Johnny van der Walt competition (Posters)

Testing the association between macrophage activation pathways and head circumference in HIV exposed and unexposed children born at Kalafong hospital.
Neisha Clemson1
Prof Theresa Rossouw2, Prof Peet du Toit1
Department of Physiology, University of Pretoria1, Department of Immunology, University of Pretoria2
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Aim: To investigate a possible association between HIV exposure, head circumference and macrophage activation pathways.
An increasing number of children born to HIV-infected mothers do not contract HIV. Studies show that HIV-exposed but uninfected (HEU) children are more likely to have growth and neurodevelopmental delay, immune dysfunction, higher morbidity and mortality than their unexposed counterparts. Head circumference (HC) is associated with brain mass and viewed as a good indicator of neurological development. Macrophages have two main phenotypes: classically activated macrophages, which are pro-inflammatory, and alternatively activated macrophages which are anti-inflammatory. An imbalance between phenotypes has been associated with various diseases and classically activated microglia and/or macrophages are known to exert cytotoxic effects on neurons and oligodendrocytes.
Methods: Fifty-five infants born to HIV-infected and uninfected mothers were recruited. Medical chart review and postnatal questionnaires provided data on maternal age and pregnancy outcomes. Macrophage activation markers (CD14, CD16, CCR2) were tested at birth and ten weeks using flow cytometry. HC was measured at birth and ten weeks and was categorised according to percentiles and z-scores. Data analysis was done in STATA at 5% significance.
Results: Thirty-three mothers were HIV infected, of those, twenty-nine were on ART. The median CD14 count for this group was 9.18 with nineteen individuals having undetectable viral load. Only one infant was HIV-infected. There was no difference between exposed and unexposed infants in terms of gestation duration, weight or length. HC was lower in HEU infants (p=0.0054) and associated with weight (p=0.006); mid-upper-arm circumference (p=0.0086) and abdominal circumference (p=0.0081) at birth. The simultaneous expression of CD14 and CCR2 was higher in HEU at birth (p=0.0005) whereas CD16 was lower at ten weeks (p=0.028). CD16 at ten weeks was lower in infants with a HC

Restoring the function of inactivating mutations of G protein-coupled receptors
Lorenzo Fenandes1
Ross Anderson2, Claire Newton2, Robert Millar1
Department of Physiology, University of Pretoria1, Department of Immunology, University of Pretoria2
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Introduction: The neuroendocrine system is a complex network of intercommunicating signal transduction cascades, that together with G protein-coupled receptors (GPCRs), are responsible for many physiological processes. Inactivating mutations of GPCRs often cause receptor proteins to become misfolded during synthesis. Misfolding is detected by the cell's quality control system, which prevents trafficking of misfolded receptors to the cell surface and targets them for degradation. The decrease in cell surface expression of GPCRs is therefore a cause of neuroendocrine dysfunction. Restoration of membrane trafficking of intracellularly retained GPCRs has been shown to be achievable using pharmacological chaperones (PCs). PCs are cell-permeant molecules which interact with misfolded proteins, promoting their correct folding and enhancing their trafficking to the cell membrane, thus “rescuing” their function. The aim of this study was to determine whether cell membrane trafficking and function of intracellularly retained mutant neuroendocrine GPCRs implicated in reproductive dysfunction, can be restored by conferring cell-permeability to endogenous peptide ligands, using cell-penetrating peptides (CPPs).
Methods:  The receptors identified through literature review for the study were the luteinising hormone receptor (LHR), the gonadotropin-releasing hormone receptor (GnRHR) and the kisspeptin receptor (GPR54). Mammalian cells were transfected with mutant receptor plasmids. An ELISA was used to quantify receptor cell surface expression in the presence of putative PCs or CPP-hormone treatment. A CRE-luciferase assay was used to measure signalling of the mutant GPCRs, to examine whether the putative PC/CPP-hormone treatments restored signalling competency of retained mutant receptors.
Results:  Several receptors were identified and cloned into expression vectors. A β-Galactosidase positive control was used to optimise the CPP delivery proteins into cells. Receptor ELISA assays confirmed poor cell surface expression of many LHR mutants. Initial experiments with CPP-hCG indicated desensitisation of the receptor upon prolonged exposure, therefore, optimisation of the cell treatment protocol was undertaken.
Conclusion: Further cellular signalling assays will be performed to determine the functionality of CPP-hormone treated mutants and these studies will be extended to include the other receptors. If successful, this proof-of-principle study could be applied to many mutant GPCRs, providing a potential novel approach to development of therapeutics targeting diseases caused by retained GPCR mutants.

