor (AR) selectively in this tissue causes androgen dependent motor deficits and muscular atrophy. The cellular and subcellular changes underlying this phenotype are unknown. Therefore, this study aimed to elucidate the ultrastructural and histologic changes accompanying myopathy and to determine the importance of androgens and overexpression level for myopathic features. Transmission electron microscopy revealed augmented mitochondrial content and reduced myofibril width in androgen exposed transgenics. Additionally, male transgenics demonstrated increased glycogen content. Histochemical analyses confirmed sex-specific changes in glycogen content and revealed a surprising loss in the proportion of oxidative fibers in symptomatic animals. However, increased mitochondrial content was confirmed by the presence of ragged red fibers. Overexpression of AR in muscle fiber results in mitochondrial pathology and dysregulation of glycogen metabolism, possibly reflecting normal but exaggerated function of androgens in skeletal muscle fibers.

No description available.

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Biotechnology is an important tool that is used to isolate and characterise genes. It is also used to produce clones that are genetically and phenotypically similar. Many Arabidopsis thaliana transcription factors have been isolated and characterised, but many have yet to be fully described. MYB proteins are members of a super-family of multifunctional transcription factors that can also interact with other transcription factors in the control of pathways. To date, more than 126 AtMYBs have been identified, but most have not been fully characterised, particularly in terms of the molecular role(s) they play in plants. Arabidopsis thaliana MYB3, MYB6, MYB7, MYB8 and MYB32 have been reported to be negative regulators of general phenylpropanoid metabolism. It has been reported that the five transcription factors mentioned above are likely to negatively regulate flavonoid biosynthesis, even though they may have different target genes. Studies on AtMYB13, AtMYB14 and AtMYB15 reported that they are likely regulators of general phenylpropanoid metabolism. The mentioned roles of the eight AtMYB transcription factors means that they can be manipulated in order to see what effect they have on primary and secondary metabolites in plants. The transcription factors ligated into the pUBI510-GRFCA vector were then used to transform sugarcane callus (Chapter 3). Sugarcane produces sucrose which makes up 70% of the sugar produced in the world, making sugarcane a commercially important and profitable plant. The sugarcane callus was transformed via particle bombardment. The transcription factors AtMYB3, AtMYB6, AtMYB7, AtMYB13 and AtMYB32 were successfully incorporated into the genomic DNA of the sugarcane callus. The data obtained for callus over-expressing AtMYB3, AtMYB13 and AtMYB32 on solid media and the callus in liquid media were contradictory (i.e callus on solid media producing more sucrose than the wildtype whereas the same transgenic line will poduce less sucrose that the wildtype in liquid media or vice versa). However, AtMYB13 transgenic lines produced more sucrose than the wildtype. Transgenic lines of AtMYB7 all produced less sucrose as compared to the wildtype both on solid and in liquid media. The transcription factors which resulted in increased production of starch when over-expressed were AtMYB7 and AtMYB13. The data obtained for AtMYB6 transgenic lines was highly inconsistent in lines grown on same media and across the two media. The effects of these transcription factors in the overall metabolism of the sugarcane callus, either on MSC3 solid or liquid media, could not be fully determined from the GC-MS analysis as there was no consistent phenotypic effect between different transgenic lines for any of the MYB transcription factors used. In Chapter 4, a micropropagation strategy was developed and phytochemicals and their biological activities were determined for the medicinal plant Salvia repens. Salvia plants have been found to be medicinally important due to the secondary metabolites, particularly the essential oils that they produce. The plant extracts have been found to have many biological activities such as antibacterial, anti-inflammatory, antioxidant and anticancer activities. Salvia repens was successfully germinated in vitro,with 60% germination being achieved in MS media containing 1x10-5 times diluted smoke water following scarification for 12 min in 75% (v/v) H2SO4. Success rates of 100% were achieved in the hardening off process when the seedlings were moved into the greenhouse. Germination of S. repens ex vitro was 100% in an autoclaved soil mixture of 1:1 (v/v) sand and vermiculite. Importantly the medicinal value of S. repens produced in vitro or ex vitro was not lost as the GC-MS metabolite analysis showed that the plants produced the chemicals that are medicinally important. Metabolite extracts of S. repens were for the first time reported to be active against fungi with MIC values lower than 1 mg/ml over 4-5 d period against four Fusarium spp. tested. Lastly (Chapter 5), transcription factors AtMYB6 and AtMYB13 were used to trasnform Salvia stenophylla via Agrobacterium-mediated transformation, in order to determine whether the over-expression of these transcription factors could up-regulate the production of medicinally and commercially important secondary metabolites in S. stenophylla. Whilst