Value of total nucleated- and CD34+ cell counts as predictors of engraftment in multiple myeloma patients: A pilot study
Simone Grobbelaar1
Durandt C2, Pepper MS2, Verwey M1, Becker P3, Gerdener T4,Mc Donald A4,Brittain D4, Mercier AE1
Department of Physiology, Faculty of Health Sciences, University of Pretoria1
Institute for Cellular and Molecular Medicine, Department of Immunology/ SAMRC Extramural Unit for Stem Cell Research and Therapy, University of Pretoria2
Research Office Biostatistics, Faculty of Health Sciences, University of Pretoria3
The Alberts Cellular Therepy Centre, Netcare Pretoria East Hospital, Pretoria4
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Introduction:  Haematopoietic stem cell transplantation (HSCT) is routinely used to treat patients diagnosed with multiple myeloma (MM), a haematological malignancy that is prevalent in South Africa. A key objective for successful HSCT is to obtain adequate engraftment. HSCs are mobilized from the bone marrow using human granulocyte colony stimulating factor (G-CSF) and are identified and enumerated using the cluster of differentiation (CD) 34 antigen. CD34+ and total nucleated cell (TNC) counts obtained from apheresis collections prior to engraftment are currently used to predict engraftment success. However, other clinical and laboratory data collected in the course of routine management have the potential to predict engraftment success. In a collaboration with the Alberts Cellular Therapy (ACT) center, this study aims to determine whether clinical and laboratory parameters generated throughout the harvesting and transplant time-line might be correlated with engraftment success in a South African patient cohort.
Methods: Epidemiological and clinical data obtained from 30 patients that underwent autologous HSCT at the ACT center during 2016 were captured. M-protein type, mobilization strategies and conditioning regiments were recorded. In addition, laboratory generated parameters not routinely evaluated in the context of engraftment success were examined, and the archived apheresis flow cytometric data re-analyzed. Flow cytometric analysis included CD45+ counts, lymphocyte counts, monocyte counts, neutrophil counts, % CD34+ HSCs smaller in size, CD34dim-expressing HSCs at various time points including at pre-harvest, mid-harvest, end-harvest and post thaw. Multiparametric statistical analysis will determine if there is a significant correlation between the captured data and engraftment success, as determined by neutrophil and platelet counts post-transplant.
Results: Data from this pilot study will be presented at this conference, and correlations between epidemiological, clinical and laboratory parameters and HSC engraftment success will be discussed. Besides CD34+ cell count, no other relevant parameters have been identified thus far.
Conclusion: Data obtained from this pilot study will assist us in the design of a larger scale study on MM patients in order to substantiate any statistical trends observed. Future studies will include ex vivo CD34 epitope mapping in an attempt to standardize CD34 isolation and enumeration protocols.

The effect of chronic ingestion of Afriplex GRT on myocardial insulin resistance and mitochondrial function - a preclinical study
Marlouw Kroukamp
Prof. Barbara Huisamen
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Obesity is an abnormal or excessive fat accumulation in the body, known to be a significant common precursor to insulin resistance and type 2 diabetes. Obesity is associated with complications such as hypertension, abnormal cholesterol levels, elevated triglycerides and insulin resistance. These can contribute to the development of cardiovascular disease (CVD) which, in turn, is associated with abnormal mitochondrial energetics. Plant-based diets have been linked to a lower risk of chronic disease development, such as CVDs. Rooibos (Aspalathuslinearis) is unique to South Africa and has known antidiabetic, anti-obesity and cardiovascular benefits. We investigated these properties with a focus on mitochondrial function, using an aspalathin enriched green rooibos extract, Afriplex GRT.
Methods: Male Wistar rats were fed a high fat diet (HFD) for 16 weeks and compared to age matched controls fed normal rat chow. Half of each group were treated with Afriplex GRT (60mg/kg/day) from weeks 11 to 16. Body weight, food and water intake were monitored and intraperitoneal (IP) fat weighed at sacrifice. After sacrifice, myocardial mitochondria were isolated. Half of these were used to measure respiration using an oxygraph (Clark-type electrode). Mitochondrial lysates were prepared from the other half for Western Blot analysis of proteins involved in autophagy. Hearts were also freeze clamped and used for Western Blots to determine levels of autophagy proteins; Beclin-1, Bnip3, LC3II.
Results: The HFD caused increased weight gain compared to controls, while ingestion of Afriplex GRT lowered this weight gain. Afriplex GRT similarly lowered the increase in IP fat in the HFD rats without affecting food intake. The GRT improved insulin sensitivity in both groups as measured by OGTTs. Neither the HFD nor Afriplex GRT had any effect on mitochondrial oxphos capacity, while autophagic flux was increased by Afriplex GRT in the HFD animals.
We conclude that Afriplex GRT caused weight loss and improved insulin sensitivity in HFD treated rats and had no discernable effect on mitochondrial function.

Influence of an estradiol antimitotic compound and subsequent glutamine deprivation in breast tumourigenic and non-tumourigenic cells
Maphuti Tebogo Lebelo1
Thandi Vuyelwa Mqoco1, Anna Margaretha Joubert1, Michelle Helen Visagie1
University of Pretoria1
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Introduction: 2-Methoxyestradiol (2ME), a 17β-estradiol metabolite, exerts anticancer- and antiangiogenic activity. Due to low bioavailability, several analogues of 2ME were synthesized including 2-ethyl-12-hydroxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6-cyclopenta[a]phenanthren-3yl sulphamate (ESE-ol). Glutamine is an essential substrate for actively proliferating cells and cell survival. Thus, there is potential use in synergistic anticancer activity in glutamine deprivation in combination with antiproliferative compounds. The aim of this study was to evaluate the influence of ESE-ol exposure followed by glutamine derivation on proliferation, oxidative stress and cell survival in breast tumourigenic (MCF-7) and non-tumourigenic (MCF-10A) cell lines.
Methods: Cell growth was evaluated using crystal violet staining (spectrophotometry). Haematoxylin and eosin staining (light microscopy) was used to observe cell morphology. Effects on hydrogen peroxide generation were demonstrated by using 2,7-dichlorofluoresceindiacetate (fluorometry). Propidium iodide staining (flow cytometry) was employed to investigate cell cycle progression and apoptosis induction was detected using Annexin V (flow cytometry).
Results: Crystal violet studies demonstrated that ESE-ol treatment induced dose-dependent antiproliferative activity. Exposure to 0.2µM ESE-ol resulted in a decrease in cell growth by 32% and 68% in MCF-7 and MCF-10A cells. However, a synergistic effect was only established in the MCF-10A cells where ESE-ol exposure followed by glutamine deprivation resulted in 68% cell growth inhibition. Light microscopy demonstrated that ESE-ol exposure and combination exposure with ESE-ol/glutamine deprivation resulted in rounded- and shrunken cells. Hydrogen peroxide production increased to 2.47 fold after exposure to ESE-ol in the MCF-10A cell line. However, combination treatment with ESE-ol/glutamine deprivation resulted only in increased hydrogen peroxide production (1.47 fold) in the MCF-7 cell line when compared to ESE-ol-treated cells. Cell cycle progression studies revealed that ESE-ol exposure followed by deprivation from glutamine resulted in a significant increase in cells occupying the Sub-G1 when compared to ESE-ol-treated cells. Exposing the cells to ESE-ol and ESE-ol/glutamine deprivation resulted in a statistically insignificant increase in early apoptosis in both cell lines.
Conclusion: This study showed that glutamine deprivation following ESE-ol treatment refrained from exerting synergistic effects on the MCF-7 cell line. This study contributes towards research studies of combination exposure conditions regarding deprivation from energy substrates and antimitotic compounds in tumourigenic cells.

Identifying potential novel pharmacoperones for the rescue of cell surface expression of follicle-stimulating hormone receptor mutants
Abigail Lightbody1,2
Ross C Anderson1,3, Robert P Millar1,2, Claire L Newton1,3
Centre for Neuroendocrinology, Faculty of Health Sciences, University of Pretoria1
Department of Physiology, Faculty of Health Sciences, University of Pretoria2
Department of Immunology, Faculty of Health Sciences, University of Pretoria3
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Introduction: Follicle-stimulating hormone functions within the hypothalamic-pituitary-gonadal axis in both male and female gamete formation as well as in the secretion of sex steroid hormones. The cognate receptor responsible for its effects, the follicle-stimulating hormone receptor (FSHR), is a G protein-coupled receptor (GPCR). Mutations in GPCRs often result in misfolding of the receptor. The misfolding of FSHR is associated with several reproductive conditions such as hypergonadotropic hypogonadism, primary or secondary amenorrhea, variable development of secondary sex characteristics and infertility due to the arrest of follicular development. We hypothesized that many FSHR mutants will be intracellularly retained by the cell’s quality control system but retain their intrinsic functionality, and that cell permeant, non-peptide ligands/ pharmacoperones (PC) able to interact with these receptors will stabilise their folding, negating these effects to promote trafficking from the endoplasmic reticulum and increased cell surface expression. This study aimed to identify FSHR mutants that cause intracellular retention, and to test existing FSHR small molecule agonists and antagonists for PC activity on intracellularly retained FSHR mutants.
Methods: Suitable FSHR mutant candidates have been identified from literature, these have been reproduced in mammalian expression vectors using site-directed mutagenesis and transfected into human embryonic kidney cells. Total and cell surface expression levels of the mutants in the presence and absence of FSHR small molecules was then measured using a cell-based receptor ELISA assay.
Results: Eight suitable, naturally occurring, human mutant candidates were identified; I160T, A189V, N191I, D224V, P348R, P159T, F591S and A575V. Thereafter, expression vectors were created and verified by Sanger sequencing. The mutant receptors had varying degrees of retention and their cell surface expression was differentially affected by the small molecule ligands tested.
Conclusion: Some mutants displayed some rescue of receptor trafficking to the cell surface, indicating PC activity of the ligand tested. Functional analyses will now be performed in order to determine whether or not receptor function is also restored.