both A. tumefaciens and A. rhizogenes strains were utilised for the transformation procedure, transformation was only achieved using A. rhizogenes and no transformants could be generated from the A. tumefaciens-treated material. Transgenic hairy roots did not produce any of the medicinally important metabolites. The GC-MS analysis of the transgenic root material identified mainly sugars and other primary metabolites. / AFRIKAANSE OPSOMMING: Biotegnologie is 'n belangrike instrument wat gebruik kan word om gene te isoleer en te karakteriseer. Dit word ook gebruik om klone wat geneties en fenotipies identies is te produseer. Baie Arabidopsis thaliana transkripsiefaktore is al geïsoleer en gekarakteriseer, maar baie moet nog volledig beskryf word. MYB proteïene is lede van 'n super-familie van multifunksionele transkripsiefaktore wat ook interaksie het met ander transkripsiefaktore tydens die beheer van metaboliese weë. Tot op hede is meer as 126 AtMYBs geïdentifiseer, maar die meeste is nie volledig gekarakteriseer nie, veral nie ten opsigtigte van die molekulêre rol(le) wat hulle in plante speel nie. Arabidopsis thaliana MYB3, MYB6, MYB7, MYB8 en MYB32 is gevind om negatiewe reguleerders van algemene fenielpropanoied-metabolisme te wees. Daar is ook berig dat dié vyf transkripsiefaktore moontlik flavenoied-biosintese negatief kan reguleer, selfs al kan hulle verskillende teikengene hê. Studies op AtMYB13, AtMYB14 en AtMYB15 het berig dat hulle waarskynlik reguleerders van algemene fenielpropanoied-metabolisme is. Die genoemde rolle van die agt AtMYB transkripsiefaktore beteken dat hulle gemanipuleer kan word om te bepaal watter effek hulle op primêre en sekondêre metaboliete in plante het. Die transkripsiefaktore, wat in die pUBI510-GRFCA vektor geligeer was, is toe gebruik om suikerriet-kallus te transformeer (Hoofstuk 3). Suikerriet vervaardig sukrose wat tot 70% van die suiker wat in die wêreld geproduseer word opmaak. Dít maak suikerriet 'n kommersieel belangrike en winsgewende plant. Die suikerriet-kallus is getransformeer deur middel van partikel-bombardering. Die transkripsiefaktore AtMYB3, AtMYB6, AtMYB7, AtMYB13 en AtMYB32 was suksesvol in die DNA van die suikerriet-kallus opgeneem. Data wat verkry was vir kallus wat AtMYB3, AtMYB13 en AtMYB32 ooruitgedruk het op soliede media en kallus in vloeibare medium was teenstrydig (m.a.w. kallus op soilede media wat meer sukrose as die wildetipe op soliede media geproduseer het, terwyl dieselfde transgeniese lyn minder sukrose as die wildetipe geproduseer het in vloeibare medium, en anders om). Nietemin, het AtMYB13 transgeniese lyne meer sukrose geproduseer as die wildetipe. Transgeniese lyne van AtMYB7 het almal minder sukrose geproduseer as die wildetipem op beide soliede en vloiebare media. Die transkriopsiefaktore wat gelei het tot 'n styging in stysel produksie wanneer hulle ooruitgedruk was was AtMYB7 en AtMYB13. Data wat verkry is van die AtMYB6 transgeniese lyne was hoogs veranderlik in lyne wat op dieselfde medium gegroie was en oor die twee media. Die effek van hierdie transkripsiefaktore op die algehele metabolisme van die suikerriet-kallus, hetsy op MSC3 soliede of vloeibaremedia, kon egter nie van die GC-MS analise ten volle bepaal word aangesien daar geen konsekwente fenotipiese effek tussen die verskillende transgeniese lyne vir enige van die gebruikte MYB transkripsiefaktore was nie. In Hoofstuk 4 was ‘n mikropropagerings strategie ontwikkel. Fitochemikalieë en hul biologiese aktiwiteite was ook bepaal vir die medisinale plant Salvia repens. Salvia plante is gevind om medisinaal belangrik te wees as gevolg van die sekondêre metaboliete, veral die essensiële olies, wat hulle produseer. Dit is ook bevind dat die plant-ekstrakte baie biologiese aktiwiteite soos anti-bakteriese, anti-inflammatoriese, anti-oksidant en anti-kanker aktiwiteite het. Salvia repens is suksesvol ontkiem in vitro, met 60% ontkieming wat bereik is in MS media met 1x10-5 maal verdunde rook-water na insnyding vir 12 min in 75% (v/v) H2SO4. Suksessyfers van 100% was behaal in die afhardingsproses wanneer die saailinge na die glashuis verskuif was. Ontkieming van S. repens ex vitro was 100% in 'n geoutoklaveerde grondmengsel van 1:1 (v/v) sand en vermikuliet. Gewigtig het die medisinale waarde van S. repens wat in vitro of ex vitro geproduseer was nie verlore gegaan nie. Die GC-MS data metaboliete analise het aangetoon dat die plante die medisinaal belangrike chemikalieë geproduseer het. Metaboliet-ekstrakte van S. repens was vir die eerste keer na berig aktief teen swamme, met MIK waardes laer as 1mg/ml oor ‘n tydperk van 4-5 d, teen vier Fusarium spp wat getoets was. Laastens (Hoofstuk 5), transkripsiefaktore AtMYB6 en AtMYB13 was gebruik om Salvia stenophylla te transformeer deur Agrobacterium-bemiddelde transformasie, om sodoende te bepaal of die ooruitdrukking van hierdie transkripsiefaktore die produksie van medisinale en kommersieël-belangrike sekondêre metaboliete in S. stenophylla kan verhoog. Alhoewel beide A. tumefaciens en A. rhizogenes stamme gebruik was vir die transformasie proses, kon transformasie slegs deur die gebruik van A. rhizogenes bereik word. Geen transformante kon gegenereer word vanuit die A. tumefaciens behandelde materiaal nie. Transgeniese harigewortels het geen van die medisinaal belangrike metaboliete vervaardig nie. Die GC-MS analise van die transgeniese wortel materiaal het hoofsaaklik suikers en ander primêre metaboliete geïdentifiseer.