The neurophysiological defects of MCA strokes, and the possible correlation between anatomical variation or pathology and stroke location
Johan Nel1
Albert van Schoor2, Peet du Toit1, M Bosman2
Department of Physiology, Faculty of Health Sciences, University of Pretoria1
Department of Anatomy, School of Medicine, Faculty of Health Sciences, University of Pretoria2
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Introduction: Strokes are one of the leading causes of death worldwide. Depending on the lateralization and localization, the expected clinical presentation and pathophysiology will vary. The middle cerebral artery (MCA) and the supplying areas will be the area of interest. Pathophysiology of strokes are based on the imbalance of the homeostatic processes that direct normal transmission of impulses via chemical or electrical systems.
Aim: To determine the extent of pathophysiology in, and locality of, MCA strokes in a sample of 30 patients form the Steve Biko Academic Hospital Radiology department. Method: CTA scans from 30 patient diagnosed with MCA strokes were collected and analysed using two cooperating computer systems. Brodmann’s map was used for cortical regions involved and for subcortical regions other sources was consulted to determine the exact affected area and the corresponding pathophysiological implications as well as clinical presentations.
Results: The area, width, length and circumference of each stroke was mapped, and we discuss the severity of physiological functional impairment of the vital regions that were involved.
Conclusion: Understanding the anatomy of the cortical and subcortical structures of the brain and the associated arterial supply, we discuss the impact of the MCA stroke, acknowledging the importance of the laterality of the stroke along with its encompassing structures.

In vitro study of the in silico designed Sirtuin 1 Inhibitor w137 on human Neuroblastoma SH-sy5y and acute Myeloid Leukemia U937 cells
Monique Otto1
Du Toit, P1,Stander, B.A1
University of Pretoria, Department of Physiology1

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Introduction: The sirtuin 1 (SIRT1) NAD+ dependent lysine deacetylase enzyme has been identified as a promising cancer treatment target due to its influence on cancer-associated gene expression and cellular protein composition. SIRT1 mediates transcription at target loci of the C-MYC oncogene and negatively influences P53 tumour suppressor activity. Through SIRT1 inhibition, over-expression of C-MYC can potentially be prevented as well as reactivation or increased expression of P53. The first aim of the study is to determine the anti-proliferative effects of the new SIRT1 inhibitor compound on two human cancer cell lines. The second aim is to provide an understanding of the cell death inducing properties, mechanistic action and other in vitro effects of the compound on two human cancer cell lines.
Methods: The In silico-designed SIRT1 inhibitor compound W137 was tested after 24 and 48 hour exposure times on the U937 and SH-SY5Y cell lines respectfully. The concentration of the compound required to inhibit 50% of the cancer cell populations (IC50) was determined through flow cytometry, crystal violet assays and spectrophotometry. Fluorescent microscopy was applied to study cell morphology. Cell cycle analyses were done using propidium iodide staining as well as cell death studies through Annexin V and propidium iodide staining to deduce the mechanism of action of the compound.
Results:  Results obtained thus far indicate IC50 values of 25 µM for U937 and 20 µM for SH-SY5Y cell lines respectively. Fluorescent microscopy using dye staining techniques indicated an increased presence of cells undergoing early and late apoptosis after exposure. A possible sub-G1 block in the cell cycle was detected in inhibitor exposed cells and an increased percentage of cells undergoing late apoptosis.
Conclusions:  The SIRT1 inhibitor W137 appears to be effective in stimulating cell death and may serve as a potential anti-cancer agent in both the U937 and SH-SY5Y cell lines respectively. Further mechanistic studies need to be conducted to understand the efficacy as well as gene- and protein studies to determine the influence of W137 exposure on the key cancer influencing genes C-MYC, P53 and BCL-2 and the subsequent protein expression.

Cholecalciferol causes Morphological abnormalities in a Metastatic Cervical cancer cell line
Yuvelia Pather1
Annie Joubert1, Rivak Punchoo1
University of Pretoria1

Introduction:  Cervical cancer affects one in forty-one women in South Africa. Studies have shown a link between vitamin D (VD) insufficiency and increased risk for various cancers. Therapeutic intervention using VD can therefore potentially ameliorate cervical cancer. The anti-cancerous properties of an early VD precursor, cholecalciferol, was investigated on a metastatic cervical epidermoid carcinoma (CaSki) cell line. Methods:  CaSki cell experimental cultures were treated with a range of 10 -1000 ng/ml of cholecalciferol and the control cultures treated with solvent-only or medium-only. All cultures were run in triplicate on three biological repeats and incubated for 96 hours. Necrosis was detected using the lactate dehydrogenase (LDH) assay. Morphology of cells was assessed using transmission and scanning electron microscopy prepared using standard methods. Statistical analysis for the LDH assay was performed using one-way ANOVA followed by a Bonferroni post-hoc test. Data was expressed as mean +/- standard error of the mean using GraphPad Prism 5. All morphological data was analysed qualitatively for representative abnormal cell growth.
Results:  Experimental cultures generally showed morphological features consistent with normal cell growth. Features of apoptosis and autophagic cell growth were observed in a limited number of experimental cells. Experimental VD treated CaSki cells also showed increased vacuolisation and mitochondrial dilation. Morphological evidence for necrosis was absent. There was a statistically non-significant LDH cytosolic release across the VD treatment range in CaSki experimental cultures compared to control cultures.
Conclusions:  This pilot study suggests cholecalciferol causes limited morphological changes to cell growth in the experimental CaSki cultures although morphological features consistent with apoptosis, autophagy and paraptosis are present. The absence of morphological necrosis is biochemically supported by statistically non-significant LDH cytosolic release. Further biochemical characterization of cell growth and modes of death can inform potential therapeutic management of cholecalciferol treatment of metastatic cervical cancer.

The Effect of Hypoxia on Protein Phosphatase 2A (PP2A) in an Immortalised Breast Cancer Cell Line
Victoria Patten1
Dr Derick van Vuuren1, Prof Anna-Mart Engelbrecht2
Department of Biomedical Sciences, Division of Medical Physiology, Faculty of Medicine and Health Sciences, Stellenbosch University1
Department of Physiological Sciences, Faculty of Natural Sciences, Stellenbosch University2
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Introduction:Malignant tumours are characterized by areas of insufficient blood supply, with resulting hypoxia and nutrient deprivation. Unperturbed, cancerous cells adapt and thrive under these conditions. Protein phosphatase 2A (PP2A) is of potential interest as a major protein phosphatase implicated as a tumour suppressor in several forms of cancer, although current literature reports contradictory results concerning its involvement. We aimed to investigate the effect of hypoxia on PP2A in a breast cancer cell line. We hypothesised that hypoxia will induce a decrease in PP2A activity, favouring phosphorylation and activation of enzymes associated with survival and proliferation.
Methods:We used a triple negative breast cancer cell line, MDA-MB-231, cultured under standard conditions. Cells were supplemented with 1% FBS, and exposed to chronic hypoxia (0.5% oxygen) over two time course experiments (24/48/72hours and 2/4/6/8hours) in a sealed hypoxic chamber. The expression and phosphorylation of ERK p42/p44 and PKB/Akt between 24 and 72 hours hypoxia was measured using Western Blotting. Concerns that these durations were not appropriate, prompted the measure of expression, phosphorylation and methylation of the catalytic subunit of PP2A at 2, 4, 6 and 8 hours hypoxia. An ATP assay was further used to measure cell viability.
Results:ERK p42 levels were lower at 48 hours hypoxia compared to 24 hours hypoxia (Hypoxia 48hrs: 0.8806 ± SD 0.3242 Arbitrary Units (AU), vs Hypoxia 24hrs: 1.306 ± SD 0.1529 AU; p<0.05), although recovered to control levels at 72 hours. No post-translational modification of PP2A seemed to be induced, with only the methyl/total ratio decreasing after 8 hours hypoxia relative to control (Control 1.00±0.001393 AU vs. Hypoxia 0.72±0.03728 AU; p<0.05, n=2). Cell viability measured via an ATP kit. ATP levels showed a decrease at 6 hours of hypoxia relative to control (Control 0.5428±0.2211 AU vs. Hypoxia 0.1614± 0.08847 AU; p<0.05).
Conclusion:There appears to be no link or evidence of association between PP2A and the duration of hypoxia in breast cancer, except for a decrease in methylation, the significance of which is unknown. However, we plan to expand on the n-numbers of the experiments, as well as to measure PP2A activity during hypoxia.