Areas of hypoxia caused by poor perfusion are a common occurrence in breast cancer. Hypoxia-inducible factors-1 and 2 (HIF1/2) drive the cellular response to hypoxia in such areas, resulting in the upregulation of genes which facilitate the survival of cancer cells and promote growth, invasion, metastasis and angiogenesis, generally leading to more aggressive tumour characteristics. Previous research has demonstrated that growth factor signalling, such as the ligand-mediated activation of HER receptors, can promote the action of HIFs in normoxia, and correlation between HER2 expression and HIFα proteins has been demonstrated in clinical samples of breast cancer. Despite this, little research has been conducted on how the growth factor-driven regulation of HIFα subunits might modify the cellular response to hypoxia. In this thesis, the role of HER2 overexpression in HIFα modulation was assessed in breast cancer cell lines and publically available clinical datasets for breast cancer with the aim of further understanding the implications of hypoxia and HIFα expression in the context of HER2-positive breast cancer. The upregulation of HIF1α and HIF2α by hypoxia was observed across breast cancer cell lines, and the role of HER2 in this process was assessed using an isogenic MCF7 cell line model overexpressing HER2. This demonstrated an increased hypoxic upregulation of HIF2α but not HIF1α when HER2 was overexpressed. The increased upregulation was shown to be facilitated by an increase in normoxic HIF2α, which is driven by a higher transcriptional rate of the EPAS1 (HIF2) gene as a direct result of HER2 overexpression. HER2 overexpression also resulted in the increased hypoxic upregulation of known hypoxia response genes in 2D and 3D culture models. This demonstrates a novel mechanism for growth-factor mediated HIFα regulation in the context of HER2 overexpression, with an important role for HIF2α. Microarray analysis of MCF7 and MCF7-HER2 cells was used to compare the global transcriptional response to acute (24 hrs) and chronic (>10 weeks) hypoxia (0.5% O2) and demonstrated a broadly increased upregulation of hypoxic response genes in the HER2 overexpressing cell line when compared to wild-type MCF7. This included an increase in previously described HIF1 and HIF2 target genes. MCF7-HER2 also illustrated an increased expression of hypoxia response genes in normoxia, and an analysis of the genes involved showed the promotion of a number of pathological processes including proliferation, invasion, angiogenesis and epithelial to mesenchymal transition. Large-scale, publically available expression datasets for breast cancer cell lines and clinical patient data were used to investigate the expression of HIF2α and hypoxia response genes in relation to HER2 expression. A set of pathologically important genes which were primed for hypoxia in MCF7-HER2 were also demonstrated to correlate with HER2 across breast cancer cell lines, suggesting that HER2 may more broadly promote a readiness to respond to hypoxia in breast cancer cells. Assessment of HIF2α in clinical samples has shown its increased expression in the HER2-positive subtype, and HIF2α was shown to be associated with worse disease-specific survival in the context of HER2-positive samples only. To investigate whether HIF2α is a potential target in HER2 overexpressing breast cancer, the effect of HIF2α inhibition through siRNA or HIF2-specific chemical inhibitors was assessed in cell lines with high or low HER2 expression, and this demonstrated an increased sensitivity of HER2 overexpressing cell lines to HIF2α inhibition. This work highlighted HER2 as an important modulator of the cellular response to hypoxia in breast cancer, demonstrating a previously overlooked role for HIF2α in this process. HIF2α expression can be directly driven by HER2 and this differs mechanistically from that previously reported for HIF1α. Finally, further work into the potential for HIF2α as a target for anti-cancer therapy is suggested, as an increased sensitivity of HER2-positive cell lines to anti-HIF2α agents was shown, as well as a HER2-specific relationship between HIF2α expression and worse prognosis. More generally, this work has shown an important interplay between growth factor receptor expression and the cellular response to hypoxia, suggesting that HER2 may promote a stronger response to hypoxia in breast cancer, which may contribute to the increased aggressiveness of HER2-positive tumours.