An investigation of coagulation and inflammatory markers in Type 2 Diabetes mellitus patients
Mary-Ann Phasha1
Prashilla Soma1, Jannet Bester1, Etheresia Pretorius1
University of Pretoria1
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Background: Type 2 diabetes mellitus (T2D) is characterized by chronic hyperglycaemia and inflammation. Studies show a link between coagulopathies and T2D due to pro-inflammatory cytokine involvement in the activation of platelets. The aim of the study was to assess the expression of molecules that are involved with coagulation and inflammation inT2D subjects.
Methods: Following ethical approval, control and T2D patients’ blood samples were collected by venipuncture. Total RNA isolated from whole blood and used to prepare cDNA. Quantitative PCR was used to assess the gene expression of tissue factor, factor XII, XIII, p-selectin, prekallikrienin(PKK),T-bet, GATA3, FOXP3, RORγ and TNF-α. Relative fold change in mRNA expression was calculated using the2^(-∆∆CT). Thromboelastography (TEG) was used to assess the reaction time (R time), clotting time (K time), alpha angle (α angle) and maximum amplitude (MA).
Results: Significant variation in gene expression of FXIII was found between T2D and controls. Factor XIII was significantly altered with a mean relative fold increase of 21.37±11.20 (p=0.042). Analysis of the T-bet/GATA3 ratio showed that 2 out of 17 T2D had a Th1 response with a mean ratio of 1.467 and 15 out of 17 T2D has a Th2 response with a mean ratio of -0.414. Furthermore, there was a decrease in the FOXP3 and TNF-α levels. The reaction time (R time) was 2-fold higher in T2D (11.0 vs. controls: 4.89; p=0.097).
Conclusion: There are variations in gene expression of coagulation factors and inflammation transcription factors in T2D. Factor XIIIa was substantially higher in T2D compared to the other factors measured and may play a role in the coagulopathies observed in T2D patients. Further investigation of reduce cytokines and inflammation transcription factors is required to determine the relationship between T-regulatory cells and coagulopathies.
Keyword: Type 2 diabetes. Coagulation. Pro-Inflammation.

Physiological stress and burnout in Crossfit participants
Jenna Robinson1
Prof Peet du Toit1, Dr Priyesh Bipath1
Department of Physiology, School of Medicine, Faculty of Health Sciences, University of Pretoria1
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Introduction:  Burnout is primarily classified as a syndrome of emotional exhaustion and physical fatigue. Longstanding burnout is said to carry an increased risk for negative health consequences such as cardiovascular, metabolic and inflammatory diseases. In addition to the effects on physical health, burnout may negatively impact on productivity, quality of life and psychological well-being in general.
Crossfit training involves the high intensity workouts of a combination of elements from several sports such as gymnastics, powerlifting, rowing and running. Mental strength and resistance to fatigue is essential, especially during competitions. Physical fatigue and emotional exhaustion due to overtraining may lead to exercise-related burnout. Apart from proper training regimens, research necessitates investigating the physiological effects of burnout to help improve quality of health.
Aim: The aim of this study was to investigate physiological stress and burnout in crossfit participants undergoing a workout programme.
Methods: Male participants (n=15; >18 years) with a mean BMI of 26.61±1.69 kg/m2 were recruited from Crossfit Brooklyn in Pretoria. The participants were required to perform a pre-determined crossfit workout in order to test the sympathetic response. Measurements included skin conductance (electrodermal response), electromyography, blood volume pulse, skin temperature, blood pressure and anthropometric indices. Physiological measurements were taken at both baseline and post workout. The Shirom-Melamed Burnout Scale and Short-form 36 (SF36) questionnaires were used to assess burnout and physical well-being respectively.
Results:  When comparing the sympathetic overload for the post workout versus baseline, there was a significant increase in blood volume pulse (126.5±1.2 vs. 73.5±9.4 beats/min; p<0.001) and electrodermal response (9.2±4.7 vs. 4.9±1.9 µSiemans; p=0.006) but not in electromyography activity (4.8±5.2 vs. 2.9±2.8 µV; p=0.241). For the physical facet 50% of participants reported feeling physically exhausted while only 21% presented with burnout on the frequency scale. The emotional exhaustion and cognitive weariness facets were distributed unevenly across the questionnaire scales. The results warrant future investigations.
Conclusion: The results present a new dynamic for understanding physiological stress and burnout in crossfit. The research is aligned to help improve the future well-being of crossfit participants in order to avoid burnout.

Effects of VEGF-VEFR-1,-2 signalling blockade in tumour growth and angiogenesis in vitro
Wihan Scholtz1
Dr. Amanda Skepu2, Prof. Annie M Joubert1, Dr. Peace Mabeta1
Department of Physiology, School of Medicine, Faculty of Health Sciences, University of Pretoria1
Biomaterials division, Mintek, RSA2
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Growing tumours have the ability to stimulate angiogenesis – the process of new blood vessel formation from existing vessels. The progression of several tumours such as melanoma is highly dependent on angiogenesis. Increased angiogenesis promotes the vertical growth of melanoma, and is associated with poor prognosis. Tumour angiogenesis is stimulated by the production of proangiogenic factors, including the most studied factor, vascular endothelial growth factor (VEGF). Vascular endothelial growth factor binds to the vascular endothelial growth factor receptors-1 and -2 (VEGFR1 and VEGFR-2). The VEGFR-2 has become a target for anti-cancer treatment.
However, signaling through VEGFR-1, and its contribution to tumour growth, has been less studied. The purpose of this study was to determine the effects of blocking VEGF receptors 1 and 2 on tumour cell growth and on angiogenesis. Murine endothelial cells (sEnd.2) that were derived from tumour vessels and B-16 (murine melanoma) cells were treated with anti-VEGFR-1, anti-VEGFR-2 or with Sunitinib – a multi-targeted receptor tyrosine kinase inhibitor that inhibits both VEGF-1 and -2. The crystal violet staining method was used to study cell viability.
Transmission electron microscopy was used to study cell morphology, while the xCELLigence system was used to study cell invasion and migration. An ex vivo aorta ring assay was used to study angiogenesis.
The crystal violet assay showed a dose-dependent decrease in melanoma and endothelial cell growth following the blocking of receptors. The xCELLigence system showed that Sunitinib had the most potent inhibitory effects compared to the control and to other treatment groups. Similarly, the aorta ring assay revealed increased inhibition of vessel formation following treatment with Sunitinib. The results showed that the targeting of multiple receptors may be a more effective therapeutic option in the treatment of melanoma and tumour angiogenesis.

Identification of novel potential pharmacoperones to rescue Neurokinin B receptor mutations
Alexis Schwulst1
Ross C Anderson1, Robert P Millar1, Claire L Newton1
Centre for Neuroendocrinology and Department of Physiology, Faculty of Health Sciences, University of Pretoria1
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Introduction: Neurokinin B (NKB) is a 10-amino acid neuropeptide that is part of the tachykinin family of proteins. It can bind to all three tachykinin receptors (TACR1, TACR2, TACR3), but it binds with highest affinity to the TACR3, also known as Neurokinin B receptor (NKBR). In the hypothalamus NKBR is involved in the control of sexual development and reproduction. Mutations in both the NKB ligand and NKBR have been shown to be implicated in reproductive disorders including hypogonadotropic hypogonadism. NKBR is a G protein-coupled receptor (GPCR). Inactivating mutations in GPCRs often cause their intracellular retention and degradation. The aim of this project is to identify potential pharmacological chaperones (PCs) able to rescue function of intracellularly retained mutants of the NKBR.
Methods: Suitable NKBR mutations have been identified through literature review. The criteria for selection were that they were naturally occurring point mutations identified in human patients that did not show evidence of ligand binding or signalling deficiency. These mutant receptors were then created in mammalian expression vectors and used to transfect HEK293T cells. A receptor ELISA assay was performed to look at cell surface (CS) and total cellular (TC) expression in order to identify any mutations that result in intracellular receptor retention and a range of small molecule NKBR selective ligands were tested for PC activity looking at effects of treatment on receptor expression profiles.
Results: The selected mutations were located in the transmembrane regions (TM) and intra and extra cellular loops (ICL/ECL) of the receptor; they are G93D-TM1, H148L-ECL1, Y256H-TM5, Y267N-TM5, P353S-TM6, Y315C-TM6, and R295S-ICL3. These mutations have been successfully introduced into mammalian expression vectors encoding NKBR and successful exogenous expression was obtained. Differences in CS expression of the mutant receptors were observed with some mutations causing retention. Different effects of the different PC treatments on the different mutants was also observed.
Conclusion: PCs can be used to rescue mutated NKBRs. Those mutants which showed rescue will now be examined to determine whether receptor functionality is also restored.

The effect of Eicosanol on Lipid metabolism and cell death in breast cancer cells in vitro
Danielle Sandra Tatchum1
Mary-Anne Phasha1, Alisa Phulukdaree1, Annie Joubert1
University of Pretoria1
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Introduction: The uncontrolled proliferation is a key characteristic of cancer. Cancer cells require building blocks such as fatty acids (FAs) for biosynthesis of cell membranes and signalling molecules. Eicosanol is a component of policosanol whose beneficial effects in the prevention of atherosclerosis have been reported. Despite evidence showing that deregulated FA and cholesterol biosynthesis are involved in cancer progression, the exact role of FA and cholesterol synthesis is not fully understood.
Materials and Methods: The cytotoxic effect of eicosanol (3.75 μg/ml to 60 μg/ml for 24 h) on the metastatic breast cancer cell line MDA-MB-231 was investigated via the methyl thiazol tetrazolium (MTT), crystal violet (CV) assays and polarization-optical differential interference contrast (PlasDIC). Quantitative polymerase chain reaction (qPCR) was performed to assess the effect of eicosanol on expression of enzymes involved in the fatty acid (fatty acid synthase (FASN)) and cholesterol (3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR)) synthesis pathways.
Results: At 60 μg/ml, 30 μg/ml, 15 μg/ml, eicosanol caused an increase in cell number (crystal violet assay) and cell metabolism (MTT assay). At lower concentrations, 7.5 μg/ml and 3.75 μg/ml, cell number and metabolism were similar to the vehicle control. PlasDIC images showed hallmarks of apoptosis namely, formation of apoptotic bodies and round cells at 3.75 μg/ml. Expression of FASN and HMGCR varied amongst the higher eicosanol treatments but not significantly, while the lowest concentration, 3.75 μg/ml caused a significant downregulation of HMGCR -2.157±0.1 (p=0.0071) and down regulation of FASN 0.5 ±0.8-fold change (p>0.05).
Conclusions: Eicosanol alters expression of key regulatory enzyme of FA and cholesterol synthesis in MDA MB 231 cells at 3.75 μg/ml. Cholesterol biosynthesis is important in transformation and cancer development. Therefore a decrease in cholesterol synthesis could provide a novel anti-cancer strategy to subvert rapid proliferation of cancer cells.

Palmitoleic acid inhibits RANKL-induced osteoclastogenesis and bone resorption by suppressing NF-ƙB and MAPK signalling pathways
Bernadette van Heerden1
Abe Edward Kasonga2, Marlena Kruger1, Magdalena Coetzee1
Department of Physiology, University of Pretoria1
School of Food and Nutrition, Massey Institute of Food Science and Technology, Massey University2
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Osteoclasts are large, multinucleated cells that are responsible for the breakdown or resorption of bone during bone remodelling. Studies have shown that certain fatty acids (FAs) can increase bone formation, reduce bone loss, and influence total bone mass. Palmitoleic acid (PLA) is a 16-carbon, monounsaturated FA that has shown anti-inflammatory properties. The effects of PLA in bone remain unexplored. Here we investigated the effects of PLA on receptor activator of nuclear factor kappa B (NF-ƙB) ligand (RANKL)-induced osteoclast formation and bone resorption in RAW264.7 murine macrophages in vitro.
Methods: RAW264.7 murine macrophages were seeded onto 96-well and 48-well plates in the presence of RANKL to stimulate osteoclast formation. Cells were then exposed to PLA (10-100µM) and cultured for five days to test the effect on osteoclastogenesis. Tartrate resistant acid phosphatase (TRAP) activity, osteoclast number, bone resorption, actin ring formation and osteoclast specific protein and gene expression were then assessed. Three independent experiments were conducted in triplicate for each test.
Results: PLA decreased the number of multinucleated TRAP positive osteoclasts and furthermore, suppressed the osteolytic capability of these osteoclasts. This was accompanied by a decrease in expression of resorption markers (TRAP, matrix metalloproteinase 9 (Mmp9), and cathepsin K (Ctsk). PLA further decreased the expression of genes involved in the formation and function of osteoclasts. Additionally, PLA inhibited NF-ƙB activity and the activation of mitogen activated protein kinases (MAPK), c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK). Moreover, PLA induced apoptosis in mature osteoclasts.
Conclusion: This study revealed that PLA inhibits RANKL-induced osteoclast formation in RAW264.7 murine macrophages through suppression of NF-ƙB and MAPK signalling pathways. This may indicate that PLA has potential as a therapeutic agent for bone diseases characterized by excessive osteoclast formation.
Keywords: palmitoleic acid; osteoclasts; bone resorption; fatty acid